Use of sip protein in preparation of capsular polysaccharide conjugate vaccine, and conjugate comprising sip protein and preparation method therefor and use thereof

Abstract

A use of a Sip protein in the preparation of a capsular polysaccharide conjugate vaccine, and a conjugate comprising the Sip protein and a preparation method therefor and a use thereof, relating to the technical field of polysaccharide conjugate vaccines. The present invention provides the use of a Sip protein in the preparation of a capsular polysaccharide conjugate vaccine. The capsular polysaccharide comprises the group B streptococcus (GBS) capsular polysaccharide. Also provided are the conjugate comprising the Sip protein and the preparation method therefor and the use thereof. By using the technical solution of the present invention, an antigen can be transformed from a T-cell independent antigen to a T-cell dependent antigen, and an antibody that generates specificity can be induced to generate a good immunoprotective effect; moreover, immunogenicity is stable, and good immunological memory can be generated; and furthermore, the protection scope of the prepared capsular polysaccharide conjugate vaccine against infections caused by different serotypes of GBS can be expanded, so that the vaccine can have multiple protections, and has good prospects for application in infections caused by uncommon serotypes of GBS.

Description

Use of Sip protein in preparing a capsular polysaccharide conjugate vaccine, conjugate containing Sip protein and preparation method and use thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of polysaccharide conjugate vaccine, and particularly relates to use of Sip protein in preparing a capsular polysaccharide conjugate vaccine, conjugate containing Sip protein and preparation method and use thereof.

[0002] The present application claims priority from Chinese patent application (application number 202410636365.7) with a filing date of May 22, 2024, the content of which is hereby incorporated by reference in its entirety. BACKGROUND

[0003] Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is a conditional pathogen that seriously threatens pregnant women, newborns and the elderly. GBS usually causes neonatal pneumonia, meningitis and sepsis, which can cause irreversible damage to the nervous system such as loss of hearing and vision, and the mortality rate reaches 5%. At present, maternal vaccine is considered to be the only feasible long-term choice for preventing GBS maternal and infant infection, and is also the preferred choice.

[0004] Firstly, polysaccharide vaccine is constructed on the basis of natural capsular polysaccharide of GBS. Capsular polysaccharide is an extracellular polysaccharide synthesized by GBS during growth, which is a macromolecular polysaccharide polymerized by repeating units of 4 monosaccharides of glucose, galactose, glucosamine and sialic acid, and has high specificity. The sialic acid residue at the end of the side chain of the polysaccharide is the most important virulence factor, which can trigger anti-complement-mediated phagocytosis by phagocytes. There are mainly 10 serotypes of capsular polysaccharide of GBS, of which more than 97% of the clinical isolates are type Ia, Ib, II, III, IV and V, and 98% of GBS clinical infections in China come from type Ia, Ib, III and V. Capsular polysaccharide vaccine shows certain effect on preventing GBS infection, but the immunogenicity of capsular polysaccharide is low, and it is a non-T cell dependent antigen, and the level of immunoglobulin produced is lower than that of the second generation vaccine later, and there is no immune memory; on the other hand, the antibodies produced by the mother are mostly IgM antibodies, which cannot pass through the placenta and can only provide short-term protection for the fetus, and have no obvious protective effect on the newborn.

[0005] In addition, a polysaccharide conjugate vaccine coupled with GBS capsular polysaccharide and tetanus toxoid (TT) or CRM197 (Cross-reacting material 197, CRM197) is also included as a second-generation vaccine. The polysaccharide conjugate vaccine can generate sufficient maternal immune response and effectively cross the placenta to reach the fetus, thereby protecting the fetus from GBS infection. However, CRM197 as a mature marketed polysaccharide conjugate vaccine carrier may have certain immune interference (CIES), leading to immune suppression of the carrier and weakening the immune response of the polysaccharide. At the same time, the polysaccharide conjugate vaccine also needs as many valences as possible to improve the coverage of clinical GBS infection.

[0006] Based on the above, it is necessary to obtain a polysaccharide conjugate vaccine with good tolerance, high immunogenicity and stability, which can generate immune memory, has multiple protective properties, and has high coverage for multiple infections.

[0007] SUMMARY

[0008] The technical problem to be solved by the present application is how to obtain a polysaccharide conjugate vaccine with good tolerance, high immunogenicity and stability, which can generate immune memory, has multiple protective properties, and has high coverage for multiple infections.

[0009] To solve the above technical problem, the application provides an application of Sip protein in the preparation of a capsular polysaccharide conjugate vaccine. The conjugate containing Sip protein and GBS capsular polysaccharide obtained by the application can convert the antigen from non-T cell dependent to T cell dependent, induce specific antibody production, and produce good immune protection. At the same time, the immunogenicity is more stable, can produce good immune memory, and can expand the protection range of the prepared capsular polysaccharide conjugate vaccine against different serotypes of GBS infection, so that the vaccine has multiple protective properties and has better application prospects for infection of less common serotypes of GBS.

[0010] The application provides an application of Sip protein in the preparation of a capsular polysaccharide conjugate vaccine.

[0011] In the application, the Sip protein can be a natural Sip protein, such as Sip protein derived from GBS, or a non-natural Sip protein, such as Sip recombinant protein, etc.

[0012] In the application, the Sip protein can be a protein described in at least one of the following A1) to A3):

[0013] A1) a protein with an amino acid sequence as shown in SEQ ID NO: 2, 2-409;

[0014] A2) a protein having 70% or more identity to the amino acid sequence of the protein of A1) and having the same function;

[0015] A3) a recombinant protein having the same function obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of A1) or A2).

[0016] In some embodiments of the present application, the Sip protein is a Sip recombinant protein having an amino acid sequence as shown in SEQ ID NO: 2.

[0017] As a preferred embodiment, the capsular polysaccharide comprises a group B streptococcal capsular polysaccharide.

[0018] The present application also provides a conjugate comprising the Sip protein, which can comprise a group B streptococcal capsular polysaccharide and the Sip protein.

[0019] Further, the active ingredient of the capsular polysaccharide conjugate vaccine can be a conjugate comprising the Sip protein, which can be obtained by conjugating a group B streptococcal capsular polysaccharide and the Sip protein.

[0020] Further, the conjugate can be used as a vaccine. Further, the vaccine can be used for preventing group B streptococcal infection.

[0021] Further, the conjugate can be used for preventing group B streptococcal infection.

[0022] The conjugate is formed by oxidizing the sialic acid group on the side chain of the capsular polysaccharide to form an aldehyde group and reducing the amine reaction to form a conjugate of group B streptococcal capsular polysaccharide and Sip protein. This treatment does not introduce new substances, and the reduced amino group is both a catalyst and a linking arm, and the yield and immunogenicity of the corresponding capsular polysaccharide conjugate vaccine obtained are better.

[0023] The group B streptococcal capsular polysaccharide used in the present application can be in its natural form, or can be modified. For example, it can be shorter than the natural capsular sugar, or it can be chemically modified.

[0024] In the present application, the group B streptococcal capsular polysaccharide preferably comprises group B streptococcal type Ia capsular polysaccharide, group B streptococcal type Ib capsular polysaccharide, group B streptococcal type III capsular polysaccharide, group B streptococcal type IV capsular polysaccharide, and group B streptococcal type V capsular polysaccharide.

[0025] As a preferred embodiment, the group B streptococcal capsular polysaccharide comprises group B streptococcal type Ia capsular polysaccharide.

[0026] The side chain groups of the polysaccharide molecule are selectively activated by sodium periodate to form aldehyde groups, which can dehydrate and condense with lysine groups on the protein molecule to form imine groups after mixing with the protein. The imine groups act as both a linker and a reducing agent to form a polysaccharide and protein conjugate molecule (i.e., a conjugate).

[0027] The application also provides a preparation method of the conjugate, which can include the following steps:

[0028] (1) Group B streptococcus capsular polysaccharide is mixed with sodium periodate at a mass ratio of 100:(4-20) to activate the group B streptococcus capsular polysaccharide and obtain activated group B streptococcus capsular polysaccharide;

[0029] (2) The obtained activated group B streptococcus capsular polysaccharide is mixed with Sip protein at a mass ratio of (2-8):1, and then sodium cyanoborohydride is added to obtain the conjugate after reaction.

[0030] Preferably, the activation time in step (1) is 1-6 h, and the activation temperature is 24-27℃.

[0031] Preferably, the mass ratio of the amount of sodium cyanoborohydride to the activated group B streptococcus capsular polysaccharide in step (2) is (3-6):(3-6).

[0032] Preferably, the reaction temperature in step (2) is 37-45℃.

[0033] The application also provides use of the conjugate in preparation of a capsular polysaccharide conjugate vaccine.

[0034] The application also provides a capsular polysaccharide conjugate vaccine comprising the conjugate.

[0035] The application also provides Sip protein for use as a drug.

[0036] Further, the Sip protein for use as a drug, wherein the drug can be a vaccine for preventing group B streptococcus infection.

[0037] The application also provides a pharmaceutical composition for preventing group B streptococcus infection, which can include the conjugate described above and a pharmaceutically acceptable carrier.

[0038] The application also provides use of the conjugate described above in preparation of a drug for inducing an immune response in a mammal.

[0039] The application also provides use of the conjugate described above in preparation of a drug for preventing group B streptococcus infection in a mammal.

[0040] The application also provides a pharmaceutical composition for use in inducing an immune response in a mammal, which can include the conjugate described above and a pharmaceutically acceptable carrier.

[0041] Further, the pharmaceutical compositions or medicaments provided herein can be immunogenic compositions, and more preferably can be vaccines. The vaccines of the present application can be prophylactic (i.e., to prevent infection) or therapeutic (i.e., to treat infection), but are generally prophylactic. Immunogenic compositions for use as vaccines comprise an immunologically effective amount of an antigen, and any other components as desired.

[0042] The pharmaceutical compositions described herein can generally be administered in combination with other immunomodulators. In particular, the pharmaceutical compositions described herein generally comprise one or more adjuvants.

[0043] The present application also provides methods of preventing a Group B streptococcal infection, comprising administering to a mammal an immunologically effective amount of a conjugate described above and / or a pharmaceutical composition described above, to elicit an immune response in the mammal.

[0044] An immunologically effective amount means an amount (either a single dose or as part of a series of doses) that is effective to treat or prevent in an individual. This amount will vary depending on the health and physical condition of the individual to be treated, the taxonomic group (e.g., non- human primate, primate, etc.) to be treated, the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the vaccine formulation, the assessment of the medical situation by the treating physician, and other relevant factors. It is expected that this amount will be in the relatively broad range of amounts that can be determined by routine experimentation. In some embodiments of the present application, the dosage administered to an 8 week old mouse is 5 μg of polysaccharide per mouse.

[0045] The present application also provides methods of eliciting an immune response in a mammal, comprising administering to the mammal an immunologically effective amount of a conjugate and / or a pharmaceutical composition described above. The immune response is preferably protective and preferably involves antibodies. The methods can elicit a booster response.

[0046] The uses and methods provided herein are preferably used to prevent and / or treat a disease caused by Group B streptococcus, such as neonatal septicemia or bacteremia, neonatal pneumonia, neonatal meningitis, endometritis, osteomyelitis, septic arthritis, etc.

[0047] The subject to be protected from disease can be different from the subject receiving the conjugate, pharmaceutical composition, or vaccine of the present application. For example, a conjugate, pharmaceutical composition, or vaccine can be administered to a woman (either pre- or peri-natally) to protect the offspring (referred to as "maternal immunization").

[0048] One method of monitoring the effectiveness of therapeutic treatment involves monitoring GBS infection after administration of the compositions of the present application. One method of monitoring the effectiveness of prophylactic treatment involves monitoring the immune response against GBS antigens after administration of the compositions.

[0049] The compositions of the present application are generally administered directly to a patient. Direct delivery can be accomplished by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or interstitial injection), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary, or other mucosal administration. Intramuscular administration in the thigh or upper arm is preferred. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 is a high performance liquid chromatogram of a purified polysaccharide solution;

[0051] Figure 2 is a nuclear magnetic H spectrum of a purified polysaccharide;

[0052] Figure 3 is a SDS-PAGE electrophoresis result of a purified Sip protein;

[0053] Figure 4 is a SDS-PAGE electrophoresis result of Ia-Sip conjugate, Sip protein, Ia-CRM197 conjugate, and CRM197 protein;

[0054] Figure 5 is a high performance liquid chromatogram of Ia-Sip, Ia-CRM197 conjugate, and corresponding raw materials (the upper graph in Figure 5 is Ia-CRM197 conjugate and corresponding raw materials, and the lower graph in Figure 5 is Ia-Sip conjugate and corresponding raw materials);

[0055] Figure 6 is the IgG titer of rabbit serum against type Ia CPS in different treatment groups;

[0056] Figure 7 is the opsonophagocytic killing of type Ia GBS cells by rabbit serum in different treatment groups after three immunizations;

[0057] Figure 8 is a survival curve of young mice injected with rabbit serum in different treatment groups after challenge with Ia515 strain;

[0058] Figure 9 is the IgG titer of mouse serum against type Ia CPS in different treatment groups;

[0059] Figure 10 is a survival curve of mice in different treatment groups after challenge with Ia515 strain after immunization;

[0060] Figure 11 is the cross-protection results of rabbit serum in different treatment groups after three immunizations against type Ib, II, III, and V strains;

[0061] Figure 12 is a passive protection experiment of type Ib, III, and V strains in different treatment groups after four immunizations;

[0062] Figure 13 is a survival curve of mice in different treatment groups after challenge with III COH1 strain or type V 2603V / R after immunization;

[0063] Figure 14 is a mouse serum typing result.

[0064] Embodiments of the present application

[0065] I. Terms used in the present application:

[0066] Any reference cited herein, including, for example, all patents, published patent applications and non-patent publications, are incorporated by reference in their entirety.

[0067] For the purpose of facilitating the understanding of the present application, several terms and abbreviations used herein are defined as follows:

[0068] In the present application, "identity" refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence (or a nucleotide sequence) can be determined using homology search sites on the internet, such as the BLAST page of the NCBI home page website. For example, the identity of an amino acid sequence can be calculated by searching using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, in Advanced BLAST 2.1, and then the value of identity (%) can be obtained.

[0069] Specifically, the 70% or more identity can be 75% or more identity. Specifically, the 75% or more identity can be 80% or more identity. Specifically, the 80% or more identity can be 85% or more identity. Specifically, the 85% or more identity can be 90% or more identity. Specifically, the 90% or more identity can be 91% or more identity, 92% or more identity, 93% or more identity, 94% or more identity, 95% or more identity, 96% or more identity, 97% or more identity, 98% or more identity, or 99% or more identity. More specifically, the 70% or more identity can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0070] The term "and / or," when used in the context of a list of two or more items, means that any single one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, the expression "A and / or B" is intended to mean either or the other of the items A and B or their combination. The expression "A, B, and / or C" is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A and B and C in combination.

[0071] The term "capsular polysaccharide conjugate vaccine" or "polysaccharide conjugate vaccine" refers to a vaccine prepared from a conjugate of a polysaccharide with a non-sugar unit (protein or short peptide, lipid) formed by covalent linkage with variable structure and number of polysaccharide units.

[0072] A "pharmaceutically acceptable carrier" generally includes any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are generally large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polyamino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known in the art. The vaccines can also contain diluents, such as water, saline, glycerol, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can be present. Sterile pyrogen-free phosphate-buffered physiologic saline is a typical carrier.

[0073] The pharmaceutical compositions of the present application can be in aqueous form (i.e., solutions or suspensions) or in dry form (e.g., lyophilized). If a lyophilized vaccine is to be used, it can be reconstituted with a liquid medium prior to injection.

[0074] "Immune response" (IR) refers to the process by which immune cells recognize, activate, proliferate and differentiate in response to antigenic stimulation, and produce immune substances to exert specific immune effects. This process is a comprehensive reflection of the physiological functions of various parts of the immune system, including antigen presentation, lymphocyte activation, immune molecule formation, and immune effect occurrence, and a series of physiological reactions. Through effective immune response, the body is able to maintain the stability of the internal environment. It is often used as a synonym for immune reaction. II. Examples

[0075] The present application is further described in detail by reference to specific embodiments given only by way of illustration. The examples provided below serve only as a guide to further improve the present technology for those of ordinary skill in the art, and do not in any way constitute a limitation on the scope of the present application.

[0076] The experimental methods in the following examples are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0077] The GBS strain information is as follows: Ia strain 515 (ATCC product, BAA-1177); Ib strain (ATCC product, 12401); type III strain COH1 (ATCC product, BAA-1176); type V strain 2603V / R (ATCC product, BAA-611).

[0078] In the following examples, the CB culture medium (Columbia Broth Medium, abbreviated as CB) is prepared as follows: 35.0 g of CB culture medium (Solarbio product, product number LA8080) is dissolved in 1 L of pure water, heated, divided, sterilized at 121°C for 15 minutes, and used.

[0079] In the following examples, the THB liquid medium is prepared as follows: 30 g of THB culture medium (BD BactoTM Todd Hewitt Broth, product number 249240) is dissolved in 1 L of pure water, sterilized at 121°C for 15 minutes, and used. The THB solid medium or THB plate is prepared by adding 15 g of agar to the THB liquid medium.

[0080] In the following examples, the THY liquid medium is prepared as follows: 30 g of THB culture medium (BD BactoTM Todd Hewitt Broth, product number 249240) and 5 g of yeast extract are dissolved in 1 L of pure water, sterilized at 121°C for 15 minutes, and used.

[0081] In the following examples, the GBS-related operations and challenge test-related operations are performed in a P2 (BSL-2) level biosafety laboratory of the Military Medical Research Institute of the Chinese People's Liberation Army.

[0082] In the following examples, the quantitative test is performed in triplicate, and the results are averaged, unless otherwise specified.

[0083] In the following examples, the data is processed using GraphPad Prism statistical software, and the experimental results are expressed as mean ± standard deviation. Two-way ANOVA is used, and * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and **** represents P<0.0001.

[0084] The application provides an application of Sip protein in preparation of a capsular polysaccharide conjugate vaccine.

[0085] In the present application, the capsular polysaccharide includes Streptococcus group B capsular polysaccharide.

[0086] The present application provides a conjugate containing the Sip protein, which includes Streptococcus group B capsular polysaccharide and the Sip protein.

[0087] In the present application, the Streptococcus group B capsular polysaccharide preferably includes Streptococcus group B type Ia capsular polysaccharide, Streptococcus group B type Ib capsular polysaccharide, Streptococcus group B type III capsular polysaccharide, Streptococcus group B type IV capsular polysaccharide, Streptococcus group B type V capsular polysaccharide, and further preferably includes Streptococcus group B type Ia capsular polysaccharide.

[0088] The present application provides a preparation method of the conjugate, which includes the following steps:

[0089] (1) mixing and activating Streptococcus group B capsular polysaccharide and sodium periodate at a mass ratio of 100:(4-20) to obtain activated Streptococcus group B capsular polysaccharide;

[0090] (2) mixing the obtained activated Streptococcus group B capsular polysaccharide and Sip protein at a mass ratio of (2-8):1, and then adding sodium cyanoborohydride to obtain the conjugate after reaction.

[0091] In the present application, the Streptococcus group B capsular polysaccharide is first dissolved in a sodium acetate buffer solution, and then sodium periodate is added for mixing. The concentration of the sodium acetate buffer solution is preferably 45-55 mM, further preferably 48-52 mM, and more preferably 50 mM; and the pH value of the sodium acetate buffer solution is preferably 4.4-4.6, and further preferably 4.5. The concentration of the polysaccharide in the present application is preferably 1.5-2.5 mg / mL, further preferably 1.8-2.3 mg / mL, and more preferably 2 mg / mL. The addition amount of the Streptococcus group B capsular polysaccharide to sodium periodate in the present application is preferably at a mass ratio of 100:(4-20), further preferably 100:(6-14), and more preferably 100:8. After uniform mixing, activation is performed in the present application. The activation time is preferably 1-6 h, further preferably 1.5-4 h, and more preferably 2 h; and the activation temperature is preferably 24-27℃, further preferably 25-26℃. The activation degree of the activated Streptococcus group B capsular polysaccharide is preferably 15-30, further preferably 20-26, and more preferably 24. In the present application, ethylene glycol is preferably added to terminate the reaction at the end of the activation reaction. The addition amount of the ethylene glycol is preferably 1 / (1.5-3) times, further preferably 1 / (1.8-2.5) times, and more preferably 1 / 2 times of the volume of sodium periodate.

[0092] In the present application, the obtained activated group B streptococcus capsular polysaccharide is first dissolved in sodium bicarbonate buffer, and then the Sip protein is added for mixing. The concentration of the sodium bicarbonate buffer in the present application is preferably 0.07-0.08 mm, further preferably 0.072-0.078 mm, and more preferably 0.075 mm; the pH value of the sodium bicarbonate buffer is preferably 8.1-8.3, and further preferably 8.2. The mass ratio of the activated group B streptococcus capsular polysaccharide to the Sip protein in the present application is preferably (2-8): 1, further preferably (3-6): 1, and more preferably 4: 1.

[0093] In the present application, after the activated group B streptococcus capsular polysaccharide is mixed with the Sip protein, sodium cyanoborohydride is added, and the conjugate is obtained after reaction. In the present application, the mass ratio of the amount of sodium cyanoborohydride added to the activated group B streptococcus capsular polysaccharide is preferably (3-6):(3-6), further preferably (4-5.5):(3.5-5), and more preferably 5:4; the reaction temperature is preferably 40-41℃, further preferably 40.2-40.7℃, and more preferably 40.5℃. In the present application, sodium borohydride is preferably added to terminate the reaction at the end of the reaction, and the amount of sodium borohydride added is preferably 1 / (2-4) times, further preferably 1 / (2.5-3.5) times, and more preferably 1 / 3 times the volume of sodium cyanoborohydride.

[0094] The present application also provides the use of the conjugate in the preparation of a capsular polysaccharide conjugate vaccine.

[0095] The present application also provides a capsular polysaccharide conjugate vaccine comprising the conjugate.

[0096] The technical solutions provided by the present application are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0097] Example 1 Extraction of polysaccharide

[0098] Capsular polysaccharide is an extracellular polysaccharide synthesized by group B streptococcus during growth and attached to the cell wall. Due to stirring during fermentation, it often detaches from the bacteria and enters the culture medium, so polysaccharide can be obtained in the culture supernatant, and then purified. Exemplary fermentation and purification steps for obtaining polysaccharide are as follows:

[0099] (1) 10L fermenter fermentation

[0100] (1) Fermenter on-tank strain culture

[0101] Take out one -80℃ glycerol frozen Ia strain 515, 37℃ fast thawing. In a sterile clean bench, take 50 μL from the frozen strain, inoculate 5mL CB medium with a micro pipette gun, shake quickly and mix, a total of 2 tubes, incubate at 37℃ for 12 hours for recovery. After incubation, take 700 μL of the bacterial solution into 70mL CB medium in a sterile clean bench, incubate at 37℃ for 5 hours.

[0102] (2) Preparation before fermentation

[0103] a) Debug the working system of the fermentation tank (AWD-ZN101).

[0104] b) Configure the fermentation broth, take 5L deionized water and add 245g Columbia broth medium to a magnetic stirrer to dissolve. Take 600g glucose and add it to 600mL deionized water, heat and stir for 2 hours on a magnetic stirrer to dissolve, transfer to a clean bench, sterilize the walls with 75% ethanol, connect a 500mL sterile filter and a vacuum pump air inlet, start the vacuum pump to make the sterile receiving flask in a low pressure state, pour the glucose solution into the sterile filter, and collect the sterile glucose solution in a 121℃ sterilized glass bottle. Configure 1.2L of 5M / L KOH and 33% phosphoric acid solution, add to the fermentation tank feed bottle.

[0105] (3) Sterilization of the fermentation tank and parameter verification

[0106] a) Sterilization parameter setting: set the air filter sterilization time to 30min, dry blowing for 40min; air sterilization for 30min, sterilization temperature 121.5℃, lower limit temperature 121.3℃; real sterilization for 20min, preheating temperature 95℃, fast exhaust delay 2min, cooling temperature 42℃.

[0107] b) Filter sterilization: reconfirm the sealing of the fermentation tank body, turn on the filter sterilization switch, and sterilize the gas filter with steam pressure not exceeding 0.2MPa.

[0108] c) Air sterilization: completely open the exhaust valve of the fermentation tank, and turn on the air sterilization switch. After air sterilization, wait until the pressure gauge drops to 0, then open the discharge port and the feed port to discharge the condensed water.

[0109] d) Parameter verification: connect the pH electrode to the data line, carefully remove the electrode protection sleeve, wash the electrode with deionized water, and then place it in a standard buffer solution with pH 7.00. Wait for about 30min for the system to stabilize and correct the neutral pH. Wash the electrode, dry it, and place it in a standard solution with pH 4.01. Wait for the pH to stabilize and correct the acidity. The corrected electrode cannot be disconnected from the data line, otherwise it needs to be rechecked.

[0110] e) Real-Off: Open the electrode port, take out the sealed bucket, carefully install the pH electrode sheath, insert the electrode port after installing the sealing ring, and tighten the nut. Close the discharge port, open the feed port, pour in the fermentation liquid and add 0.1% defoaming agent, and tighten the feed port. Turn on the fermentation tank stirring, speed 300 rpm, turn on the real-off switch.

[0111] f) Real-Off End: Turn off the steam generator, turn on the stirring, wait for the fermentation tank cooling program to cool down to the set temperature, open the fermentation tank exhaust valve to adjust the tank pressure to 0.02 MPa, open the drain valve to the sewer, and close the drain valve to the room.

[0112] (4) Fermentation

[0113] a) Set the fermentation parameters: temperature 37°C, pH 7.00, stirring speed 300 rpm, aeration rate 5 L / h, tank pressure 0.02 MPa, fermentation time 16 hours.

[0114] b) Take out the feed tube of the feed bottle, clamp the silicone tube into the acid / base pump, unscrew the feed port sealing cap, and insert the needle end of the feed tube into the feed hole after sterilizing it with a flame.

[0115] c) Pour pure alcohol into the feed port and place alcohol cotton, ignite the flame, adjust the tank pressure to less than 0.01 MPa, unscrew the feed port, pour the glucose solution filtered by a sterile filter and the 5-hour cultured inoculum into the fermentation tank, close the feed port, adjust the tank pressure, and start fermentation.

[0116] d) Sampling: First open the lower valve of the discharge port, then quickly open the upper valve, after a small amount of liquid flows out, connect the sample. First close the upper valve, then quickly close the lower valve. The entire sampling process should be operated quickly to avoid contamination.

[0117] (II) Fermentation liquid treatment: separation of supernatant and bacterial cells

[0118] a) Collect all the fermentation liquid and transfer it to the clean bench, balance it with an electronic balance, use a Hitachi centrifuge at 10000 rpm for 20 min, carefully pour out the supernatant to avoid pouring out the bacterial cells, the supernatant should be transparent and clear, if there are signs of turbidity, it should be re-balanced and the bacterial cells should be separated again.

[0119] b) rinse the triple-solvent filter and 3L receiver with deionized water, and then connect the triple-solvent filter, receiver and vacuum pump inlet in order. Take the 0.45 μm water-phase filter membrane and place it between the filter cup and stainless steel filter head, so that the filter membrane completely covers the column stainless steel filter head, and then clamp and fix it with a spring clamp. Turn on the vacuum pump and check whether there is any air leakage in the filtration system, and confirm that the filter membrane is tightly attached to the stainless steel filter head. Pour the supernatant of the centrifuged fermentation broth into the filter cup and start the filtration. After the filtration of the 0.45 μm filter membrane is completed, wash the filtration system with deionized water, take the 0.22 μm water-phase filter membrane and repeat the filtration process to ensure that the bacterial cells and insoluble impurities are completely removed.

[0120] (III) Purification of polysaccharide

[0121] For example, the operation process of polysaccharide purification can refer to "Study on the separation and purification process of group B streptococcus capsular polysaccharide antigen" (Yang Yufang, Liang Linlin, Sun Xuenan, et al. Study on the separation and purification process of group B streptococcus capsular polysaccharide antigen [J]. Chinese Journal of Biochemical Pharmacy, 2006, Vol. 27, No. 3).

[0122] The purified crude sugar solution prepared by the above method is determined by high performance liquid chromatography, and the structure of the polysaccharide is identified.

[0123] As can be seen from Figure 1, there is a peak with a retention time of 26.5 minutes in the high performance liquid chromatogram, which is speculated to be a molecule greater than 100 KDa according to the retention time of the dextran standard, which is basically consistent with the molecular weight of polysaccharide la. In addition to the main peak, there is a small peak of about 5 KDa, which may be produced by spontaneous degradation of polysaccharide during the test. No large molecule peak other than 100 KDa is found in the high performance liquid chromatography result, that is, the obtained product is a single large molecule polysaccharide.

[0124] The purified polysaccharide dissolved in water for injection was directly freeze-dried by a freeze dryer, and was sent to a company for H nuclear magnetic spectrum, and the structure of the polysaccharide was verified by experiments. The results are shown in FIG. 2. The characteristic peaks in the sample are completely consistent with the polysaccharide Ia polysaccharide recorded in the literature (Pinto V, Berti F. Exploring the Group B Streptococcus capsular polysaccharides: The structural diversity provides the basis for development of NMR-based identity assays [J]. Journal of Pharmaceutical and Biomedical Analysis, 2014, 98: 9-15.) and no obvious impurity peak is found, which indicates that the obtained product polysaccharide is Ia polysaccharide, the structure is correct and the purity is high.

[0125] The water for injection is ultrapure water without heat source.

[0126] Example 2 Coupling of Capsular Polysaccharide and Sip Protein

[0127] (I) Extraction and purification of Sip protein

[0128] The DNA molecule (Sip protein coding gene without tag and signal peptide) with nucleotide sequence of SEQ ID NO: 1 is used to replace the fragment between NcoI and XhoI enzyme digestion recognition sites of pET28a(+) vector (where the first five nucleotides CCATG of NcoI enzyme digestion recognition site and the whole XhoI enzyme digestion recognition site are retained), to obtain a recombinant expression vector pET28a-Sip. The recombinant expression vector is constructed into E. coli BL21(DE3) super competent cells (Beijing Quanshi Gold Biotechnology Co., Ltd., BL21(DE3)) to obtain a recombinant expression microorganism BL21 / pET28a-Sip. The recombinant expression microorganism BL21 / pET28a-Sip is cultured using a culture medium and is induced for expression using IPTG. The obtained bacteria are collected by ultrasonic disruption, and the supernatant after disruption of the bacteria is collected and purified by a nickel column to obtain Sip recombinant protein. The amino acid sequence of the Sip recombinant protein is shown in SEQ ID NO: 2, and the 2nd to 409th positions of SEQ ID NO: 2 are the amino acid sequence of Sip protein; the 410th to 417th positions are the amino acid sequence of the pET28a(+) vector sequence transcribed and translated, and the 412th to 417th positions are the amino acid sequence of His tag.

[0129] Table 1 Amino acid sequence of Sip recombinant protein and its coding gene

[0130] The specific operation is as follows:

[0131] The same protein expression strain is taken out, inoculated into LB liquid medium containing kana resistance at 1% in a sterile clean bench, and incubated at 37°C and 180 rpm for 12 hours as seed bacteria. After successful seed bacteria culture, transfer to a sterile clean bench, inoculate into LB liquid medium containing kana resistance at 1%, and incubate at 37°C and 180 rpm for about 2.5 hours. Sample in a sterile clean bench to measure the OD of the bacterial solution 600 nm Add inducer IPTG between 0.6-0.8, incubate at 37°C and 180 rpm for 4 hours. Centrifuge the bacterial solution at 4°C and 10000 rpm for 10 min, discard the supernatant and collect the bacterial body.

[0132] Resuspend the bacterial body with 1 / 10 volume of loading solution, place it on ice and break the cells with an ultrasonic wave disruptor for half an hour until the liquid changes from turbidity to clarity (open 5s and stop 3s, 20KHz x 25%). Centrifuge at 4°C and 10000 rpm for 10 min, filter the supernatant with a 0.45μm water phase filter to collect the protein solution. Turn on the purification instrument, connect the purification software, clean the purification instrument pipeline with elution solution and loading solution respectively, connect the nickel column, confirm that there is no liquid leakage in the system, equilibrate the column with 2-3 times the column volume of loading solution, 5mL / min, until the UV and conductivity detection show that the pH and conductivity are consistent with the loading solution, set the baseline. Replace the loading solution with the protein solution, 3mL / min, after loading is completed, replace it with the loading solution, equilibrate the column with 2-3 times the column volume, 15mL / min, until the detection conductivity and UV return to the baseline. Set the elution program, sequentially elute the elution solution in the mobile phase from 0 to 100% in 60 min, 3mL / min, collect the target product according to the UV280 peak of the elution curve, i.e. Sip protein.

[0133] Loading solution: 20mM PB buffer (pH7.4) with 0.5M NaCl and 20mM imidazole;

[0134] Elution solution: 20mM PB buffer (pH7.4) with 0.5M NaCl and 0.5M imidazole.

[0135] The collected Sip protein is measured for protein concentration with nanodrop, and observed by 10% precast gel electrophoresis staining. As can be seen from Figure 3, the protein is concentrated between 40KDa-60 KDa, and the original length of Sip protein is known to be 45KDa, the results show relatively few miscellaneous bands and the molecular weight is less than the target band, which may be due to the rupture of the protein during boiling. It is considered that Sip protein is expressed and purified with high purity, which can be used for subsequent coupling experiments.

[0136] (ii) Polysaccharide protein coupling

[0137] After the extracted polysaccharide Ia is activated by high iodine, an aldehyde group is formed at the side chain sialic acid. The reaction solvent is replaced and the broken polysaccharide in the activation process is removed by 30KDa ultrafiltration. A certain amount of Sip protein is added to the collected polysaccharide after activation, and the coupling is realized after the reaction is completed under specific conditions. The specific operation is as follows.

[0138] 1. Activation of polysaccharide:

[0139] Dissolve 2mg / mL polysaccharide Ia in 50mm sodium acetate buffer solution (pH 4.5), add sodium periodate according to the mass ratio of polysaccharide to sodium periodate 100:8, slowly rotate and mix in the dark at room temperature, activate for 2 hours, add half of the glycol of the added sodium periodate to terminate the reaction, and the mass of the added sodium periodate should be such that the activation degree of the activated polysaccharide is 24 at the end of the reaction. Replace the solution with 50 times the volume of water for injection by 30KDa ultrafiltration tube.

[0140] Determination of polysaccharide activation degree: AHMT method is to use the condensation reaction of aldehyde group and AHMT in alkaline environment, and the product is oxidized to red purple compound by potassium permanganate, which has the highest absorption peak at 540nm. Dissolve DL-glyceraldehyde dimer with ultrapure water to prepare a stock solution of 1mmol / L, dilute 5 times, and then use a micropipette to take 0, 100, 200, 300, 400, 500μL into a 2mL EP tube, respectively, and add ultrapure water to a final volume of 1mL, shake well, then use a micropipette to take 500μL into a 5mL EP tube, respectively, and use a micropipette to add 750μL of 1% purpald solution to each tube, mix well, and place in a 37℃ incubator with the lid open at 180rpm for 30min. Immediately after the reaction is completed, use a pipette to add 1.75mL of 0.2% sodium borohydride solution (purchased from Sigma) to quickly mix and terminate the reaction, and take 200μL from each tube into an enzyme-labeled tube to determine the absorbance at 540nm. Take DL-glyceraldehyde dimer as the abscissa and OD 540nm as the ordinate to draw a standard curve. Each time the sample is measured, a new standard curve is drawn. Dilute the polysaccharide sample with ultrapure water, dilute 10, 50, 100 times, and then add the same reagents as in the standard curve preparation process and treat them in the same way. Then take 200μL of sample from each tube and add it to an enzyme-labeled tube, and measure the absorbance at 540nm. Find the aldehyde group content corresponding to this absorbance on the standard curve.

[0141] Activation degree = sugar concentration / aldehyde group concentration x 1000

[0142] Table 2 Treatment of different groups of samples

[0143] 2. Activation of polysaccharide and coupling with carrier protein: 8 mg / mL of activated polysaccharide was dissolved in 0.075 mm sodium bicarbonate buffer (pH 8.2), and carrier protein was added in a polysaccharide: protein mass ratio of 4:1, and finally sodium cyanoborohydride (purchased from Sigma) was added in a polysaccharide: sodium cyanoborohydride mass ratio of 4:5, and slowly mixed in a 40.5°C incubator in the dark, and 1 / 3 volume of 2 mg / mL sodium borohydride stock solution was added to terminate the reaction. 50 times the volume of water for injection was added to the 100 KDa ultrafiltration tube to replace the solution and remove unreacted polysaccharide and protein. The product was obtained.

[0144] (III) SDS-PAGE electrophoresis

[0145] The polysaccharide protein coupling product obtained above was subjected to SDS-PAGE electrophoresis, and the specific steps were as follows:

[0146] The precast gel plate was clamped vertically into the electrophoresis tank, mops buffer solution was poured into the tank, and the buffer solution between the two precast gel plates was ensured not to communicate with each other, and the comb was carefully removed; the protein sample was added to the loading buffer, mixed well, and then boiled in water bath for 10 min to fully decompose the protein into a single chain; 20 μL of the treated protein sample and 5 μL of the pre-stained protein marker were added to the sample well using a micropipette, and electrophoresis was performed at 180 V constant voltage for about 40 min until the bromophenol blue overflowed from the bottom of the precast gel plate, and then the electrophoresis was stopped; the gel plate was opened, the precast gel was taken out, and was placed in a staining machine for staining, and the electrophoresis was photographed and saved. The results are shown in FIG. 4.

[0147] As can be seen from FIG. 4, the polysaccharide protein coupling product band is concentrated in the sample well, which is due to the large molecular weight of the polysaccharide protein coupling product, which is difficult to enter the gel and finally accumulates in the sample well. The band outside the sample well is speculated to be due to the fragmentation of the coupling product molecules during the boiling water bath denaturation before loading.

[0148] (IV) Detection of polysaccharide and protein concentrations in different polysaccharide coupling products

[0149] 1. The polysaccharide and protein concentrations of the obtained polysaccharide protein coupling product (Ia-Sip coupling product) and Ia-CRM197 were determined respectively.

[0150] The Ia-CRM197 was prepared according to the preparation method of the Ia-Sip coupling product described above by using the Ia polysaccharide prepared above and the CRM197 purchased from Pfenex company.

[0151] The results are shown in Table 3.

[0152] Table 3 Polysaccharide and protein concentrations in different polysaccharide coupling products

[0153] According to Table 6, the polysaccharide protein ratio in the two polysaccharide conjugates is similar, and can be used for subsequent evaluation of immunogenicity.

[0154] 2. The molecular weight of the obtained polysaccharide protein conjugate la-Sip and la-CRM197, and the respective raw polysaccharide and protein, was determined, respectively. The results are shown in Figure 5.

[0155] As can be seen from Figure 5, the high performance liquid chromatogram shows that the polysaccharide protein conjugate has the shortest retention time and the largest molecular weight. The retention times of the polysaccharide and the protein also reflect the molecular size of the substances. The retention time of the polysaccharide is less than that of the protein, which corresponds to the fact that the polysaccharide with a molecular weight of 100 KDa is larger than the protein with a molecular weight of 45 KDa.

[0156] Example 3 Immunogenicity

[0157] (I) Determination of la antibody titer

[0158] In this experiment, New Zealand rabbits were used as immunized rabbits (purchased from Beijing Jinmuyang Experimental Animal Breeding Co., Ltd.), each weighing 3 kg, and were divided into la polysaccharide group, la-Sip conjugate group, la-CRM197 conjugate group, Sip protein group, and control group, 2 rabbits in each group. Each group was injected with the corresponding la polysaccharide, la-Sip conjugate, la-CRM197 conjugate, and Sip protein, respectively, at a dose of 50 μg / rabbit (polysaccharide for polysaccharide, and no polysaccharide for protein). The control group was injected with sterile PBS. The primary immunization was mixed with the sample at a ratio of 1:1 using Freund's complete adjuvant and injected subcutaneously at four points on the back of the rabbit on the first day of the immunization process. The booster immunization was mixed with the sample at a ratio of 1:1 using Freund's incomplete adjuvant and injected intramuscularly in the thigh of the rabbit on days 21 and 42 of the immunization process. Before the start of the immunization process and on days 28 and 49, the rabbit ear vein was bled, and 10 mL of blood was collected from each rabbit. The collected blood was placed in a 37°C incubator for 1 hour, and then placed in a 4°C refrigerator for 1 hour to allow the rabbit serum to fully separate. The treated rabbit blood was centrifuged at 8000 rpm for 10 min at 4°C, and the upper serum was collected and stored at -80°C.

[0159] The serum of rabbits immunized with Ia polysaccharide, Ia-Sip conjugate and Ia-CRM197 conjugate respectively was taken for Elisa test to determine the antibody titer, and the antigen was Ia polysaccharide purified as the raw material of the conjugate, and the primary antibody was the serum obtained by taking blood from the ear vein 7 days after the second and third immunization. The specific operation of the Elisa test is as follows: a) antigen coating: Ia polysaccharide was diluted to 1 / 3 μg / mL with 1x coating solution (Solabio, item C1050), and the diluted polysaccharide was added to the enzyme-labeled plate at 100 μL / well, and the plate was coated at 4°C overnight; the coated plate was placed in a plate washer and washed with PBST for 5 times. b) antigen blocking: 3% BSA solution diluted with PBST was added to the plate at 300 μL / well, and the plate was blocked at 4°C overnight; the blocked plate was placed in a plate washer and washed with PBST for 5 times. c) incubation of primary antibody: the pre-diluted antiserum at 100, 1000, 10000 and 100000 times was added to the plate at 100 μL / well, and each sample was repeated twice, and the plate was incubated in a 37°C incubator for 30 min; the plate after incubation of the primary antibody was placed in a plate washer and washed with PBST for 5 times. d) incubation of secondary antibody: the finished product of goat anti-rabbit antibody was diluted 10000 times with PBST, and the diluted secondary antibody was added to the plate at 100 μL / well, and the plate was incubated in a 37°C incubator for 30 min; the plate after incubation of the secondary antibody was placed in a plate washer and washed with PBST for 5 times. e) color development: TMB color developing solution was added to the plate at 100 μL / well, and the plate was reacted at room temperature for 5-10 min, and 50 μL of stop solution was added to each well to stop the reaction. f) reading: the plate was placed in an enzyme-labeled instrument, and the OD 450nm absorbance was read, and the blank well was used as a comparison.

[0160] The results are shown in Figure 6. Compared with the second immunization, the titers of Ia-Sip conjugate and Ia-CRM197 conjugate increased after the third immunization. Compared with the polysaccharide group, the serum titers of the conjugate group were relatively high after each round of immunization.

[0161] (II) Determination of opsonophagocytic effect

[0162] The specific antibodies produced by the conjugate immunized rabbits are the key to the opsonophagocytic effect in vivo. The pre-immune serum and the serum 7 days after the third immunization of the Ia polysaccharide, Sip protein, Ia-Sip conjugate and Ia-CRM197 conjugate groups were taken for in vitro opsonophagocytic test, and the presence and relative concentration of the neutralizing antibodies in the serum were evidenced by the bactericidal rate.

[0163] A primary HL60 cell bank was established (HL60 cells were purchased from Procella, catalog number CL-0110). The original cells were transferred to 15 mL sterile centrifuge tubes and centrifuged at 800 rpm for 5 min, discarding the supernatant. The HL60 cells were resuspended in 8 mL of Procella culture medium (purchased from Procella, catalog number CM-0110) and transferred to a 25 cm... 2 Cultured in cell culture flasks at 37°C in a cell culture incubator until the cell counter reading reaches 5 × 10⁻⁶. 5 Harvest cells and transfer them to 15 mL sterile centrifuge tubes. Centrifuge at 800 rpm for 5 min, discard the supernatant, and resuspend the HL60 cells in cryopreservation buffer. Transfer the resuspended cells to cryovials and freeze at -80°C for 24 hours, then transfer to liquid nitrogen for storage. For HL60 working cell culture and passage, take one HL60 cell line frozen in liquid nitrogen, rapidly thaw it in a 42°C water bath, and add it to a 25 cm³ agar containing 5 mL of the specific culture medium. 2 Cultured in cell culture flasks at 37°C in a cell culture incubator until the cell counter reading reaches 2 × 10⁻⁶. 6 To meet the requirements for subsequent experiments, HL60 cells were passaged and induced. Cells were transferred to 15 mL sterile centrifuge tubes and centrifuged at 800 rpm for 5 min. The cells were resuspended in dedicated culture medium, diluted 5-fold, and then transferred to 25 cm⁻¹ centrifuge tubes. 2 Cells were cultured in cell culture flasks at 37°C in a cell culture incubator. HL60 induction was performed by transferring HL60 cells to 15 mL sterile centrifuge tubes and centrifuging at 800 rpm for 5 min. The supernatant was discarded, and the HL60 cells were resuspended in antibiotic-free medium and incubated at 25 cm⁻¹. 2 Dilute to 5×10 in cell culture flasks 5 Then add 1% sterile DMSO (dimethyl sulfoxide). Incubate at 37°C for 2-3 days, then transfer to 15mL sterile centrifuge tubes and centrifuge at 800rpm for 5min. Resuspend in antibiotic-free medium and adjust the concentration to 1×10⁻⁶. 6 For experimental use.

[0164] Take one vial of strain Ia 515, frozen at -80℃ with glycerol, thaw at room temperature, streak on a THB plate, and incubate at 37℃ for 24 hours to obtain single colonies. Pick a single colony and add it to a test tube containing 5mL of THB liquid medium, shake to mix, and incubate at 37℃ for 3 hours. After incubation, transfer to a sterile laminar flow hood, inoculate 1% into a test tube containing 5mL of THB liquid medium, incubate at 37℃ for 4.5 hours, centrifuge, resuspend in antibiotic-free medium, and adjust the bacterial concentration to OD0.05. 600nm To approximately 0.3. Take a sterile ELISA plate and add 10 μL of diluted bacterial solution (diluted 10 μL to 10 μL) to each well in a sterile laminar flow hood. 5The enzyme-labeled plate was mixed after adding sample, and was placed in a room temperature shaker for 30 min. After shaking, the enzyme-labeled plate was transferred to a sterile clean bench, 10 μL of young rabbit complement (purchased from Pel Freez) and 40 μL of activated HL60 cells were added to each well, and the mixture was mixed and placed in a 37°C bacterial incubator for rotation mixing for 30 min. 70 μL was taken from each well and coated on a THB plate, and the THB plate was cultured in a 37°C bacterial incubator overnight. The number of colonies on the THB plate was counted, and the bactericidal rate was calculated. The calculation formula of the bactericidal rate is as follows:

[0165] Bactericidal rate (%) = (number of colonies before immunization - number of colonies after immunization) / number of colonies before immunization x 100.

[0166] As can be seen from FIG. 7, neutralizing antibodies were produced in the four groups of rabbit serum, the bactericidal rate of group la was the lowest, the concentration of neutralizing antibodies in the serum was the lowest, the bactericidal rate of la-Sip conjugate group was the highest, and the concentration of neutralizing antibodies was the highest. The concentration of neutralizing antibodies produced by the conjugated vaccine is higher than that of the polysaccharide and protein as raw materials in the body. The results meet the expectations of the test, indicating that the modification of polysaccharide is successful; and the bactericidal rate of the conjugate with Sip as the carrier is higher than that of the conjugate with CRM197 as the carrier, indicating that the conjugate vaccine with surface protein as the carrier for la 515 strain has higher immunogenicity and stronger ability to induce specific antibodies to protect the body.

[0167] (Three) Determination of passive protection effect on young mice

[0168] The 4-week-old SPF level young mice theoretically have no specific antibodies against group B streptococcus. The young mice were injected intraperitoneally with pre-immune and post-immune rabbit serum, and a lethal dose of la type GBS bacterial solution was injected after a period of time. The death of the young mice was recorded, and the survival curve was drawn to evaluate the ability of the serum to neutralize la 515 strain in the young mice.

[0169] The mice used in this experiment were 4-week-old SPF level CD1 female mice (purchased from Spafas (Beijing) Biotechnology Co., Ltd.). Each group had 6 mice, and 40 μL of immune rabbit serum was injected into the test group 12 hours before challenge, and the same dose of pre-immune rabbit serum was injected into the control group.

[0170] In a sterile laminar flow hood, pick a single clone of bacterial strain Ia 515 from a THB plate and inoculate it into 5 mL of THB liquid medium. Shake well and incubate at 37°C for 3 hours. After incubation, shake well and transfer to a sterile laminar flow hood. Inoculate with a 1% inoculum into a sufficient volume of THB liquid medium, mix well, and incubate at 37°C for 4.5 hours. Centrifuge the culture at 10,000 rpm for 5 minutes, discard the supernatant, resuspend the culture in THB liquid medium, and adjust the bacterial concentration to achieve the desired OD value. 600nm To a concentration of 0.3, use THY liquid medium for serial dilution, taking the required dilution increments at 1 mL / animal (1.15 × 10⁻⁶). 5 Mice that had been injected with serum 12 hours prior were given an intraperitoneal injection at a dose of ( / mL) to observe the protective effect of the serum and plot survival curves.

[0171] As shown in Figure 8, rabbit serum from the Ia polysaccharide group showed no protective effect against pups, indicating poor antigen clearance in the pups. The Sip protein, Ia-Sip conjugate, and Ia-CRM197 conjugate groups effectively protected pups from lethal doses of bacterial infection, with significant differences in survival rates before and after immunization. While pups in the pre-immunization rabbit serum group exhibited infection symptoms and gradually died, mice in the Sip protein, Ia-Sip conjugate, and Ia-CRM197 conjugate groups remained healthy and showed almost no mortality. Survival curves of young mice injected with rabbit serum showed that the rabbit serum containing the Sip protein, Ia-Sip conjugate, and Ia-CRM197 conjugate groups contained a large number of neutralizing antibodies against Ia polysaccharide. These antibodies mediated the clearance of type Ia GBS strains in young mice, thus protecting them from death. This demonstrates that the conjugates can produce high concentrations of specific antibodies, effectively protecting the body from infection by type Ia GBS strains, indicating that the Ia-Sip conjugate is a valuable candidate for a conjugate vaccine.

[0172] (iv) Challenge Test

[0173] Mice were immunized three times with a mixture of Ia polysaccharide, Sip protein, Ia-Sip conjugate, Ia-CRM197 conjugate, and sterile PBS, thoroughly mixed with Freund's adjuvant (purchased from Sigma). Twelve mice were immunized in each group. Seven days after the third immunization, a lethal concentration of Ia515 bacterial suspension was injected intraperitoneally. Mice mortality was recorded, and survival curves were plotted. The specific procedures are as follows:

[0174] Immunize mice, this experiment uses 8-week-old CD1 female mice, polysaccharide group, Ia-Sip conjugate group, Ia-CRM197 conjugate group, Sip protein group, 12 in each group, each time with 5 μg / one (with polysaccharide, without polysaccharide, with protein) dose of mice, with sterile PBS as control group. The first immunization with Freund's complete adjuvant and sample mixed in the immune process of the first day of intraperitoneal injection of mice, with Freund's incomplete adjuvant and sample mixed in the immune process of 21, 42 days intraperitoneal injection of mice.

[0175] On the 0th day, the 28th day, the 50th day of the immune process, the mice were irradiated with infrared lamp to speed up the blood circulation of the mice, and then the mice were fixed in the sleeve to cut the tail vein blood, about 500 μL of blood was collected from each mouse, the collected blood was placed in a 37℃ incubator for 1 hour, and then placed in a 4℃ refrigerator for 1 hour, so that the mouse serum was fully separated. The treated mouse blood was centrifuged at 8000 rpm at 4℃ for 10 min, the upper serum was sucked, and the antibody titer was verified by Elisa.

[0176] 1. Take the Ia polysaccharide, Ia-Sip conjugate, Ia-CRM197 conjugate immunized mouse serum to determine the antibody titer by Elisa test, the antigen is the purified Ia polysaccharide as the raw material of the conjugate, the primary antibody is the serum obtained by tail vein blood before immunization, 7 days after the second and third immunization, and the secondary antibody is goat anti-mouse antibody. The results are shown in Figure 9.

[0177] From Figure 9, it can be seen that after vaccination of Ia polysaccharide, Ia-Sip conjugate, Ia-CRM197 conjugate, the serum titer is increased, and it is significantly higher than that of the control group of mice inoculated with PBS, which is basically consistent with the rabbit serum. The results show that the serum titer can reach a high level after the second immunization, and the subsequent immunization enhancement effect is not obvious. The serum titers of Ia-Sip conjugate and Ia-CRM197 conjugate are similar, indicating that the immune response caused by Ia-Sip conjugate is not weaker than that of the vaccine constructed with traditional CRM197 as carrier.

[0178] 2. In a sterile clean bench, single clone bacteria were picked from THB plate and inoculated into 5 mL of THB liquid medium, shaken evenly, and placed in a 37℃ incubator for 3 hours. After taking out, shake evenly, transfer to a sterile clean bench, inoculate into sufficient THB liquid medium at 1%, mix evenly, and place in a 37℃ bacterial incubator for 4.5 hours. The cultured bacteria were centrifuged at 10000 rpm for 5 min, the supernatant was discarded, and the bacteria were resuspended with THY liquid medium. Adjust the concentration of the bacterial solution to about 0.3 OD, dilute with THY liquid medium, take the required dilution gradient, and inoculate 1 mL / one (3.5×10 600 8 ​The mice were injected intraperitoneally with the dose of 1 mL of the vaccine, and the survival of the mice was observed to draw the survival curve.

[0179] As shown in Fig. 10, the mice inoculated with the protein and the conjugate survived from the lethal dose of the bacteria. The control group inoculated with sterile PBS all died within 20 hours, the mice inoculated with the polysaccharide Ia only had one mouse alive and in a poor condition with straight hair after 24 hours, the survival rate of the mice inoculated with the Sip protein, the Ia-CRM197 conjugate reached more than 85% after 24 hours, 10 out of 12 mice survived, and all the mice in the group inoculated with the Ia-Sip conjugate survived after 24 hours and were in good condition without any sign of weakness. The results show that the Ia-Sip conjugate can induce a very effective specific immune response, and the protection effect on the body is much better than that of the polysaccharide Ia before conjugation, which can completely protect the mice from the damage of the lethal dose of the Ia515 bacteria. The results of the Sip protein group show that the Ia515 strain has Sip protein on the surface, and the conjugation of the polysaccharide Ia and the Sip protein further enhances the immunogenicity, so that the immune protection effect on the Ia515 strain is better than that of the traditional Ia-CRM197 scheme.

[0180] (V) Cross-protection

[0181] 1. Take the serum of the rabbits in the Ia-Sip conjugate and Sip protein groups before immunization and 7 days after the third immunization, and perform in vitro opsonophagocytosis test against the type Ib, II, III and V strains (operation same as the phagocytosis test above, only replace the type Ia strain), calculate the bactericidal rate, and verify the cross-protection effect of the antibodies.

[0182] As shown in Fig. 11, the serum obtained after the rabbits are immunized with the Sip protein and the Ia-Sip conjugate also has bactericidal effect on the type Ib, II, III and V strains, and the neutralizing antibodies in the serum of the rabbits after immunization can mediate the opsonophagocytosis of the type Ib, II, III and V strains. Since the bactericidal effect of the serum of the two groups on the type Ib, II, III and V strains is derived from the Sip protein, and the Sip protein group immunizes animals according to the protein concentration and the Ia-Sip conjugate group immunizes animals according to the polysaccharide concentration, the actual injection of antigen of the Sip protein group is 3.82 times that of the Ia-Sip conjugate group, and the bactericidal effect of the Sip protein group is basically slightly better than that of the Ia-Sip conjugate group, which is in line with the expectation. The results confirm that the Ia-Sip conjugate as an antigen has the ability to cause cross-protection, which can cover non-type Ia strains containing Sip surface protein, and can provide a larger protection range compared with the conjugate with CRM197 as the carrier, which embodies the advantages of the Ia-Sip conjugate as a candidate vaccine.

[0183] 2. Serum protection effect test for type Ib, type III and type V strains (operation refers to "(three) determination of passive protection effect of young mice", the only difference is that type Ia strain is replaced). After a period of time (12 hours) after intraperitoneal injection of pre-immune and post-three immunization rabbit serum into young mice, a lethal dose of type Ib, type III and type V bacterial solution is injected, the death of young mice is recorded, the survival curve is drawn, and the ability of serum to neutralize type Ib, type III and type V strains in young mice is evaluated.

[0184] wherein the injected amount of type Ib is 7.9 x 10 5 / mL; the injected amount of type III is 1.325 x 10 5 / mL; and the injected amount of type V is 5.75 x 10 5 / mL.

[0185] As can be seen from FIG. 12, the survival rate of mice injected with serum of the polysaccharide Ia group and the Ia-Sip conjugate group is slightly higher than that of the control group injected with pre-immune serum, indicating that the serum can provide certain cross-protection ability. Since the bactericidal effect of the two groups of serum on type Ib, type III and type V strains is derived from Sip protein, and the Sip protein group immunizes animals according to the protein concentration, and the Ia-Sip conjugate group immunizes animals according to the polysaccharide concentration, the actual injected antigen of the Sip protein group is 3.82 times that of the Ia-Sip conjugate group, and the number of specific antibodies against Sip protein produced by the Ia-Sip conjugate group is less, and the effect of clearing antigens in the body is relatively worse than that of the Sip protein group, which is basically consistent with the in vitro opsonophagocytosis result. The Ia-Sip conjugate can bring certain cross-protection effect, which is the greatest advantage of the conjugate as a candidate vaccine.

[0186] 3. Challenge test for type III or V strains (operation refers to "(four) challenge test", the only difference is that type Ia strain is replaced). Sip protein, Ia-Sip conjugate, Ia-CRM197 conjugate and sterile PBS mixed with Freund's adjuvant are used for three rounds of immunization of mice, 12 in each group, and 1 mL of lethal concentration of type III COH1 bacterial solution or type V 2603V / R bacterial solution is injected intraperitoneally 7 days after the third immunization, the death of mice is recorded, and the survival curve is drawn. The injected amount of type III COH1 strain is 6.85 x 10 8 / mL; and the injected amount of type V 2603V / R strain is 1.95 x 10 8 / mL.

[0187] As can be seen from Fig. 13, the survival rates of the Sip protein and Ia-Sip conjugate groups were slightly higher than that of the control group, and the immunoprotective effect of the Sip protein group was the strongest. The survival rate of the Ia-Sip conjugate group was slightly higher than that of the sterile PBS control group, indicating that the Ia-Sip conjugate group could provide certain cross-protection effect, but since the immunoprotection was derived from the Sip protein as a carrier, a higher concentration of immunization dose needs to be tried to produce sufficient immune response. The Ia-CRM197 conjugate group had no protective effect on type III strains, and all the mice died around 53 hours. The presence of the Sip protein carrier enabled the Ia-Sip conjugate to produce specific antibodies against non-Ia type strains, expanding the protection range of the candidate vaccine, and proving that the candidate vaccine prepared by using the B group streptococcal surface protein Sip as a carrier has a special advantage.

[0188] (VI) IgG antibody typing

[0189] The IgG (purchased from Abeam) typing antibody was used to replace the secondary antibody in the above-mentioned Elisa test. The serum of the mice immunized with Ia polysaccharide, Sip protein, Ia-Sip conjugate and Ia-CRM197 conjugate after the third immunization was detected by IgG typing antibody, and the OD 450nm readings of the same serum dilution were recorded, and the results were summarized and compared.

[0190] As can be seen from Fig. 14, the level of IgG antibody produced by mice inoculated with polysaccharide or protein was relatively low, while the serum of mice inoculated with conjugate had high IgG1 and a small amount of IgG2b. Non-T cell dependent antigen does not produce IgG, and one of the markers of T cell dependent antigen is to produce IgG antibody mainly in IgG1, and the typing results of the conjugate and polysaccharide, protein are consistent with the existing records, which verifies the content of "transforming antigen into T cell dependent antigen by conjugating carrier protein to induce stronger immune response and produce immune memory". The test results confirmed that the modification of Ia polysaccharide was successful. The effect of the Ia-Sip group was better than that of the Ia-CRM197 group, which more strongly supported the feasibility of the Ia-Sip conjugate constructed in the experiment as an effective vaccine.

[0191] In summary, the Ia-Sip conjugate group constructed by the technical solution of the present application shows excellent Ia antibody titer in mice and New Zealand rabbits, and has no significant difference with the Ia-CRM197 conjugate group, and is significantly higher than the Ia polysaccharide immunization group. The serum after immunization of New Zealand rabbits was used for in vitro cell opsonophagocytosis test, and both conjugates showed high bactericidal effect on Ia, and were better than the Ia polysaccharide group. The mouse challenge test shows that both conjugates can protect mice from a lethal dose of Ia type bacteria. This shows that the constructed Ia-Sip candidate vaccine shows good immunogenicity. In addition, the Ia-Sip group is subjected to in vitro opsonophagocytosis and challenge with Ia, II, III and V type strains, and shows certain immunoprotective effect, which shows that the specific antibodies produced by the constructed Ia-Sip are not limited to Ia, but have broad cross-protection, and have better application prospect for multiple infections.

[0192] The present application has been described in detail above. For those skilled in the art, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present application, and without unnecessary experiments. Although specific examples are given in the present application, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, the present application is intended to include any changes, uses or improvements of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application. Industrial applicability

[0193] 1. The conjugate vaccine obtained by coupling Sip protein and group B streptococcus capsular polysaccharide in the technical solution of the present application can change the antigen from non-T cell dependent to T cell dependent, avoid the weakening of polysaccharide immune response caused by carrier effect, induce specific antibody production to produce better immune protection, make the immunogenicity more stable, and can produce better immune memory. At the same time, it also expands the protection range of single vaccine to multiple serotype infections, so that the vaccine has multiple protection.

[0194] 2. In the technical solution of the present application, the aldehyde group is formed by selectively oxidizing the sialic acid group on the side chain of the capsular polysaccharide, and the B group streptococcus capsular polysaccharide and Sip protein form a conjugate through reductive amination reaction. This treatment does not introduce new substances, and the reduced amino group is both a catalyst and a linking arm, and the yield and immunogenicity of the corresponding capsular polysaccharide conjugate vaccine obtained are better.

[0195] 3. The application shows that the constructed Ia-Sip candidate vaccine has good immunogenicity by antibody titer, cell opsonophagocytosis, passive protection and challenge test. The Ia-Sip group shows certain immune protection effect in in vitro opsonophagocytosis and challenge with type Ib, III and V strains, without being limited to Ia, and can realize cross protection, and has the feasibility of becoming an effective vaccine.

Claims

1. Use of Sip protein in the preparation of a capsular polysaccharide conjugate vaccine.

2. Use according to claim 1, characterized in that, The capsular polysaccharide comprises a group B streptococcal capsular polysaccharide.

3. A conjugate comprising the Sip protein of claim 1, wherein The conjugate comprises a group B streptococcal capsular polysaccharide and the Sip protein.

4. The conjugate of claim 3, wherein, The group B streptococcal capsular polysaccharide comprises a group B streptococcal type la capsular polysaccharide.

5. A method for preparing the conjugate of claim 3 or 4, characterized in that, The method comprises the following steps: (1) mixing and activating a group B streptococcal capsular polysaccharide with sodium periodate at a mass ratio of 100:(4-20) to obtain an activated group B streptococcal capsular polysaccharide; (2) mixing the obtained activated group B streptococcal capsular polysaccharide with Sip protein at a mass ratio of (2-8):1, then adding sodium cyanoborohydride, and obtaining the conjugate after reaction.

6. The production method according to claim 5, wherein The activation time in step (1) is 1-6h, and the activation temperature is 24-27℃.

7. The preparation method according to claim 5, characterized in that, The addition amount of sodium cyanoborohydride and activated group B streptococcal capsular polysaccharide in step (2) is (3-6):(3-6) in mass ratio.

8. The preparation method according to claim 5, characterized in that, The reaction temperature in step (2) is 37-45℃.

9. Use of the conjugate of claim 3 or 4 in the preparation of a capsular polysaccharide conjugate vaccine.

10. A capsular polysaccharide conjugate vaccine comprising the conjugate of claim 3 or 4.

11. Use according to claim 1 or 2, characterized in that, The active ingredient of the capsular polysaccharide conjugate vaccine comprises the conjugate of the Sip protein, which is obtained by conjugating the group B streptococcal capsular polysaccharide of claim 3 or 4 and the Sip protein.

12. The conjugate of claim 3 or 4, wherein, The conjugate is used as a vaccine.

13. The conjugate of claim 3 or 4, wherein, The conjugate is used for preventing group B streptococcal infection.

14. The conjugate of claim 3 or 4, wherein, The conjugate is prepared according to the preparation method of any one of claims 5-8.

15. Sip protein for use as a medicament.

16. The Sip protein for use as a medicament according to claim 15, characterized in that, The medicament is a vaccine for preventing group B streptococcal infection.

17. A pharmaceutical composition for preventing a group B streptococcal infection, characterized by, The pharmaceutical composition comprises the conjugate of claim 3 or 4 and a pharmaceutically acceptable carrier.

18. A method for preventing group B streptococcal infection, the method comprising administering an immunologically effective amount of the conjugate of claim 3 or 4 and / or the pharmaceutical composition of claim 17 to a mammal to prevent group B streptococcal infection.

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