CN110652585B - Polysaccharide-protein conjugate immune preparation and application thereof - Google Patents

Abstract

The invention provides a polysaccharide-protein conjugate immune preparation, and a preparation method and application thereof. In the polysaccharide-protein conjugate, the carrier protein is Ag473 or Ag473 protein fragment with reserved antigen activity. The polysaccharide-protein conjugate of the invention can not only prevent infectious diseases caused by bacteria corresponding to capsular polysaccharide contained in the conjugate, but also prevent infectious diseases caused by serogroup B neisseria encephalitis. The combined immune preparation of the invention has stable physicochemical properties, has no interference among all components, and can achieve the broad-spectrum antibacterial effect. The Ag473 is used as the carrier protein, so that the phenomenon of epitope inhibition by using TT and DT as the carrier protein and unsafe risks such as toxicity reversion, anaphylactic reaction and the like can be avoided to a certain extent.

Description

Polysaccharide-protein conjugate immune preparation and application thereof

Technical Field

The invention relates to a polysaccharide-protein conjugate immune preparation, and preparation and application thereof.

Background

Bacterial infectious diseases are diseases which seriously damage human health, at present, medicines clinically used for treating bacterial infectious diseases are mainly antibacterial medicines, but abuse of the antibacterial medicines leads to rapid increase of drug-resistant bacteria, particularly multi-drug-resistant bacteria, so that the drug-resistant bacteria cannot effectively control infection, become a difficult problem of clinical treatment, and bring a heavy economic burden to society. The bacterial vaccine can improve the resistance of susceptible people to pathogenic bacteria, reduce the incidence rate of pathogenic bacteria infection and is beneficial to the control of infectious diseases, so the development of relevant bacterial vaccines is always a research hotspot in the field. Practice proves that the bacterial vaccine has a refulgent effect in the development of more than one century, and plays a great role in preventing bacterial infectious diseases; common bacterial vaccines such as streptococcus pneumoniae vaccine, neisseria meningitidis vaccine, haemophilus influenzae type B (Hib) vaccine, and the like.

Neisseria meningitidis is responsible for epidemic cerebrospinal meningitis and there are many data indicating that neisseria meningitidis can act as a pathogen for primary lower respiratory tract infections, causing primary neisseria meningitidis pneumonia. Neisseria meningitidis is hidden in nasal and pharyngeal secretions of patients or carriers, is directly transmitted from air by spray mainly through cough, sneeze, speaking and other modes, and enters respiratory tract to cause infection, so that the infection is strong, the epidemic area is extremely wide, and the whole world is wide. The incidence rate of children under 7 years is highest, and areas with concentrated crowd such as schools, construction sites and markets are easy to develop. Neisseria meningitidis can be divided into a total of 13 serotypes of pathogenic bacteria A, B, C, D, 29E, H, I, K, L, W, 135, X, Y, Z, according to the specificity of its capsular polysaccharide. Among them, five serogroups of group A, group B, group C, group Y and group W135 are those responsible for almost all individuals suffering from human diseases. Currently, meningococcal polysaccharide or polysaccharide conjugate vaccines that have been marketed include monovalent vaccines, such as group a meningococcal polysaccharide vaccine and group C meningococcal polysaccharide-protein conjugate vaccine, and the like; and multivalent vaccines such as group a group C meningococcal polysaccharide vaccine, group a group C meningococcal polysaccharide-protein conjugate vaccine, actyw 135 meningococcal polysaccharide vaccine, and actyw 135 meningococcal polysaccharide-protein conjugate vaccine, and combination vaccine of AC group meningococcal polysaccharide-protein conjugate vaccine and Hib conjugate vaccine, etc. Group B neisseria meningitidis (MenB) capsular polysaccharides are not suitable as vaccine candidates because the capsular polysaccharide of MenB is composed of polysialidase, which resembles carbohydrate moieties on developing human nervous tissue, which are recognized as self-antigens and thus poorly immunogenic to humans. Currently, two vaccine against MenB are marketed, namely Trumenba (pyroxene) and Bexservo (NoHua), and the antigen component of Trumenba is mainly bivalent rLP2086; the antigen active ingredients of Bexsero are mainly NadA, NHBA, fHbp and OMV-NZ.

Since neisseria meningitidis capsular polysaccharide is a T cell independent antigen and the immune system of children under two years of age is not yet mature, the immune response to most capsular polysaccharides is weak and the antibody level required for protecting the human body cannot be achieved; meanwhile, the capsular polysaccharide can not induce immune memory in vivo, so that the residence time of the antibody in vivo is short. Thus, neisseria meningitidis capsular polysaccharide vaccines are only suitable for children over 5 years of age and cannot be used for conventional vaccination of children under 2 years of age. The combination of neisseria meningitidis capsular polysaccharide with carrier protein converts capsular polysaccharide into T-cell dependent antigen, thereby stimulating infant T-cell dependent antibody synthesis and producing enhanced response, while also increasing immunoglobulin (IgG) antibody ratio and antibody affinity maturation. The polysaccharide conjugate vaccine is not only suitable for adults, but also suitable for infants.

And (II) streptococcus pneumoniae (Streptococcus pneumoniae), namely pneumococcus, can cause diseases such as pneumonia, meningitis, septicemia, tympanitis and the like, and the streptococcus pneumoniae which is found to have more than 90 serotypes currently is carried in respiratory tracts such as nasopharynx of 5% -10% of healthy adults and 20% -40% of children. The diseases caused by streptococcus pneumoniae have been a global serious public health problem with high morbidity and mortality worldwide, especially for children under 2 years of age and the elderly. The currently marketed streptococcus pneumoniae capsular saccharide vaccines and capsular glycoprotein conjugate vaccines, all of which are designed based on streptococcus pneumoniae capsular polysaccharides, encompass the most common serotypes responsible for streptococcus pneumoniae-based diseases. Pneumococcal polysaccharide antigen is a non-T cell dependent antigen, primary immunity can induce the generation of antibodies with a protection level, and secondary immunity can not induce the generation of immune memory; streptococcus pneumoniae capsular saccharide vaccines fail to elicit an effective protective antibody response in children under 2 years of age. The existing streptococcus pneumoniae capsular glycoprotein conjugate vaccine uses diphtheria or tetanus toxoid as a protein carrier, and the polysaccharide conjugate vaccine is not only suitable for adults, but also suitable for infants.

Haemophilus influenzae type (Hib) is a common symbiotic bacterium in the nasopharynx of children and can cause pneumonia and meningitis in children, the incidence and death of Hib are most common in developing countries, and the disease burden is most serious in children of 4-18 months of age, but also in children of less than 3 months and more than 5 years of age. In the non-immune population, hib is the primary cause of non-epidemic bacterial meningitis in children within 1 year of age. Even with timely administration of sufficient antibiotic treatment, 3-20% of Hib meningitis patients die. The most effective preventive measure for Hib infection is intramuscular injection of Hib conjugate vaccine, which can greatly reduce the incidence of pneumonia and meningitis. Although Hib capsular polysaccharide has certain immunogenicity, the immune response to polysaccharide is weak because of the abnormal development of the immune system of children, so that the Hib capsular polysaccharide and carrier protein are conjugated for immunization, and DT immune paths in infants are stimulated to enhance the immune response of infants to polysaccharide. The Hib vaccine currently marketed in China is a conjugate vaccine of Hib capsular polysaccharide and TT.

In summary, TT or DT is used as carrier protein in the meningococcal polysaccharide conjugate vaccine, the streptococcus pneumoniae capsular glycoprotein conjugate vaccine and the Hib polysaccharide conjugate vaccine on the market at present, and about 95% of children receive DT and TT vaccine, so that the proportion of people with pre-existing immunity to DT and TT is very high. When the patient is pre-vaccinated with DT or TT, epitope suppression is largely generated if DT or TT is further used as a protein carrier. In addition, TT and DT are carrier proteins, and may have unsafe risks such as toxicity reversion, anaphylactic reaction and the like. Meanwhile, due to the specificity of the group B neisseria meningitidis, the existing meningococcal combined vaccine does not contain antigen components aiming at the group B neisseria meningitidis, and cannot achieve the effect of broad-spectrum anti-neisseria meningitidis.

In addition, the development of the combined vaccine is beneficial to saving the times of vaccination, simplifying the immunization program, reducing the immunization cost and improving the immunization coverage rate, and can provide a broad-spectrum immunization effect, so the development of the combined vaccine has important significance. Streptococcus pneumoniae vaccine, neisseria meningitidis vaccine and haemophilus influenzae type B vaccine have wide variety receiving population and close inoculation procedures, so that combined vaccine can be further developed to achieve the effect of broad-spectrum anti-meningitis and/or pneumonia.

Disclosure of Invention

The invention provides a polysaccharide-protein conjugate immune preparation, and preparation and application thereof, aiming at overcoming the technical problems. The polysaccharide-protein conjugate of the invention can not only prevent infectious diseases caused by bacteria (such as streptococcus pneumoniae, neisseria encephalitis and Hib) corresponding to capsular polysaccharide contained in the conjugate, but also prevent infectious diseases caused by serogroup B neisseria encephalitis by using Ag473 as a carrier protein. The combined immune preparation (the combined immune preparation generally refers to an immune preparation comprising at least two polysaccharide-protein conjugates) has stable physicochemical properties, has no interference among the components, can effectively prevent diseases caused by the bacteria, reduces the number of times of vaccination, simplifies the immunization program, reduces the immunization cost and improves the immunization coverage rate; further can achieve broad-spectrum antibacterial effect. And Ag473 is used as carrier protein, so that the phenomenon of epitope inhibition by TT and DT as carrier proteins and unsafe risks such as toxicity reversion and anaphylactic reaction can be avoided to a certain extent.

The first aspect of the present invention provides a polysaccharide-protein conjugate, wherein the carrier protein is Ag473 protein or an Ag473 fragment that retains antigen activity.

Ag473 is a surface protein from Neisseria meningitidis serotype B that is specifically recognized by monoclonal antibodies (mAb) 4-7-3 (described in U.S. Pat. No. 5,172,B 2 and Hsu C A, linW R, li J C, et al immunoproteomic identification of the hypothetical protein NMB1468as a novel lipoprotein ubiquitous in Neisseria meningitidis with vaccine potential [ J ]. Proteomics,2010,8 (10): 2115-2125). In the application, lipidated Ag473 (L-Ag 473 for short) or non-lipidated Ag473 (NL-Ag 473 for short) can be used as a carrier protein; the amino acid sequence of L-Ag473 is an amino acid sequence (structure shown in figure 1) with at least 90 percent (preferably 95 percent, 96 percent, 97 percent, 98 percent, 99 percent or 100 percent) homology with SEQ ID NO.1, the triple structure of lipoprotein signal sequence, and the N-region has at least two positively charged residues; the H-region or hydrophobic region consists of 7 to 22 residues which are predominantly hydrophobic and uncharged; the C-region has a consensus [ LVI ] [ ASTIV ] [ GAS ] [ C ] sequence, known as a lipid cassette. The amino acid sequence of NL-Ag473 is shown as an amino acid sequence having at least 90% (preferably, e.g. 95%, 96%, 97%, 98%, 99% or 100%) homology with SEQ ID NO.2, L-Ag473 and NL-Ag473 are shown as Ching-Liang C, yon-Ling Y, yueh-Chen K, et al Immunomodulatory Activity of Meningococcal Lipoprotein Ag473Depends on the Conformation Made up of the Lipid and Protein Moieties [ J ]. PLoS ONE,2012,7 (7): e40873-.

SEQ ID NO.1：

MKKLLIAAMMAAALAACSQEAKQEVKEAVQAVESDVKDTAASAAESAASAVEEAKDQVKDAAADAKASAE(EAVTEAK) _1～4 EAVTEAAKDTLNKAADATQEAADKMKDAAK

7 amino acid sequences in SEQ ID NO.1EAVTEAKThere may be 1 or 2-4 repeats, forming 4 variants of L-Ag473-1 (107 aa, 1468 putative protein from serotype B Neisseria meningitidis strain MC 58), L-Ag473-2 (114 aa), L-Ag473-3 (121 aa) or L-Ag473-4 (128 aa), respectively.

Lipidated Ag473 generally refers to L-Ag473 having the sulfo group of cysteine (Cys, abbreviated C) at position 17 at the N-terminus modified with a diacyl glycerol group linked by a thioether bond and/or the amino group modified with a fatty acid acylation, as shown in the structure of formula I; r is R ₁ 、R ₂ Or R is ₃ Branched or straight-chain saturated or unsaturated hydrocarbon groups of 1 to 30 carbon atoms respectively, further branched or straight-chain alkyl or alkene of 14 to 20 carbon atomsRadical, further, R ₁ Or R is ₃ Is- (CH) ₂ ) ₁₄ CH ₃ ；R ₂ Is- (CH) ₂ ) ₇ -CH＝CH-(CH ₂ ) ₅ CH ₃ 。

SEQ ID NO.2：

MWRSQEAKQEVKEAVQAVESDVKDTAASAAESAASAVEEAKDQVKDAAADAKASAE(EAVTEAK) _{1～ 4} EAVTEAAKDTLNKAA DATQEAADKM KDAAK

7 amino acid sequences in SEQ ID NO.2EAVTEAKThere may be 1 or 2-4 repeats, forming 4 variants of NL-Ag473-1 (93 aa), NL-Ag473-2 (100 aa), NL-Ag473-3 (107 aa), or NL-Ag473-4 (114 aa), respectively.

SEQ ID NO.2 homologous amino acid sequence such as SEQ ID NO.3:

MCSQEAKQEVKEAVQAVESDVKDTAASAAESAASAVEEAKDQVKDAAADAKASAEEAVTEAKEAVTEAKEAVTEAKEAVTEAAKDTLNKAADATQEAADKMKDAAK

in some embodiments of the invention, the amino acid sequence of the Ag473 protein fragment retaining antigenic activity of the invention is: the N-terminal starts from any one of amino acids 18 to 40 in SEQ ID NO.1, and the C-terminal ends at the last amino acid.

The amino acid sequence of the Ag473 protein fragment retaining the antigen activity is shown in SEQ ID NO.4 (18 th to 121 th residues of the N end of SEQ ID NO.1 are the amino acid sequence of Ag473 mature protein):

SQEAKQEVK EAVQAVESDV KDTAASAAES AASAVEEAKD QVKDAAADAK ASAEEAVTEAKEAVTEAKEA VTEAKEAVTEAAKDTLNKAA DATQEAADKM KDAAK。

ag473 can be produced by biological, biochemical, etc. methods, and can be prepared by reference to chirg-Liang, chu, et al, "The Immunomodulatory Activity of Meningococcal Lipoprotein Ag473Depends on the Conformation Made up of the Lipid and Protein moieties," PLoS ONE 7.7 (2012): e40873 ".

Ag473 of the present invention may be expressed using E.coli including, but not limited to, C43 (DE 3), C41 (DE 3), DK8 (DE 3) S or C2014 (DE 3); expression vectors include, but are not limited to, pET21 or pET22b. Culture media include, but are not limited to, luria-Bertani broth (LB), super Broth (SB), terrific Broth (TB), 2XYT, M63 or M9.

In some embodiments of the invention, the polysaccharide in the polysaccharide-protein conjugate is selected from the group consisting of: streptococcus pneumoniae capsular polysaccharide, neisseria encephalitis (n.menningitidis) capsular polysaccharide or Hib (haemophilus influenzae type B) capsular polysaccharide.

The neisseria encephalitis may be selected from the group A, B, C, D, 29E, H, I, K, L, W135, X, Y or Z serogroup. Preferably, the neisseria encephalitis is selected from the A, C, W, X or Y serogroup.

The definition or preparation of the neisseria encephalitis capsular polysaccharide can be found in technical standards or publications known to those skilled in the art. Definition and preparation of capsular polysaccharides of neisseria encephalitis serogroup a (e.g., CMCC 29201), C (e.g., CMCC 29205), W135 (CMCC 29037) or Y (CMCC 29028) are described in the chinese pharmacopoeia 2015.

In some embodiments of the invention, the neisseria meningitidis capsular polysaccharide (e.g., the A, C, W, X or Y serogroup neisseria meningitidis) is partially depolymerized to an average size of less than 500,000 daltons, such as an average size of about 5,000 to 100,000 daltons, further still 8,000 to 75,000 daltons, and still further still 12,000 to 35,000 daltons. Further, in some embodiments of the invention, the capsular polysaccharide of each serogroup neisseria meningitidis may be partially depolymerized to an average size of about 12,000 to 22,000 daltons.

In some embodiments of the invention, the average ratio of the single serogroup neisseria meningitidis capsular polysaccharide to carrier protein is from 20:1 to 1:50 (w/w), and further may be from 10:1 to 1:20 (w/w). Further, in some embodiments of the invention, the average ratio of capsular polysaccharide to carrier protein of the single serogroup neisseria meningitidis is from 5:1 to 1:10 (w/w), further may be from 3:1 to 1:5 (w/w), such as 2:1 (w/w), 1:1 (w/w), 1:2 (w/w), 1:3 (w/w), 1:4 (w/w), etc.

Definition and preparation of Hib capsular polysaccharides may be found in the technical standards or publications known to those skilled in the art. Definition and preparation of Hib (e.g. CMCC 58534) capsular polysaccharides are described in the chinese pharmacopoeia of version 2015.

In some embodiments of the invention, the Hib capsular polysaccharide may also be partially depolymerized to a capsular polysaccharide having a degree of polymerization of about 10 to 35. Further, in some embodiments of the invention, the capsular polysaccharide is partially depolymerized to about 15-35 degrees of polymerization.

In some embodiments of the invention, the average ratio of Hib capsular polysaccharide to carrier protein may be from 20:1 to 1:50 (w/w), further from 10:1 to 1:20 (w/w). Further, in some embodiments of the invention, the average ratio of Hib capsular polysaccharide to carrier protein is from 5:1 to 1:10 (w/w), further may be from 3:1 to 1:5 (w/w), such as 2:1 (w/w), 1:1 (w/w), 1:2 (w/w), 1:3 (w/w), 1:4 (w/w), etc.

The streptococcus pneumoniae may be selected from the following serotypes: 1. 2, 3, 4, 5, 6A, 6B, 6C, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 36, 37, 38, 39, 40, 41F, 42, 43, 44, 45, 47, 48A, or 48. Preferred in the present invention are pathogenic streptococcus pneumoniae serotypes.

Preferably, the streptococcus pneumoniae serotype may be selected from the following serotypes: 1. 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23A, 23B, 23F, or 33F.

The streptococcus pneumoniae capsular polysaccharides may be defined or prepared by techniques known to those skilled in the art. The preparation and purification of partially defined streptococcus pneumoniae capsular polysaccharides is disclosed, for example, in patent WO2006110381 a.

In some embodiments of the invention, the average ratio of the single serotype streptococcus pneumoniae capsular polysaccharide to carrier protein is from 20:1 to 1:50 (w/w), further may be from 10:1 to 1:20 (w/w). Further, in some embodiments of the invention, the average ratio of capsular polysaccharide to carrier protein for each serogroup neisseria meningitidis is 5:1 to 1:10 (w/w), further may be 3:1 to 1:5 (w/w), such as 2:1 (w/w), 1:1 (w/w), 1:2 (w/w), 1:3 (w/w), 1:4 (w/w), etc.

In some embodiments of the invention, a single carrier protein may carry more than one polysaccharide antigen in the polysaccharide-protein conjugate. Preferably, the polysaccharide antigen is a pathogenic serogroup/type. For example, a single carrier protein may be conjugated to a capsular polysaccharide selected from any one of the following bacterial combinations: menA and MenC; menA and MenW135; menA and MenX; menA and MenY; menC and MenW135; menC and MenX; menC and MenY; menW135 and MenX; menW135 and MenY; menX and MenY; menA, menC, and MenW135; menA, menC, and MenX; menA, menC, and MenY; menA, menW135 and MenX; menA, menW135 and MenY; menC, menW135 and MenX; menC, menW135 and MenY; menW135, menX and MenY; menA, menC, menW135 and MenX; menA, menC, menW135 and MenY; menC, menW135, menX and MenY; hib and MenA; hib and MenC; hib and MenW135; hib and MenX; hib and MenY; hib, menA and MenC; hib, menA, and MenW135; hib, menA and MenX; hib, menA and MenY; hib, menC and MenW135; hib, menC and MenX; hib, menC and MenY; hib, menX and MenY; hib, menW and MenY; hib, menA, menC and MenW135; hib, menA, menC and MenX; hib, menA, menC and MenY; hib, menA, menW135 and MenY; hib, menA, menX and MenY; hib, menC, menW135 and MenY; hib, menC, menX and MenY; hib, menW135, menX and MenY; hib, menA, menC, menW135 and MenY; hib, menA, menC, menX and MenY; hib, menC, menW135, menX and MenY; or Hib, menA, menC, menW, menX and MenY; streptococcus pneumoniae of serotypes 4, 6B, 9V, 14, 18C, 19F and 23F; streptococcus pneumoniae of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F; streptococcus pneumoniae of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F; streptococcus pneumoniae of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F and 23F; streptococcus pneumoniae of serotypes 1, 2, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 18C, 19A, 19F and 23F; streptococcus pneumoniae of serotypes 1, 2, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F; streptococcus pneumoniae of serotypes 1, 2, 3, 4, 5, 6B, 7, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20F, 23F and 33F; streptococcus pneumoniae of serotypes 4, 6B, 9V, 14, 18C, 19F and 23F and Hib; streptococcus pneumoniae of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F and Hib; serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae and Hib; serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F and 23F streptococcus pneumoniae and Hib; serotypes 1, 2, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae and Hib; serotypes 1, 2, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F streptococcus pneumoniae and Hib; serotypes 1, 2, 3, 4, 5, 6B, 7, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20F, 23F and 33F streptococcus pneumoniae and Hib; streptococcus pneumoniae of serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, hib, menA and MenC; streptococcus pneumoniae of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F and Hib, menA and MenC; serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA and MenC; serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA and MenC; serotypes 1, 2, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA and MenC; serotypes 1, 2, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F streptococcus pneumoniae, hib, menA and MenC; serotypes 1, 2, 3, 4, 5, 6B, 7, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20F, 23F and 33F streptococcus pneumoniae and Hib, menA and MenC; streptococcus pneumoniae of serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, hib, menA, menC, menW and MenY; streptococcus pneumoniae of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F, hib, menA, menC, menW135 and MenY; serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA, menC, menW and MenY; serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA, menC, menW and MenY; serotypes 1, 2, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA, menC, menW135 and MenY; serotypes 1, 2, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F streptococcus pneumoniae, hib, menA, menC, menW and MenY; serotypes 1, 2, 3, 4, 5, 6B, 7, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20F, 23F and 33F streptococcus pneumoniae and Hib, menA, menC, menW135 and MenY; streptococcus pneumoniae of serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, hib, menA, menC, menW, menX and MenY; streptococcus pneumoniae of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F, hib, menA, menC, menW, menX and MenY; serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA, menC, menW, menX and MenY; serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA, menC, menW, menX and MenY; serotypes 1, 2, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA, menC, menW, menX and MenY; serotypes 1, 2, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F streptococcus pneumoniae, hib, menA, menC, menW, menX and MenY; serotypes 1, 2, 3, 4, 5, 6B, 7, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20F, 23F and 33F streptococcus pneumoniae and Hib, menA, menC, menW, menX and MenY.

In some embodiments of the present invention, when a single carrier protein carries more than one polysaccharide antigen, the average ratio of the total content of polysaccharide antigen to carrier protein may be 20:1 to 1:50 (w/w), and further may be 10:1 to 1:20 (w/w). Further, in some embodiments of the invention, the average ratio of Hib capsular polysaccharide to carrier protein is from 5:1 to 1:10 (w/w), further may be from 3:1 to 1:5 (w/w), such as 2:1 (w/w), 1:1 (w/w), 1:2 (w/w), 1:3 (w/w), 1:4 (w/w), etc.

In some embodiments of the invention, the polysaccharide-protein conjugate may have a content ratio of 1 (w/w) or more (e.g., 2,3,4 or higher) between polysaccharide antigens when a single carrier protein carries more than one polysaccharide antigen.

In a second aspect the invention provides a method of preparing a polysaccharide-protein conjugate as described above, comprising conjugating a purified polysaccharide to a carrier protein.

Streptococcus pneumoniae, meningococcal and Hib capsular polysaccharides may be prepared by standard techniques or literature reports known to those skilled in the art. In some embodiments of the invention, the purified polysaccharide may be depolymerized prior to attachment to the carrier protein, for example, by partial depolymerization of capsular polysaccharides from neisseria meningitidis serotypes using mild oxidation conditions. And depolymerizing the HIb capsular polysaccharide under acidic conditions.

Conjugation of the polysaccharide of the invention to a carrier protein may be made by conventional coupling techniques in the art, and the polysaccharide may be attached to the carrier protein directly or via spacer groups (e.g., ADH, B-acrylamido (WO 00/10599), nitrophenyl-ethylamine (Gever et al (1979) Med. Microbiol. Immunol.165; 171-288), haloalkyl halides (US 4057685), glycosidic linkages (US 4673574, US 4808700), hexamethylenediamine, 6-aminocaproic acid (US 4459286), etc.). In some embodiments of the invention, conjugation of the purified polysaccharide to the carrier protein may be directly via covalent bonds. Typically, the polysaccharide is conjugated to a carrier protein after activation, "activation" refers to chemical treatment of the polysaccharide to provide chemical groups capable of reacting with the carrier protein, and the methods of activation and conjugation can be performed with reference to conventional methods in the art; for example, meningococcal capsular polysaccharides can be treated with adipic acid dihydrazide in physiological saline at 15 to 30 ℃ and pH 5.0±0.1; about 2 hours.

In a third aspect the present invention provides the use of a polysaccharide-protein conjugate as described above for the preparation of an immune formulation. The immune preparation of the invention is preferably a vaccine.

The invention also provides application of the polysaccharide-protein conjugate in preparing a medicament for preventing and/or treating diseases caused by streptococcus pneumoniae and/or neisseria encephalitis and/or haemophilus influenzae type B. The diseases caused by streptococcus pneumoniae include, but are not limited to, pneumonia, meningitis, septicemia, otitis media and the like; such diseases caused by streptococcus pneumoniae include, but are not limited to, epidemic cerebrospinal meningitis, primary neisseria meningitidis pneumonia, and the like; the diseases caused by the haemophilus influenzae type B include, but are not limited to, pneumonia, meningitis and the like.

The fourth aspect of the invention also provides an immunological formulation comprising at least one polysaccharide-protein conjugate as described above.

Preferably, the immunizing formulation comprises a polypeptide selected from the group consisting of: serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23A, 23B, 23F, and 33F streptococcus pneumoniae capsular polysaccharide-protein conjugates; A. c, W135, group X and Y N.meningitidis capsular polysaccharide-protein conjugates; and at least one, at least two, at least three, at least four, at least five, at least six … …, at least thirty-one of the Hib capsular polysaccharide-protein conjugates.

In some embodiments of the invention, when the immune formulation comprises at least two or more polysaccharide-protein conjugates, wherein the polysaccharide may be selected from any one of the following bacterial capsular polysaccharides in combination: menA and MenC; menA and MenW135; menA and MenX; menA and MenY; menC and MenW135; menC and MenX; menC and MenY; menW135 and MenX; menW135 and MenY; menX and MenY; menA, menC, and MenW135; menA, menC, and MenX; menA, menC, and MenY; menA, menW135 and MenX; menA, menW135 and MenY; menC, menW135 and MenX; menC, menW135 and MenY; menW135, menX and MenY; menA, menC, menW135 and MenX; menA, menC, menW135 and MenY; menC, menW135, menX and MenY; hib and MenA; hib and MenC; hib and MenW135; hib and MenX; hib and MenY; hib, menA and MenC; hib, menA, and MenW135; hib, menA and MenX; hib, menA and MenY; hib, menC and MenW135; hib, menC and MenX; hib, menC and MenY; hib, menX and MenY; hib, menW and MenY; hib, menA, menC and MenW135; hib, menA, menC and MenX; hib, menA, menC and MenY; hib, menA, menW135 and MenY; hib, menA, menX and MenY; hib, menC, menW135 and MenY; hib, menC, menX and MenY; hib, menW135, menX and MenY; hib, menA, menC, menW135 and MenY; hib, menA, menC, menX and MenY; hib, menC, menW135, menX and MenY; hib, menA, menC, menW135, menX and MenY; streptococcus pneumoniae of serotypes 4, 6B, 9V, 14, 18C, 19F and 23F; streptococcus pneumoniae of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F; streptococcus pneumoniae of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F; streptococcus pneumoniae of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F and 23F; streptococcus pneumoniae of serotypes 1, 2, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 18C, 19A, 19F and 23F; streptococcus pneumoniae of serotypes 1, 2, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F; streptococcus pneumoniae of serotypes 1, 2, 3, 4, 5, 6B, 7, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20F, 23F and 33F; streptococcus pneumoniae of serotypes 4, 6B, 9V, 14, 18C, 19F and 23F and Hib; streptococcus pneumoniae of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F and Hib; serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae and Hib; serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F and 23F streptococcus pneumoniae and Hib; serotypes 1, 2, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae and Hib; serotypes 1, 2, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F streptococcus pneumoniae and Hib; serotypes 1, 2, 3, 4, 5, 6B, 7, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20F, 23F and 33F streptococcus pneumoniae and Hib; streptococcus pneumoniae of serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, hib, menA and MenC; streptococcus pneumoniae of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F and Hib, menA and MenC; serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA and MenC; serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA and MenC; serotypes 1, 2, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA and MenC; serotypes 1, 2, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F streptococcus pneumoniae, hib, menA and MenC; serotypes 1, 2, 3, 4, 5, 6B, 7, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20F, 23F and 33F streptococcus pneumoniae and Hib, menA and MenC; streptococcus pneumoniae of serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, hib, menA, menC, menW and MenY; streptococcus pneumoniae of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F, hib, menA, menC, menW135 and MenY; serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA, menC, menW and MenY; serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA, menC, menW and MenY; serotypes 1, 2, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA, menC, menW135 and MenY; serotypes 1, 2, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F streptococcus pneumoniae, hib, menA, menC, menW and MenY; serotypes 1, 2, 3, 4, 5, 6B, 7, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20F, 23F and 33F streptococcus pneumoniae and Hib, menA, menC, menW135 and MenY; streptococcus pneumoniae of serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, hib, menA, menC, menW, menX and MenY; streptococcus pneumoniae of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F, hib, menA, menC, menW, menX and MenY; serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA, menC, menW, menX and MenY; serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA, menC, menW, menX and MenY; serotypes 1, 2, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 18C, 19A, 19F and 23F streptococcus pneumoniae, hib, menA, menC, menW, menX and MenY; serotypes 1, 2, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F streptococcus pneumoniae, hib, menA, menC, menW, menX and MenY; serotypes 1, 2, 3, 4, 5, 6B, 7, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20F, 23F and 33F streptococcus pneumoniae and Hib, menA, menC, menW, menX and MenY.

In some embodiments of the invention, when the immune formulation comprises at least two or more polysaccharide-protein conjugates, the immune formulation is a mixture of the polysaccharide-protein conjugates alone.

In some embodiments of the invention, the mass content of capsular polysaccharide of a single serogroup neisseria encephalitis in the single dose is 0.5-15 ug.

Further, in some embodiments of the invention, the mass content of capsular polysaccharide of a single serogroup neisseria encephalitis in the immunizing agent may be 2 ug-10 ug, and further may be 5ug.

In some embodiments of the invention, the mass content of capsular polysaccharide of a single serogroup neisseria encephalitis in a single dose may vary from 10% to 20% in the immunizing formulation.

In some embodiments of the invention, the mass content of Hib capsular polysaccharide in a single dose in the immunizing formulation is 2-20 ug.

Further, in some embodiments of the invention, the mass content of Hib capsular polysaccharide in the immunizing formulation is 2.5 ug-10 ug in a single dose.

In some embodiments of the invention, the mass content of Hib capsular polysaccharide in a single dose may be within a range of 10% to 20% error in the immunizing formulation.

In some embodiments of the invention, the mass content of capsular polysaccharide of a single serogroup streptococcus pneumoniae in the single dose is 0.5-15 ug.

Further, in some embodiments of the invention, the mass content of capsular polysaccharide of a single serotype streptococcus pneumoniae in the immunizing formulation in a single dose is 2 ug-10 ug, further may be 4ug-8ug.

In some embodiments of the invention, the mass content of capsular polysaccharide of a single serotype streptococcus pneumoniae in a single dose may vary from 10% to 20% in the immunizing formulation.

In some embodiments of the invention, the capsular polysaccharide of serotype 6B streptococcus pneumoniae is 2 times the mass content of capsular polysaccharides of other serotypes of streptococcus pneumoniae.

In some embodiments of the invention, the total mass content of carrier protein in a single dose in the immune formulation is between 10ug and 200ug.

Further, in some embodiments of the invention, the total mass content of carrier protein in the immunizing formulation in a single dose is 20 ug-100 ug, and further may be 50 ug-80 ug.

In some embodiments of the invention, the total mass content of carrier protein in a single dose may be within a range of 10% to 20% error in the immunizing formulation.

The immune formulation may further comprise a pharmaceutically acceptable carrier and/or adjuvant.

The adjuvant may be selected from aluminum adjuvants, BAY, DC-chol, pcpp, TLR agonists, monophosphoryl lipid a, cpG, QS-21, MF57, immunostimulatory complexes (ISCOMs), cholera toxin formyl methionyl peptides, etc.

The aluminum adjuvant may be selected from aluminum phosphate, aluminum sulfate or aluminum hydroxide.

In some embodiments of the invention, an adjuvant may or may not be added when L-Ag473 is employed as the carrier protein; the adjuvant is preferably added when NL-Ag473 is used as carrier protein.

In some embodiments of the invention, the single dose of the immunizing formulation contains 0.2mg to 0.8mg elemental aluminum adjuvant. In some embodiments of the invention, the mass content of elemental aluminum in a single dose in the immunizing formulation may be 10% -50% error.

Further, in some embodiments of the invention, the single dose of the immunizing formulation contains 0.4mg±20% elemental aluminum adjuvant.

Further, in some embodiments of the invention, the single immunization dose of the immunization formulation may be from 0.5ml to 1ml.

The immunizing formulations of the present invention may be administered in a single dose or in a series of doses (prime-boost procedure, may be "one boost" or "several boosts"). The prime-boost procedure involves administering a first immune composition (priming vaccine) to a subject, followed by a second immune composition (booster vaccine) to induce an optimal immune response. The appropriate time interval between primary and booster immunizations will be appreciated by those skilled in the art.

The immunological formulation of the present invention comprises: liquid preparations (such as suspensions, syrups or elixirs) for administration through a nozzle, for example, the mouth, nose, anus, vagina, mouth, stomach, mucous membranes (e.g., lingual, alveolar, gingival, olfactory or respiratory mucosa), etc.; and formulations for parenteral, subcutaneous, intradermal, intramuscular, intraperitoneal or intravenous administration (e.g., injection administration), such as sterile suspensions or emulsions. Intravenous and parenteral administration is preferred. The immunological formulation may be mixed with a suitable carrier, diluent or excipient such as sterile water, physiological saline, dextrose and the like. The immune formulation may also be lyophilized. Depending on the desired route of administration and formulation form, the immune formulation may contain adjuvants such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity-enhancing additives, preservatives, flavouring agents, colouring agents and the like. Those skilled in the art will recognize that: the selected components of the immunological formulation must be non-chemically reactive with the immunologically active ingredient (i.e., the polysaccharide-protein carrier conjugate as described above).

The dosage used will depend on the immune component, the route of administration, the target population and other factors. The clinical trial staff will determine the appropriate dosage of each immune component and the effective immunization regimen based on their knowledge. A single administration is sufficient or requires multiple administrations with single and/or combined immunogens.

Further, in one embodiment of the invention, the route of administration of the immune formulation is intramuscular or subcutaneous, preferably a intramuscular route of administration. Administration may be by injection or by alternative delivery means.

In a fifth aspect the invention provides a method of preparing the immune formulation comprising mixing the polysaccharide-protein conjugate.

In some embodiments of the invention, a method of preparing the immune formulation comprises the steps of:

(1) Preparing the polysaccharide-protein conjugates respectively, and performing aluminum adsorption respectively;

(2) Mixing the aluminum-adsorbed polysaccharide-protein conjugate prepared in the step (1).

In a sixth aspect, the present invention provides the use of an immunological formulation as described in the manufacture of a medicament for the prophylaxis and/or treatment of a disease caused by streptococcus pneumoniae and/or neisseria encephalitis and/or haemophilus influenzae type B. The diseases caused by streptococcus pneumoniae include, but are not limited to, pneumonia, meningitis, septicemia, otitis media and the like; such diseases caused by streptococcus pneumoniae include, but are not limited to, epidemic cerebrospinal meningitis, primary neisseria meningitidis pneumonia, and the like; the diseases caused by the haemophilus influenzae type B include, but are not limited to, pneumonia, meningitis and the like.

The polysaccharide-protein conjugate and the immune preparation containing the polysaccharide-protein conjugate have the following technical advantages:

the polysaccharide-protein conjugate of the invention can not only prevent infectious diseases caused by bacteria (such as streptococcus pneumoniae, neisseria encephalitis and Hib) corresponding to capsular polysaccharide, but also prevent infectious diseases caused by serogroup B neisseria encephalitis by using Ag473 as a carrier protein. The combined immune preparation (the combined immune preparation generally refers to an immune preparation comprising at least two polysaccharide-protein conjugates) has stable physicochemical properties, has no interference among the components, can effectively prevent diseases caused by the bacteria, reduces the number of times of vaccination, simplifies the immunization program, reduces the immunization cost and improves the immunization coverage rate. Further, the vaccine can simultaneously achieve the effect of immunization against the most common strains A, C, W, X, Y and B neisseria meningitidis causing meningitidis epidemic, and can achieve the effect of broad-spectrum neisseria meningitidis resistance. On the basis of increasing Hib polysaccharide protein and/or streptococcus pneumoniae polysaccharide protein, the composition can further prevent meningitis caused by other bacteria, and can also prevent other diseases caused by Hib and/or streptococcus pneumoniae, such as pneumonia and the like.

The immune preparation of the invention is not only suitable for adults, but also suitable for infants, and has wider audience range.

And thirdly, ag473 is used as carrier protein, so that the phenomenon that TT and DT are epitope inhibition of the carrier protein and unsafe risks such as toxicity reversion and anaphylactic reaction can be avoided to a certain extent.

Drawings

FIG. 1 is a diagram showing the amino acid structure of lipidated Ag473 with 90% homology;

FIG. 2 is a SDS-PAGE of samples of the collection in example 4;

FIG. 3 is a SDS-PAGE of samples of the collection fluid analyzed in example 5.

Detailed Description

The present invention will be described in further detail and fully with reference to the following examples. The following examples are illustrative only and are not to be construed as limiting the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the examples described below were all commercially available unless otherwise specified.

EXAMPLE 1 preparation of purified capsular polysaccharide of neisseria meningitidis serotypes A, C, W, Y

Separately, frozen wet seed cultures of neisseria meningitidis serotypes A, C, W135 and Y were thawed and reconstituted with the aid of liquid Watson Scherp medium and then inoculated into Blake flasks containing Mueller Hinton agar medium, CO ₂ Incubate for 15 to 19 hours at 35 to 37℃in the atmosphere. After incubation, the growth was again sequentially amplified to 4L and 50L in shake flasks containing Watson Scherp medium, which were incubated on a horizontal shaker at 35 to 37 ℃. After incubation, the polysaccharide paste was collected by centrifugation using a Cetavlon precipitate.

The polysaccharide paste is subjected to suspension centrifugation for 2-4 times with 0.9M calcium chloride, the supernatant is collected, and the supernatant is subjected to super concentration by an ultrafiltration membrane with a molecular weight cut-off of 10-30 kD. Adding magnesium chloride into the concentrated solution, adjusting pH to 7.2-7.5, adding DNase and RNase for incubation, adding ethanol to make the final concentration of the concentrated solution be 30% -50%, centrifuging to remove nucleic acid and protein, recovering supernatant, adding ethanol to make the final concentration be 80%, centrifuging, collecting precipitate, and washing the precipitate polysaccharide with ethanol and acetone respectively. Re-dissolving the precipitated polysaccharide with sodium acetate solution, adding magnesium chloride, adjusting pH to 7.2-7.5, adding DNase and RNase, incubating to remove residual nucleic acid, adding equal volume of sodium acetate-phenol solution into polysaccharide-enzyme mixture after enzyme incubation, mixing, centrifuging, removing protein and endotoxin, and collecting water phase. The combined aqueous phases were diluted with water and subjected to ultrafiltration dialysis (dialysis), the dialysate was taken up in ethanol to 80% and used for precipitation at 1 to 5 ℃, the ethanol supernatant was discarded, and the polysaccharide precipitate was collected by centrifugation and dried.

The purified capsular polysaccharide powder was reconstituted by addition of sterile 50mM sodium acetate buffer (pH 6.0) to a polysaccharide concentration of 1.25g/L and the polysaccharide solution was heated to 55 ℃ + -0.1. To the reaction mixture was added 30% hydrogen peroxide to a reaction concentration of 1%. Monitoring the change of the molecular size of the polysaccharide every 15 to 20 minutes in the reaction process, rapidly cooling the polysaccharide solution to 5 ℃ when the molecular size of the polysaccharide reaches the target molecular size, concentrating the depolymerized polysaccharide solution to 15g/L, ultrafiltering and dialyzing the concentrated depolymerized polysaccharide solution with 10 times of sterile physiological saline (0.85% sodium chloride) to obtain depolymerized polysaccharide for later use, and measuring the average sizes of neisseria meningitidis serotype A, C, W135 and Y polysaccharide molecular weights of 15,000-22,000 daltons respectively.

EXAMPLE 2 preparation of Hib capsular polysaccharide

The haemophilus influenzae type B is derived from the China medical Collection center, the working seeds are amplified, cultured and heat-inactivated on a Hib comprehensive culture medium, and then the supernatant is centrifugally collected and concentrated and ultrafiltered by an ultrafiltration membrane bag with a molecular weight cut-off of 100 KD.

Ultrafiltering with phosphate buffer solution of pH7.0, adding absolute ethanol to reach ethanol final concentration of 30%, stirring, and centrifuging to remove nucleic acid.

Adding ethanol into the supernatant to reach a final concentration of 70%, stirring, centrifuging, and collecting precipitate.

And (3) after the precipitate is redissolved by water for injection, adding phenol with the volume of 2 times of the solution to extract for 4-6 times, and removing protein.

Ethanol is added into the phenol extract to lead the final concentration to reach 65-75 percent, the precipitate is collected after centrifugation, the precipitate is redissolved by injection water, and the solution is sterilized and filtered by a sterile filter with the diameter of 0.22 mu m to obtain the haemophilus influenzae type B capsular polysaccharide stock solution.

EXAMPLE 3 preparation of Streptococcus pneumoniae polysaccharide

1. 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F serotype pneumococcal strains are passaged to establish an original seed library, a main seed library and a working seed library, and the times are respectively as follows: the original seed lot is the 1 st generation, the main seed lot is the 4 th generation, and the working seed lot is the 8 th generation. After the working seed batch is started, the working seed batch is cultured in an inoculation fermentation tank, and the passage should not exceed 10 generations. And each generation of seeds adopts milk powder as a freeze-drying protective agent, and is freeze-dried and preserved. And (3) streaking and picking strains from working seeds in a soytone solid culture medium, inoculating the strains to 5ml of liquid comprehensive culture medium, culturing at 35 ℃, transferring the strains to 200ml of culture solution, culturing, and amplifying the strains to 2L of culture solution and 30L of culture solution. Stopping culturing at late stage of logarithmic growth phase or early stage of stationary phase, sampling, checking for pure bacteria, sterilizing with formalin solution for two hours, adding appropriate amount of deoxycholate sodium to final concentration of 0.3%, stirring for two hours to lyse bacteria and release capsular polysaccharide. The lysed culture broth was centrifuged and the supernatant was collected and concentrated 10-fold by ultrafiltration using a 100KD molecular weight cut-off ultrafiltration membrane.

Ultrafiltering with phosphate buffer solution of pH7.0, adding absolute ethanol to reach ethanol final concentration of 30%, stirring, and centrifuging to remove nucleic acid.

Adding ethanol into the supernatant to reach a final concentration of 70%, stirring, centrifuging, removing the supernatant, and collecting precipitate.

Adding water to dissolve the precipitate, adding phenol with the volume of 2 times of the solution volume for multiple extraction, and removing protein.

Ethanol is added into the supernatant to reach the final concentration of 55%, the precipitate is collected after centrifugation, the precipitate is redissolved by injection water, and the pneumococcal capsular polysaccharide stock solution of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F is obtained after aseptic filtration by a sterile filter of 0.22 mu m.

Example 4 preparation of L-Ag473 protein

1. Construction of recombinant plasmids

Designing and synthesizing a primer by referring to the nucleic acid sequence of the L-Ag473, introducing an NdeI restriction site into the upstream primer, and introducing an XhoI restriction site into the downstream primer to perform total gene synthesis.

The amino acid sequence of the L-Ag473 is shown in SEQ ID NO. 5:

MKKLLIAAMMAAALAACSQEAKQEVKEAVQAVESDVKDTAASAAESAASAVEEAKDQVKDAAADAKASAEEAVTEAKEAVTEAKEAVTEAKEAVTEAAKDTLNKAADATQEAADKMKDAAK

primer I is shown as SEQ ID NO. 6: GGATCCCATGAAAAAATTATTGATTGCCGCAATG

Primer II is shown as SEQ ID NO: 7: AAGCTTGGCGGCATCTTTCATTTTGTCTGCCG

The PCR amplification is carried out to obtain the target gene of the coding L-Ag473, the NdeI and XhoI sites are utilized to clone the PCR product into a pET21 vector, the PCR product is connected and then is transformed into an E.coli C41 (DE 3) cell, a recombinant is selected, and a plasmid with correct sequencing is named pET21-L-Ag473-His.

2. Expression and purification of proteins of interest

Single colonies of recombinants sequenced correctly were inoculated into 5ml M9 (supplemented with 50. Mu.g/ml benzyl penicillin) medium, shake-cultured overnight at 37℃with 220 rps; 1ml of the culture medium was transferred to 100ml of M9 (supplemented with 50. Mu.g/ml benzyl penicillin), cultured under the same conditions until the OD was about 0.5, and L-Ag473 was induced by adding 0.5mM IPTG, and after 4 hours, the induced bacterial liquid was centrifuged at 10000rpm for 10min to collect bacterial cells.

The cells were resuspended in homogenization buffer (20 mM Tris-Cl,500mM NaCl,10% glycerol, 50mM sucrose, 10mM imidazole, pH 8.0), disrupted by high pressure homogenizer, and the supernatant was collected by centrifugation. Supernatant fluid is passed through

After filtration through a membrane, the solution was loaded on a 20ml nickel-NTA column (2.2 cm. I.D..times.5.3 cm) and purified, and L-Ag473 was collected by washing with a homogeneous buffer, then with a 50mm imidazole-containing buffer, and finally eluting with a 500mm imidazole-containing buffer. The collected liquid samples were analyzed by SDS-PAGE, and the SDS-PAGE diagram is shown in FIG. 2. The N-terminal sequencing result of the Edman degradation method shows that the N-terminal of the L-Ag473 is blocked, which indicates that the protein may undergo lipidation modification.

Example 5 preparation of NL-Ag473

1. Construction of recombinant plasmids

With reference to the nucleic acid sequence of NL-Ag473 (SEQ ID NO: 5), primers were designed and synthesized, bamHI cleavage sites were introduced into the upstream primer, and HindIII cleavage sites were introduced into the downstream primer, and total gene synthesis was performed.

The NL-Ag473 amino acid sequence is shown in SEQ ID NO. 8:

MCSQEAKQEVKEAVQAVESDVKDTAASAAESAASAVEEAKDQVKDAAADAKASAEEAVTEAKEAVTEAKEAVTEAKEAVTEAAKDTLNKAADATQEAADKMKDAAK

primer I' is shown as SEQ ID NO: 9: GGAATTCCATATGAAAAAATTATTGATTGC

Primer iI' is shown as SEQ ID NO: 10:

CCGCTCGAGGTGATGATGTTTGGCGGCATCTTTCATTTTG

the target gene encoding NL-Ag473 is obtained through PCR amplification, the PCR product is cloned into a pET21 vector by utilizing BamHI and HindIII sites, and is transformed into an escherichia coli BL21 (DE 3) cell after connection, recombinants are selected, and a plasmid with correct sequencing is named pET21-NL-Ag473-His.

2. Expression and purification of proteins of interest

Single colonies of recombinants sequenced correctly were inoculated into 5ml LB (50. Mu.g/ml ampicillin) medium, shake-cultured overnight at 37℃with 220 rps; 1ml of the culture medium was transferred to 100ml of LB (50. Mu.g/ml ampicillin) medium, cultured under the same conditions, when the OD value was 0.5-0.8, the culture medium was added with 0.5mM IPTG to induce NL-Ag473 to express, after 4 hours, the induced bacterial liquid was centrifuged at 10000rpm for 10 minutes, and bacterial cells were collected.

The cells were resuspended in homogenization buffer (20 mM Tris-Cl,500mM NaCl,10% glycerol, 50mM sucrose, 10mM imidazole, pH 8.0), disrupted by high pressure homogenizer, and the supernatant was collected by centrifugation. The supernatant was filtered through a 0.45mM membrane, and then loaded on a 20ml nickel-NTA column (2.2 cm I.D.×5.3 cm) for purification, and washed with a homogeneous buffer, followed by a 50mM imidazole-containing buffer, and finally eluted with a 500mM imidazole-containing buffer, and NL-Ag473 was collected. The collected liquid samples were analyzed by SDS-PAGE, and the SDS-PAGE pattern is shown in FIG. 3. The N-terminal sequencing result of the Edman degradation method shows that the N-terminal amino acid sequence of NL-Ag473 is MCSQEA.

EXAMPLE 6 preparation of Hib capsular polysaccharide protein conjugate

The Ag473 protein (prepared by NL-Ag473 and L-Ag473 respectively, the concentration is 10mg/ml, the solvent is PBS) is mixed with adipoyl hydrazine (100 mg/ml, the solvent is sodium bicarbonate aqueous solution), and the feeding mass of the Ag473 protein and the adipoyl hydrazine is 1:10; EDAC (1 mol/L, solvent: water) was added to the mixture until the final concentrations were 0.1mol/L, and the mixture was reacted at room temperature for 2 hours, followed by ultrafiltration dialysis with phosphate buffer at pH7.0 or so to obtain Ag473-ADH derivatives (NL-Ag 473-ADH derivatives and L-Ag473-ADH derivatives, respectively).

To a Hib polysaccharide solution (prepared in example 2, polysaccharide concentration 10 mg/ml) was added CDAP (100 mg/ml, solvent acetonitrile) at a mass ratio of Hib polysaccharide to CDAP (1-cyano-4-dimethylamino-pyridine tetrafluoroboric acid) of 1:0.75, and the pH was controlled to 9.2, and the reaction was carried out at room temperature for about 2 minutes. According to the mass ratio of Hib polysaccharide to Ag473-ADH derivative of 1:1, making the pH be 9-9.5, adding glycine (mass ratio of glycine to CDAP of 1:1) to make reaction for 3 hr to terminate binding reaction, using ultrafiltration dialysis and gel chromatography to purify the conjugate to remove free protein, free polysaccharide and reaction chemical reagent, using 0.2 micrometer filter to make sterilization filtration so as to obtain Hib polysaccharide-L-Ag 473 protein conjugate and Hib polysaccharide-NL-Ag 473 protein conjugate stock solution.

(2) The purified Hib polysaccharide-L-Ag 473 protein conjugate and Hib polysaccharide-NL-Ag 473 protein conjugate stock solution are adsorbed with aluminium phosphate adjuvant to make the Hib polysaccharide content be 20 mug/ml and aluminium phosphate adjuvant final concentration be 2.72mg/ml.

EXAMPLE 7 preparation of Neisseria meningitidis polysaccharide protein conjugate

Starting materials were neisseria meningitidis serotypes A, C, W-135 prepared in example 1 and Y depolymerized capsular polysaccharides, each conjugated separately.

CDAP (100 mg/ml, solvent acetonitrile) was added to a solution of Neisseria meningitidis polysaccharide (concentration: 6mg/ml, solvent: water for injection) at a mass ratio of Neisseria meningitidis polysaccharide to CDAP of 1:0.75, and the pH was controlled to 9.2, and the reaction was carried out at room temperature for about 2 minutes. The combination reaction is carried out according to the mass ratio of neisseria meningitidis polysaccharide to L-Ag473-ADH derivative (prepared in example 6) of 1:1, the PH is 9-9.5, glycine (the mass ratio of glycine to CDAP is 1:1) is added to react for 3 hours to stop the combination reaction, the combination is subjected to ultrafiltration dialysis and gel chromatography purification to remove free protein, free polysaccharide and reaction chemical reagent, and the solution is subjected to sterilization filtration by a 0.2 mu m filter to obtain neisseria meningitidis polysaccharide protein conjugate stock solution.

EXAMPLE 8 preparation of Streptococcus pneumoniae polysaccharide protein

CDAP-activated polysaccharide, was added to 13 pneumococcal capsular polysaccharide stock solutions prepared in example 3, respectively: adding L-Ag473-ADH (prepared in example 6) in a weight ratio of protein=1:1 for a binding reaction, wherein the PH is 9-9.5, adding glycine for 3 hours for stopping the binding reaction to prepare pneumococcal capsular polysaccharide conjugate, performing ultrafiltration dialysis on the conjugate to remove unbound protein and polysaccharide, and performing sterilization filtration on the conjugate by using a 0.2 mu m filter after purification to obtain the pneumococcal capsular polysaccharide protein monovalent conjugate.

Characterization data of the prepared polysaccharide protein conjugate are obtained by detection according to a method specified in pharmacopoeia as follows:

EXAMPLE 9 immune Effect experiment of Hib capsular polysaccharide protein conjugate

The BALB/c mice of 12-14 g are randomly grouped according to the weight, 6 of the BALB/c mice are injected into a negative control group, 0.5ml of sterile normal saline is injected into the BALB/c mice, the Hib polysaccharide content of an experimental group is 10 ug/dose, and the mice are immunized 1 time every 2 weeks, 0.5ml of the mice are totally 3 times. Blood was collected from the orbital venous plexus at 6 weeks, and the content of IgG of the anti-Hib polysaccharide-specific antibody and the anti-Ag 473 antibody in the serum of each mouse was detected by ELISA.

TABLE 1 antibody levels (GM, 95% CI)

Conclusion: as can be seen from the antibody levels in table 1, both lipidated and non-lipidated Ag473 combined with Hib polysaccharide had good immune effects, demonstrating that Ag473 was conjugated to Hib as a carrier protein. The lipidated Ag473 has a better immune effect than non-lipidated Ag473.

EXAMPLE 10 immune Effect experiment of meningococcal polysaccharide protein

The experimental group was (1) serotype A, C, W and Y monovalent meningococcal polysaccharide protein, (2)A, C, W135 and Y meningococcal polysaccharide protein conjugate composition, (3) hib polysaccharide protein conjugate, and A, C meningococcal polysaccharide protein conjugate composition; wherein, the content of each meningococcal polysaccharide is 5 ug/dose; the Hib polysaccharide is 5 ug/dose, and the carrier proteins are L-Ag473.

12-14 g BALB/c mice are randomly grouped according to body weight, 6 of the mice in each group are injected with 0.5ml of sterile physiological saline, and the mice are immunized 1 time every 2 weeks, 0.5ml of the mice in each group, and the total amount of the mice is 3 times. The blood was collected from the orbital venous plexus at 6 weeks, and the content of IgG against the corresponding polysaccharide-specific antibody and the anti-Ag 473 antibody in the serum of each mouse was measured by ELISA, and the data are shown in tables 2 and 3.

TABLE 2 anti-polysaccharide specific antibody levels (GMT, 95% CI)

TABLE 3 anti-Ag 473 antibody levels (GMT, 95% CI)

Example 11

12-14 g BALB/c mice are randomly grouped according to weight, 6 mice in each group are injected with 0.5ml sterile physiological saline in a negative control group; mixing 13 Streptococcus pneumoniae polysaccharide protein conjugates prepared in example 8 to obtain 13-valent compositions as experimental groups, and determining pH value to be in the range of 6-7, wherein each serotype Streptococcus pneumoniae polysaccharide content is 2 ug/dose; immunization was performed 1 time every 2 weeks, 0.5ml each time, 3 times total. The serum of each mouse was collected from the orbital venous plexus at 6 weeks and tested for the content of IgG against streptococcus pneumoniae polysaccharide-specific antibodies and against Ag473 antibodies by ELISA, as shown in table 4.

TABLE 4 Table 4

Conclusion: as can be seen from the data in table 4, conjugation of pneumococcal polysaccharide and Ag473 carrier protein was good, conjugation of pneumococcal polysaccharide and Ag473 each serotype polysaccharide could elicit immune responses for more than 6 weeks, and antibody titers were maintained at high levels.

Finally, what is necessary here is: the above embodiments are only for further detailed description of the technical solutions of the present invention, and should not be construed as limiting the scope of the present invention, and some insubstantial modifications and adjustments made by those skilled in the art from the above description of the present invention are all within the scope of the present invention.

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Claims (5)

1. A polysaccharide-protein conjugate, characterized in that the carrier protein is Ag473, the polysaccharide being selected from the group consisting of: streptococcus pneumoniae capsular polysaccharide, neisseria encephalitis capsular polysaccharide or Hib capsular polysaccharide; the polysaccharide-protein conjugate can prevent infectious diseases caused by bacteria corresponding to capsular polysaccharide contained in the conjugate and can be used for preventing infectious diseases caused by serogroup B neisseria encephalitis; when Ag473 is a lipidated recombinant protein on the surface of Neisseria meningitidis, the amino acid sequence of the protein is shown as SEQ ID NO.5, and when Ag473 is a non-lipidated recombinant protein on the surface of Neisseria meningitidis, the amino acid sequence of the protein is shown as SEQ ID NO. 8; wherein neisseria encephalitis is selected from serogroup A, C, W, X or Y, the average ratio of neisseria meningitidis capsular polysaccharide to carrier protein is from 20:1 to 1:50 (w/w), the neisseria meningitidis capsular polysaccharide is depolymerised to an average size of 12,000-22,000 daltons, and the streptococcus pneumoniae is selected from the following serotypes: 1. 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23A, 23B, 23F or 33F, the average ratio of single serotype streptococcus pneumoniae capsular polysaccharide to carrier protein being from 3:1 to 1:5 (w/w), the average ratio of total polysaccharide antigen content to carrier protein being from 3:1 to 1:5 (w/w).

2. A method of preparing the polysaccharide-protein conjugate of claim 1, comprising conjugating the purified polysaccharide to a carrier protein.

3. Use of the polysaccharide-protein conjugate of claim 1 for the preparation of an immune formulation.

4. An immunological formulation comprising at least one polysaccharide-protein conjugate of claim 1.

5. A method of preparing an immunological formulation as claimed in claim 4 wherein the polysaccharide-protein conjugate is admixed.

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