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CN-122005775-A - Group B streptococcus oligosaccharide protein conjugate and preparation method and application thereof

CN122005775ACN 122005775 ACN122005775 ACN 122005775ACN-122005775-A

Abstract

The invention relates to a group B streptococcus oligosaccharide protein conjugate, a preparation method and application thereof, belonging to the technical field of anti-group B streptococcus vaccine development. The invention takes inactivated group A streptococcus C5a peptidase ScpA193 as a carrier, and is connected with group B streptococcus oligosaccharide antigen to prepare the oligosaccharide protein conjugate for resisting group B streptococcus, wherein the oligosaccharide antigen is oligosaccharide trisaccharide derivative, oligosaccharide pentasaccharide derivative or oligosaccharide octasaccharide derivative of polysaccharide on the cell wall surface of group B streptococcus. The oligosaccharide protein conjugate prepared by the invention can induce two types of antibodies with different targets to cooperatively target group B streptococcus at the same time, and is a compound vaccine. The vaccine has better applicability, can effectively inhibit group B streptococcus, and is not limited by the problem of bacterial drug resistance caused by the use of antibiotics in a large amount.

Inventors

  • GU GUOFENG
  • SUN CHONGZHEN
  • WANG GUIRONG
  • ZHAO JIELIN

Assignees

  • 山东大学

Dates

Publication Date
20260512
Application Date
20260119

Claims (10)

  1. 1. The group B streptococcus oligosaccharide protein conjugate is characterized in that the structural general formula is shown in a formula (I): Formula (I) Wherein the GBC oligosaccharide antigen is an oligosaccharide trisaccharide derivative, an oligosaccharide pentasaccharide derivative or an oligosaccharide octasaccharide derivative of the polysaccharide on the cell wall surface of the group B streptococcus; The connecting arm is succinimide, glutarimide or adipoimide; The novel carrier protein ScpA193 is a group A streptococcus virulence factor C5a peptidase inactivated by directed mutation, and is structurally characterized in that histidine at position 193 is mutated into alanine; m is the number of GBC oligosaccharide antigens linked to the novel carrier protein ScpA193 and is any integer from 1 to 30.
  2. 2. The group B streptococcus oligosaccharide protein conjugate of claim 1, wherein the GBC oligosaccharide antigen is an oligosaccharide trisaccharide derivative or oligosaccharide pentasaccharide derivative of group B streptococcus cell wall surface polysaccharide.
  3. 3. Group B streptococcus oligosaccharide protein conjugate according to claim 1, wherein the oligosaccharide trisaccharide derivative is 2-aminoethyl α -L-rhamnosyl- (1→3) - α -D-galactopyranosyl- (1→3) -2-deoxy-2-acetamido- β -D-glucopyranoside.
  4. 4. Group B streptococcus oligosaccharide protein conjugate according to claim 1, wherein the oligosaccharide pentasaccharide derivative is α -L-rhamnosyl- (1→2) - α -L-rhamnosyl- (1- > 1) - [ Α -L-rhamnosyl- (1→3) ] -6-oxo-aminoethylphosphine-D-glucitol.
  5. 5. The group B streptococcus oligosaccharide protein conjugate of claim 1, wherein the oligosaccharide octasaccharide derivative is α -L-rhamnosyl- (1→2) - [ α -L-rhamnosyl- (1→3) - α -D-galactopyranosyl- (1→3) -2-deoxy-2-acetamido- β -D-glucopyranosyl- (1→4) - ] - α -L-rhamnosyl- (1→2) - α -L-rhamnosyl- (1- > 1) - [ Α -L-rhamnosyl- (1→3) ] -6-oxo-aminoethylphosphine-D-glucitol.
  6. 6. Group B streptococcus oligosaccharide protein conjugate according to claim 1, wherein the group B streptococcus oligosaccharide protein conjugate has the following structural general formula (II), (III), (IV): Formula (II) Formula (III) (IV) Wherein in the formula (II), the formula (III) and the formula (IV), m is any integer from 1 to 30.
  7. 7. A method of preparing a group B streptococcus oligosaccharide protein conjugate according to claim 1, comprising the steps of: (1) The GBC oligosaccharide antigen is activated, namely the GBC oligosaccharide antigen is dissolved in a mixed solution of N, N-dimethylformamide and phosphate buffer solution, the concentration of the GBC oligosaccharide antigen in the mixed solution is 0.1-10 mg/mL, bissuccinimidyl glutarate is added into the mixed solution under the stirring condition, the molar equivalent ratio of the bissuccinimidyl glutarate to the GBC oligosaccharide antigen is 10-15:1, the mixture is stirred at room temperature for 4 hours, white powdery solid is obtained through reduced pressure distillation, the solid is washed by ethyl acetate, and the activated GBC oligosaccharide antigen is obtained after drying; (2) And (3) synthesizing the conjugate, namely dissolving the activated GBC oligosaccharide antigen and the novel carrier protein ScpA193 in a phosphate buffer solution according to a molar ratio of 5-30:1, stirring overnight at room temperature, removing small molecular substances by a dialysis method to obtain an aqueous solution of the oligosaccharide protein conjugate, and freeze-drying the aqueous solution of the oligosaccharide protein conjugate to obtain the group B streptococcus oligosaccharide protein conjugate.
  8. 8. The method according to claim 7, wherein the volume ratio of the N, N-dimethylformamide to the phosphate buffer solution in the mixed solution in the step (1) is 4:1, wherein the phosphate buffer solution has a composition of Na 2 HPO 4 0.1M,KH 2 PO 4 0.1m, ph 8; Preferably, the concentration of the activated GBC oligosaccharide antigen in the step (2) in the phosphate buffer is 0.1-10 mg/mL.
  9. 9. Use of a group B streptococcus oligosaccharide protein conjugate according to claim 1 in the manufacture of a medicament for the treatment/prophylaxis of group B streptococcus infection.
  10. 10. The use according to claim 9, wherein the antibody induced by the oligosaccharide protein conjugate binds to group B streptococcus of type Ia, type II, type III and type V serotypes; Preferably, the pharmaceutical product comprises a vaccine.

Description

Group B streptococcus oligosaccharide protein conjugate and preparation method and application thereof Technical Field The invention relates to a group B streptococcus oligosaccharide protein conjugate, a preparation method and application thereof, belonging to the technical field of anti-group B streptococcus vaccine development. Background Group B Streptococcus (GBS) is a gram positive bacterium, coated with a capsule, colonized in the gastrointestinal and genital tracts of humans, and the principal pathogenic bacteria for invasive bacterial infections in neonates and infants worldwide. GBS infection often causes severe disease such as neonatal pneumonia, sepsis, and meningitis, with high morbidity and mortality. Meanwhile, GBS is also identified as a new pathogen for adult susceptible populations, particularly pregnant women, the elderly and immunocompromised adults. Vaccination is the most effective means of preventing bacterial infection diseases and is the most cost-effective means of social life saving. Therefore, the development of safe and effective GBS vaccines is of great significance for the prevention and treatment of GBS infectious diseases. The ideal anti-group B streptococcus vaccine should be characterized by (1) conservation of its antigenic determinants in all group B streptococcus strains and (2) high immunogenicity, which can well induce strong immune memory in humans, such as induction of serum immune protein G (IgG) and mucosal immune protein A (IgA) responses, while not cross-immunoreaction with human tissues. GBS cell wall polysaccharides (Group B carbohydrate, GBC) were found to have highly conserved expression across all serotypes and are therefore considered ideal target molecules for the development of GBS saccharide antibacterial vaccines. However, there are limited studies on the chemical synthesis and antigenicity of GBC oligosaccharide components. Jennings et al report chemical synthesis of various GBC oligosaccharide fragments including disaccharide, trisaccharide and tetrasaccharide derivatives, and revealed that GBC oligosaccharide fragment structures such as α -L-Rha- (1- > 2) - α -L-Rha and α -L-Rha- (1- > 3) - α -D-Gal- (1- > 3) - β -D-GlcNAc, etc., were able to specifically bind anti-GBC polyclonal antibodies by competition inhibition experiments (Infection and Immunity 1991, 59, 1690)1696.). The Cod e task combines different oligosaccharide structures of GBC, and immunizes mice after coupling the same with CRM197 protein, and discovers that the functional GBC specific antibody produced by mouse serum is induced, can efficiently identify different GBS strains and mediate opsonophagocytosis to kill GBS bacteria (JACS Au 2022, 2, 1724)1735). The results show that GBC oligosaccharides have great application potential as immune antigen molecules. The carrier proteins used in the above studies are, however, common carrier proteins such as diphtheria toxin mutant (CRM 197), tetanus toxoid protein (TT) and the like. The glycoprotein conjugates prepared by repeatedly using the carrier proteins for a long time have been reported to generate some adverse immune effects during immunization, which are mainly reflected in two aspects of antigen competition and carrier-induced antigen epitope inhibition (Human Vaccines & Immunotherapeutics 2013, 9 (12), 2505-2523). The ScpA193 protein is derived from a virulence protein Ca5 peptidase mutant of group A streptococcus, and is proved to be a carrier molecule which can well present the immunogenicity of oligosaccharide antigens (RSC adv.2017, 7, 42056; bacterines 2020, 9, 139), meanwhile, ca5 peptidase is highly expressed in all streptococcus genera in a conservation way, and has high homology in structure, an antibody which is induced by the ScpA193 protein can well identify GBS bacteria (bacterines 2020, 9, 139), and therefore, igG antibodies induced by the ScpA193 protein in a human body are expected to have a neutralization and killing effect on GBS bacteria. At present, the ScpA193 is used as a carrier to be connected with GBS oligosaccharide antigen to prepare the group B streptococcus oligosaccharide conjugate vaccine, and related literature reports are not yet available. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a group B streptococcus oligosaccharide protein conjugate, a preparation method and application thereof, wherein an inactivated group A streptococcus C5a peptidase ScpA193 is taken as a carrier, and is connected with group B streptococcus oligosaccharide antigen to prepare the group B streptococcus oligosaccharide protein conjugate, wherein the oligosaccharide antigen is an oligosaccharide derivative of group B streptococcus cell wall surface polysaccharide. Description of the terminology: Room temperature is defined as having a meaning conventional in the art, generally 25±2 ℃. The invention is realized by the following technical scheme: group B streptococcus oligosaccharide