Polysaccharide-protein conjugate, nanoparticles thereof and vaccine composition

Abstract

The present application discloses a polysaccharide-protein conjugate comprising a group A streptococcus polysaccharide or a variant thereof lacking an immunodominant N-acetylglucosamine (GlcNAc) side chain, and a streptococcus hemolysin O protein or a variant thereof, a nanoparticle thereof, and a vaccine composition, the polysaccharide-protein conjugate comprising a group A streptococcus polysaccharide or a variant thereof lacking an immunodominant N-acetylglucosamine (GlcNAc) side chain, and a streptococcus hemolysin O protein or a variant thereof, 101 amino acid residues at the C terminal of the streptolysin O are deleted. The steps for preparing the GACPR-SLO polysaccharide-protein conjugate are simple, the cost is low, and the immune protection effect of the GACPR-SLO (deltaC101) can be remarkably improved by displaying the GACPR-SLO (deltaC101) itself or displaying the GACPR-SLO (deltaC101) on particles.

Description

Technical Field

[0001] The present invention relates to biomedicine, and in particular to a polysaccharide-protein conjugate, nanoparticles and a vaccine composition. Background Art

[0002] Group A Streptococcus (GAS) is an important Gram-positive pathogen and one of the important pathogens in human bacterial infections. People infected with this bacterium may show a variety of clinical symptoms. Direct infection of the skin or soft tissue can cause acute pharyngitis, acute tonsillitis, pyoderma, etc. GAS can cause scarlet fever and necrotizing fasciitis, and in severe cases can cause streptococcal toxic shock syndrome, leading to death. In addition, GAS infection can also cause a series of immune-mediated diseases, such as acute glomerulitis, rheumatic fever, rheumatoid arthritis, etc. Among them, rheumatic fever, if not effectively treated, can easily develop into rheumatic heart disease, which is an important cause of death caused by GAS infection. GAS infection has a high incidence and mortality rate worldwide, especially in developing countries and regions. It is estimated that it can cause 800 million infections and 639,000 deaths each year, causing a huge economic burden on society.

[0003] The development of GAS vaccines has always been the focus of attention and research, but there is still no safe and reliable vaccine available. The main reason is that there are many serotypes of GAS strains, and the genomes of each serotype are quite different, making it difficult to prepare vaccines for all prevalent strains. In addition, during the development of GAS vaccines, it was found that some highly immunogenic surface proteins of GAS cross-reacted with human tissue proteins, posing a risk of inducing autoimmune diseases. At present, GAS vaccine research is still mainly based on recombinant protein vaccines. The core strategy is to use reverse vaccinology and bioinformatics to screen relatively conservative surface proteins and remove protein fragments that may cross-react with human tissues, and to achieve coverage of multiple GAS serotypes by using multiple proteins in combination. For example, in 2020, Pastural et al. reported a StreptAnova protein vaccine based on M protein, which was composed of 30 M protein N-terminal sequences. In 2019, Walker et al. reported a Combe5 protein vaccine based on non-M protein, which was composed of five GAS surface highly immunogenic proteins. However, on the one hand, this multi-component protein vaccine still finds it difficult to ensure full coverage of epidemic strains. On the other hand, the complex protein composition also causes great trouble for vaccine safety assessment and quality control.

[0004] Polysaccharide conjugate vaccines are considered to be one of the most successful forms of bacterial vaccines at present and have played a huge role in the prevention and control of human infectious diseases. Group A streptococcal polysaccharide (Group A carbohydrate, GAC) consists of a group of trisaccharide repeating units. The main chain of its structure is composed of L-rhamnose (L-Rha) alternately linked by α-1,2 and α-1,3. N-acetylglucosamine (GlcNAc) appears at the free 3-position of rhamnose at intervals through β-(1→3) linkage, forming a branched structure of the main chain. GAC is the main component of the cell wall of this bacterium, with a rich content, accounting for 30-50% of the cell wall weight, and is highly conserved in almost all Group A streptococci. Due to its extremely high proportion in the cell wall and the highly conserved structure among various serotypes, GAC is considered an effective candidate component for GAS vaccines. However, as a candidate component for GAS vaccines, there are two problems that need to be solved:

[0005] 1. There is a risk of triggering autoimmunity with GAC. The research by Goldstein et al. showed that the glycoproteins on the surface of human heart valve cells contain abundant GlcNAc, which has the same immunogenicity as the GlcNAc in the GAC side chain. Therefore, the GlcNAc in the GAC side chain is considered to have the risk of triggering human autoimmune diseases. To solve this problem, researchers obtained GAC (GACPolyrhamnose Backbone, GAC PR ), which contains no GlcNAc side chain and only a rhamnose backbone, by knocking out the GlcNAc side chain synthesis gene GacI. Victor Nizet et al. conjugated GAC PR with Streptococcus pneumoniae SP_0435 protein and immunized New Zealand rabbits. The results of flow cytometry showed that the immune sera of New Zealand rabbits could bind to various serotype GAS strains, and could promote the killing effect of human whole blood and neutrophils on various serotype GAS strains. In the mouse passive protection infection model, it could reduce the mortality rate of mice after infection with GAS, and the protective effect was comparable to that of the original GAC. Therefore, by removing the GlcNAc side chain, the occurrence of autoimmune diseases in the body can be avoided while ensuring the immune protection effect.

[0006] 2. GAC PR has a simple structure and low immunogenicity. Alone, GAC PRIt is a T cell-independent antigen that cannot generate memory immunity and requires conjugation to a carrier protein with T cell epitopes to enhance the immunoprotective effect of polysaccharides. One solution is to use a protein antigen derived from a pathogen as the carrier protein, which has both antigenic properties and immunostimulatory effects, enabling the construction of a novel vaccine containing two different types of antigens, thus achieving a "two birds with one stone" effect. Streptolysin O (SLO) is a hemolytic exotoxin secreted and expressed by GAS and is an important virulence factor of this bacterium. Previous studies have shown that by introducing amino acid point mutations, it is possible to lose the hemolytic activity of SLO while stimulating the body to produce neutralizing antibodies, which is considered an effective vaccine candidate component of this bacterium. Research has shown that after removing the 101 amino acids at the C-terminus of SLO (SLO(ΔC101)), it can still effectively stimulate the body to generate neutralizing antibodies against GAS and help the body resist GAS infection. In addition, in recent years, nanoparticle carriers represented by ferritin have been shown to facilitate the maturation of antigen-presenting cells and stimulate strong B cell and T cell responses, thus effectively enhancing the antibody titer of subunit vaccines, providing new ideas for the development of polysaccharide conjugate vaccines.

[0007] In the related research of previous group A streptococcal polysaccharide conjugate vaccines, in most studies, GAC was prepared by chemical extraction after strain fermentation or direct chemical synthesis of oligosaccharides. On the one hand, the chemical extraction method requires a large amount of cultivation of GAS strains, which has certain biosafety risks. On the other hand, in the process of polysaccharide purification, a small amount of highly immunogenic proteins on the surface of group A streptococcal cells often remain, and the residual M protein may lead to the occurrence of autoimmune diseases. The method of directly chemically synthesizing oligosaccharides has only reported the synthesis of 3-4 trisaccharide repeating units so far, while the real GAC is composed of 18 trisaccharide repeating units. It is difficult to simulate the real conformation of GAC by the method of directly chemically synthesizing oligosaccharides, and it involves the treatment of harmful chemical reagents. In recent years, with the discovery of glycosylation modification in bacteria and the development of synthetic biology, the biosynthesis method of polysaccharide conjugate vaccines has developed rapidly. The oligosaccharide transferase synthesis gene and sugar synthesis gene cluster are transformed into Escherichia coli. Through induced expression, the synthesis and coupling of polysaccharides and proteins are completed by the bacteria themselves. After ultrasonic disruption of the bacteria, the polysaccharide-protein can be purified to complete. The polysaccharide conjugate vaccine prepared by the biosynthesis method is more controllable in terms of carrier protein design, polysaccharide chain length control, and glycoprotein molar ratio design, which is an important direction for the future development and optimization of polysaccharide conjugate vaccines. In January 2025, Dorfmueller et al. published online in the journal npj vaccines the biosynthesis of GAC in Escherichia coli using the PglB oligosaccharide transferase system. However, the PglB system cannot recognize and transport natural GAC polysaccharides, and the recognition and transport of GAC can only be achieved by first modifying the structure of the reducing end of the polysaccharide, and the steps are relatively cumbersome.

[0008] SnoopCatcher-SnoopTag is a protein ligation system developed from pneumococcal pili proteins. Catalyzed by glutamate (E803) in SnoopCatcher, an isopeptide bond can form between aspartic acid (N854) in SnoopCatcher and lysine (K742) in SnoopTag, thus constructing a covalently stable multi-protein complex, which has high application value in bacterial outer membrane protein localization, increasing protein stability, and vaccine development. Summary of the Invention

[0009] In view of this, the present invention provides a polysaccharide-protein conjugate, which comprises group A streptococcal polysaccharide or its variant and streptolysin O protein or its variant. Preferably, the group A streptococcal polysaccharide or its variant lacks the immunodominant N-acetylglucosamine (GlcNAc) side chain, and the streptolysin O lacks 101 amino acid residues at the C-terminus. More preferably, the SLO(ΔC101) protein has the PglL glycosylation motif PilE4573 and / or the SnoopCatcher tag sequence. In addition, the polysaccharide-protein conjugate does not contain unnatural amino acid residues.

[0010] According to another aspect of the present invention, there is provided a method for preparing the polysaccharide-protein conjugate, wherein the group A streptococcal polysaccharide or its variant and the streptolysin O protein are linked through the PglL system.

[0011] According to another aspect of the present invention, there is provided a nanoparticle, on the surface of which the polysaccharide-protein conjugate is adsorbed. Preferably, the nanoparticle is an AP205 nanoparticle, a Qβ nanoparticle or a Ferritin nanoparticle.

[0012] According to another aspect of the present invention, there is provided a method for preparing the Ferritin nanoparticle of the polysaccharide-protein conjugate, wherein the polysaccharide-protein conjugate has the SnoopCatcher tag sequence.

[0013] According to another aspect of the present invention, there is provided an immunogenic vaccine composition, which comprises the polysaccharide-protein conjugate or the nanoparticle.

[0014] According to another aspect of the present invention, there is provided a vaccine, which comprises the immunogenic vaccine composition.

[0015] The present invention prepares GAC PR -SLO polysaccharide-protein conjugate with simple steps and low cost. In addition, GAC PR -SLO(ΔC101) itself or when displayed on the nanoparticle will significantly improve its immunoprotective effect.

[0016] Other features and advantages of the present application will be described in detail in the subsequent specific implementation section. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings forming a part of this application are used to provide a further understanding of this application. The schematic embodiments and descriptions thereof are used to explain this application. In the drawings:

[0018] Figure 1 For the construction and verification of the GAC PR expression plasmid.

[0019] Figure 2 Western blot detection of GAC PR -SLO glycoprotein expression results.

[0020] Figure 3 Shows the results of the preliminary purification of GAC PR -SLO protein purifier (a) and nickel beads (b).

[0021] Figure 4 Shows the detection results of SnoopTag-Ferritin ferritin nanoparticles.

[0022] Figure 5 Shows the detection results of SnoopTag-Ferritin ferritin nanoparticle polysaccharide-protein dual antigen nanoparticles.

[0023] Figure 6 Shows the results of the characterization of the nanoparticles.

[0024] Figure 7 Shows the safety evaluation results of the nanoparticle vaccine.

[0025] Figure 8 Shows that the nanoparticle vaccine can effectively stimulate the generation of specific antibodies in mice.

[0026] Figure 9 Shows that the nanoparticles can reduce the bacterial load and inflammatory response in organs.

[0027] Figure 10 Shows that the nanoparticles can improve the survival rate of mice. Specific implementation manners

[0028] It should be noted that, without conflict, the implementation manners in this application and the features in each implementation manner can be combined with each other.

[0029] The present application will be described in detail below with reference to the accompanying drawings and in combination with the implementation manners.

[0030] I. Preparation of polysaccharide-protein dual antigen nanoparticle vaccine

[0031] 1. Construction of polysaccharide expression plasmid

[0032] Materials: Group A Streptococcus MGAS5005 (ATCC, BAA 947), preserved in the laboratory; Escherichia coli W3110 wild strain (ATCC, 27325), preserved in the laboratory; pBBR1MCS-2 plasmid (Wuhan Miaoling Biology); PrimeSTAR GXL DNA Polymerase (Takara); NEB builder HiFi DNA assembly mix (NEB); GAC specific antibody (Abcam).

[0033] Methods: Group A Streptococcus GAC PR The polysaccharide synthesis gene cluster is the GacA-G gene, with a full length of 7835 bp (GenBank: CP000017.2, 604904..612738). Revive the GAS standard strain MGAS5005 (ATCC, BAA 947), culture the strain to the mid-logarithmic phase, and extract high-quality genomic DNA using the precipitation method. Use PrimeSTAR GXL DNA Polymerase from Takara to amplify the full-length fragment of the gene cluster, and use the NEB builder HiFi DNA assembly mix reagent from NEB to ligate the amplified full-length fragment to the pBBR1MCS-2 plasmid by homologous recombination. Extract the ligated plasmid and send it to the company for second-generation sequencing to ensure that the plasmid sequence is correct.

[0034] The Escherichia coli W3110 wild strain cannot synthesize LPS (lipopolysaccharide) by itself due to the natural deletion of the wbbL gene. Electrotransform the constructed expression plasmid into the W3110 wild strain. After overnight culturing of the strain, extract the LPS of the strain by the boiling method and perform WB detection using the GAC specific antibody. Compared with the control group, obvious LPS bands can be detected in the experimental group, proving that the constructed sugar expression plasmid can correctly express GAC in Escherichia coli PR 。 Figure 1 Shows GAC PR Construction and verification of the GAC expression plasmid, where (a) shows the amplification of the polysaccharide gene cluster, and figure (b) WB verifies GAC PR Can be correctly expressed in Escherichia coli.

[0035] 2. Design, expression, and preparation of polysaccharide protein

[0036] Materials: pET-32a(+) plasmid, preserved in the laboratory; Escherichia coli W3110ΔwaaLΔwbbh-L, preserved in the laboratory; Anti-His antibody (Abclonal Biotechnology), GAC specific antibody (Abcam);

[0037] Method: First, design the substrate protein sequence. Using SLO(ΔC101) (whose amino acid sequence is shown in SEQ ID NO 1) as the substrate protein, in order to achieve spontaneous binding to ferritin nanoparticles subsequently, the SnoopCatcher tag sequence (whose amino acid sequence is shown in SEQ ID NO 2) was fusion-expressed at the C-terminus of SLO(ΔC101). In order to achieve the directional coupling of GAC PR with SLO(ΔC101), the oligosaccharyltransferase PglL glycosylation motif PilE4573 (whose amino acid sequence is shown in SEQ ID NO 3) was fusion-expressed at the C-terminus of SnoopCatcher, and the final substrate protein obtained was SLO(ΔC101)-SnoopCatcher-PilE4573 (whose amino acid sequence is shown in SEQ ID NO 4).

[0038] To reduce the number of plasmids for subsequent polysaccharide-protein expression and purification, the oligosaccharyltransferase PglL and the substrate protein SLO(ΔC101)-SnoopCatcher-PilE4573 were co-expressed on the pET-32a(+) plasmid. This plasmid and the polysaccharide expression plasmid were co-electroporated into Escherichia coli W3110ΔwaaLΔwbbh-L. In the LB liquid medium with Kana and Amp double resistance, it was cultured with shaking at 37°C and 180 rpm until OD 600 = 0.6, IPTG was added to a final concentration of 0.5 mM, and it was cultured with shaking at 28°C and 180 rpm for 12 h. 1 mL of the bacterial solution was centrifuged at 12000 rpm for 1 min, the supernatant was discarded, and the cells were resuspended in 300 μL of hypertonic liquid (20 mM Tris-HCl pH = 8, 20% sucrose, 1 mM EDTA), and incubated with rotation at 4°C for 1 h. Then it was centrifuged at 12000 rpm for 3 min, the supernatant was completely aspirated, and the cells were resuspended in 300 μL of hypotonic liquid (20 mM Tris-HCl pH = 8, 1% sodium deoxycholate) and incubated with rotation at 4°C for 1 h. Then it was centrifuged at 12000 rpm for 3 min, and the supernatant was aspirated to obtain the periplasmic protein solution of bacteria. This solution was detected by Western Blot. Using His antibody and commercial GAC antibody for WB detection, compared with the control group, after transferring the GAC sugar plasmid, obvious glycosylated bands could be detected, proving that the synthesis of GAC PR -SLO glycoprotein can be achieved in the engineered strain W3110ΔwaaLΔwbbh-L. The results are as Figure 2 shown. Figure 2 Figure shows the Western blot detection of GAC PR -SLO glycoprotein expression results, showing that GAC PR -SLO glycoprotein is abundantly expressed.

[0039] The constructed glycoprotein-expressing strain was expanded to 5 L. After overnight induction of expression, the bacteria were harvested by high-speed centrifugation. After disrupting the bacterial cells with a high-pressure homogenizer, the His-tag of the fusion expression was used, and a protein purifier and nickel column were used for preliminary purification. Figure 3 (Left) shows the result of preliminary purification using a GAC PR -SLO protein purifier and nickel column. To improve the purity of the polysaccharide protein, the target glycoprotein solutions were combined and ultrafiltered and concentrated to 5 mL using an ultrafiltration tube, and further purified using a Superdex 200 molecular sieve. The results are as shown in Figure 3 (Right), showing that a GAC PR -SLO glycoprotein with relatively high purity can be obtained.

[0040] The steps for preparing GAC PR -SLO glycoprotein in the present invention are simple and low-cost. During the synthesis of SLO(ΔC101) protein, unnatural amino acids such as p-azidomethylphenylalanine (pAMF) were not incorporated, reducing the cost. In addition, the method of the present invention only requires large-scale culturing of the glycoprotein-expressing engineering strain, and the GAC PR -SLO(ΔC101) product can be obtained through one-step purification, and the preparation method is simple.

[0041] 3. Expression and preparation of SnoopTag-Ferritin nanoparticles

[0042] To enable spontaneous ligation with the prepared polysaccharide protein, SnoopTag was fused to the N-terminus of ferritin from Pyrococcus furiosus, linked with a (GGGGS) 3 linker. The designed sequence was entrusted to Shanghai Sangon Biotech Co., Ltd. for full gene synthesis and ligated to the pET-28a(+) plasmid backbone. Since ferritin nanoparticles have strong heat resistance and are convenient for purification, no His-tag was ligated for subsequent practical applications. The synthesized ferritin nanoparticle expression plasmid was transformed into BL21(DE3) competent cells, and 1 L was expanded and cultured. After induction of expression, preliminary purification was performed using a HiTrap Q HP anion exchange column. The purification results are shown in Figure 4 a. To further improve the purity of the nanoparticles, the target product was concentrated using an ultrafiltration tube and further purified using a Superdex 200 molecular sieve. The results are as shown in Figure 4 b, showing that ferritin nanoparticles with relatively high purity can be obtained.

[0043] 4. Preparation of polysaccharide protein dual-antigen nanoparticles

[0044] The prepared polysaccharide protein and ferritin nanoparticles were mixed in a PBS buffer system at a molar ratio of 2:1 and reacted overnight at 4°C. The SDS-PAGE results showed that after the reaction between the polysaccharide protein and ferritin nanoparticles, the molecular weight increased significantly, indicating an effective binding between the two ( Figure 5 a). Subsequently, verification was carried out again using the Western blot method. His antibody ( Figure 5 b) and GAC antibody ( Figure 5 c) could both react with the polysaccharide protein and nanoparticles, and the band positions corresponded, indicating the successful preparation of polysaccharide nanoparticles. The results are as shown in Figure 5 . ( Figure 5 a) shows the Coomassie brilliant blue staining results. ( Figure 5 b) and ( Figure 5 c) show the Western blot detection results.

[0045] For the characterization of the prepared nanoparticles, we comprehensively analyzed the elution peak images of the molecular sieve during the purification process, determined the particle size through a particle size analyzer, and observed through an electron microscope. The results are as shown in Figure 6 . From the elution peak results of the molecular sieve, the combined vaccine with the largest molecular weight was filtered out first, followed by the nanoparticles and finally the glycoprotein, which was consistent with our expectations ( Figure 6 a). The particle size was detected using dynamic light scattering, and the results showed that the particle size was 25.02 nm ( Figure 6 b). Compared with before binding, the particle size increased significantly. We observed the prepared nanoparticles by negative staining with a transmission electron microscope ( Figure 6 c). The sample particle size was evenly distributed, further proving that the polysaccharide protein bound to the surface of the ferritin nanoparticles, and the polysaccharide protein nanoparticles were successfully prepared.

[0046] II. Evaluation of the immune protection effect of the polysaccharide protein dual-antigen nanoparticles vaccine

[0047] 1. Safety evaluation

[0048] The prepared glycan protein nanoparticles were used to immunize mice. There were two experimental groups and a negative control group, with 10 mice in each group. Each mouse in the experimental group was subcutaneously immunized with 2.5 μg of polysaccharide content on the back of the neck. Subcutaneous injections were performed on the neck at 0 d, 21 d, and 35 d. Blood was taken and the supernatant was collected at 36 d, 38 d, and 40 d after immunization. According to the instructions of the mouse ELISA kit (IL-6, TNF-α, IFN-γ), the cytokine levels in the mouse serum were detected. The aspartate aminotransferase (AST) ( Figure 7 e) and alanine aminotransferase (ALT) ( Figure 7d), Lactate dehydrogenase (LDH) ( Figure 7 f) and Blood urea nitrogen (BUN) ( Figure 7 g). The combined vaccine immunization group did not cause an increase in IFN-γ ( Figure 7 a), TNF-α ( Figure 7 b), IL-6 ( Figure 7 c) when combined with the negative control group. From the perspective of serum biochemical indexes, compared with the negative control group, the combined vaccine immunization group did not have a significant impact on the biochemical indexes of myocardial function (LDH), liver function (ALT, AST), and kidney function (BUN), and all were within the normal range, indicating that the combined vaccine has good biosafety. The safety evaluation results of the nanoparticle vaccine are as Figure 7 shown.

[0049] 2. Specific antibody evaluation

[0050] To verify whether the prepared combined vaccine can stimulate mice to produce corresponding antibodies, an immunization experiment was designed, and the production of IgG ( Figure 8 a) and IgG1 ( Figure 8 b) antibodies was detected by ELISA. In the anti-SLO ELISA experiment, compared with the PBS group, Fer group, and polysaccharide-protein group, the total IgG antibody titer and IgG1 antibody titer in the polysaccharide nanoparticle group were significantly increased; the same conclusion was obtained in the anti-GAC ELISA experiment ( Figure 8 c and Figure 8 d). In summary, the prepared polysaccharide nanoparticles can effectively induce humoral immune responses, produce specific antibodies against GAC and SLO, and induce the production of IgG and IgG1 antibodies.

[0051] 3. Evaluation of the protective effect against challenge

[0052] Ten days after three-dose immunization of each group of mice, Streptococcus pyogenes MGAS5005 was used as the target bacterium for intraperitoneal infection, and each mouse was infected with 1.0×10 8 CFU. Twelve hours later, the control group mice showed listlessness, loss of appetite and water intake, body tremors, piloerection, etc.; the combined vaccine group had less impact and remained in a normal state. To further prove the protective effect of the nanoparticles, the organs were taken 3 days after infection to detect the bacterial colonization amount. Compared with other groups, the livers ( Figure 9 a) and kidneys ( Figure 9The bacterial loads in (b) were significantly reduced, indicating that the prepared polysaccharide nanoparticles had good immunoprotective effects. The TNF-α levels in the sera of mice three days after infection were further analyzed and measured using an ELISA kit. The TNF-α level in the sera of the nanoparticle group was around 30 pg / mL, while that in the sera of the PBS group was around 50 pg / mL ( Figure 9 c), suggesting that the immunized mice with the prepared polysaccharide nanoparticles could be protected from the inflammatory response caused by Streptococcus pyogenes infection.

[0053] To further evaluate the protective effect of the glycoprotein nanoparticles, 10 days after three immunizations, we intraperitoneally infected the mice with the Streptococcus pyogenes MGAS5005 strain, with each mouse infected with 5.0×10 8 CFU, and continuously observed. The results showed that the polysaccharide nanoparticles could produce good protective effects, with the survival rate of the mice being 90%, while the survival rates of the mice in the PBS group, Fer group, and glycoprotein group were 10%, 20%, and 50% respectively ( Figure 10 ). This indicates that the prepared polysaccharide nanoparticles can resist high-dose Streptococcus pyogenes infection and produce good protective effects.

[0054] From the experimental results, whether it is the antibody production level, the bacterial load in the organs, or the survival rate after challenge, displaying GAC PR -SLO(ΔC101) on the Ferrtin particles can significantly improve their immunoprotective effects.

[0055] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A polysaccharide-protein conjugate comprising a group A streptococcal polysaccharide or a variant thereof and a streptolysin O protein or a variant thereof.

2. The polysaccharide-protein conjugate according to claim 1, wherein The group A streptococcal polysaccharide or its variant lacks immunodominant N-acetylglucosamine (GlcNAc) side chain, and the streptolysin O lacks 101 amino acid residues at the C-terminus.

3. The polysaccharide-protein conjugate according to claim 1, wherein The SLO (ΔC101) protein has a PglL glycosylation motif PilE4573 and / or a SnoopCatcher tag sequence.

4. The polysaccharide-protein conjugate according to claim 1, wherein The polysaccharide-protein conjugate does not contain non-natural amino acid residues.

5. A method for preparing the polysaccharide-protein conjugate according to any one of claims 1 to 4, wherein: The group A streptococcal polysaccharide or variant thereof and the streptolysin O protein are linked via the PglL system.

6. A nanoparticle, the polysaccharide-protein conjugate according to any one of claims 1 to 4 being adsorbed on the surface of the nanoparticle.

7. The nanoparticle according to claim 6, wherein The nanoparticles are AP205 nanoparticles, Qβ nanoparticles or Ferritin nanoparticles.

8. The method for preparing the Ferritin nanoparticles of the polysaccharide-protein conjugate according to claim 7, wherein: The polysaccharide-protein conjugate has a SnoopCatcher tag sequence.

9. An immunogenic vaccine composition comprising the polysaccharide-protein conjugate according to any one of 1 to 4 or the nanoparticle according to claim 6 or 7.

10. A vaccine comprising the immunogenic vaccine composition of claim 9.

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