A composition of pneumococcal polysaccharide-protein conjugate, its preparation method and application

Abstract

This invention relates to the field of biomedical technology, specifically to a composition of pneumococcal polysaccharide-protein conjugate and its preparation method, as well as the application of this composition in the preparation of vaccines or drugs for the prevention or treatment of diseases caused by bacterial and / or viral infections. The pneumococcal polysaccharide-protein conjugate composition of this invention comprises at least one first carrier protein bound to capsular polysaccharides from different pneumococcal serotypes and at least one second carrier protein bound to capsular polysaccharides from different pneumococcal serotypes. The first carrier protein is selected from respiratory syncytial virus (RSV) Pre-F protein, and the second carrier protein is selected from varicella-zoster virus (VZV) gE protein. This invention, through an innovative dual-carrier system design, screens and integrates bacterial pneumococcal capsular polysaccharide antigens conjugated to RSV and VZV structural proteins respectively, achieving synergistic prevention of bacterial (pneumococcal) and viral (VZV, RSV) respiratory infections.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a composition of pneumococcal polysaccharide-protein conjugate and its preparation method, as well as the use of the composition in the preparation of vaccines or drugs for the prevention or treatment of diseases caused by bacterial and / or viral infections. Background Technology

[0002] Streptococcus pneumoniae is a capsule-forming Gram-positive diplococcus with over 90 different serotypes. Capsular polysaccharides are its most important virulence factor. This bacterium colonizes the human nasopharynx and can cause invasive diseases such as meningitis and pneumonia, as well as non-invasive diseases such as otitis media, when the body's immunity is weakened. Statistics show that in 2019, pneumococcal infection caused 829,000 deaths globally, ranking third among bacterial infections and first in terms of lost years of life. The majority of deaths are among children under 5 years old (75% of invasive pneumococcal disease and 83% of meningitis occur in children under 2 years old) and people over 60 years old. With the increasing aging of the global population (the UN's 2023 World Society Report projects that the population aged 65 and over will increase from 761 million in 2021 to 1.6 billion in 2050), the disease burden is becoming increasingly severe. In terms of treatment, pneumococcus is showing increasing resistance to commonly used antibiotics, and vaccination is the most economical and effective preventative measure. Currently available vaccines include pneumococcal polysaccharide vaccines (PPV) and conjugate vaccines (PCV). The early product, PPV23 (Wyeth), achieved 90% coverage of dominant serotypes, but failed to induce an effective immune response in infants. Further development revealed that PCV, by covalently binding polysaccharides to carrier proteins, can convert non-T-cell-dependent antigens into T-cell-dependent antigens. Vaccination at 2 months of age promotes a stronger immune response and memory response. Studies have found that conjugate vaccines can be used at any age and have a longer-lasting and more potent effect than polysaccharide vaccines. However, the carrier proteins used in existing conjugate vaccines (such as tetanus toxoid and diphtheria toxoid) only consider enhancing the immunogenicity of the polysaccharides, without considering the immunogenicity of the carrier protein itself. Therefore, while increasing vaccine potency and expanding coverage, the selection of carrier proteins is also a crucial consideration.

[0003] Varicella-zoster virus (VZV) belongs to the Alphaherpesvirinae subfamily of the Herpesviridae family and is composed of linear double-stranded DNA encapsulated in a nucleocapsid. The virus mainly contains six key glycoproteins (gB, gC, gE, gH, gI, and gL), with gE protein showing the highest expression level in infected cells. It adsorbs to gI non-covalently and can specifically bind to the Fc fragment of IgG antibodies, becoming an important target for viral immune evasion and neutralizing antibodies. VZV is distributed globally and infects only humans. Primary infection manifests as chickenpox (most commonly in childhood). After infection, the virus can remain latent in sensory neurons for a long time. With age or impaired immune function, weakened cellular immune responses can lead to viral reactivation, resulting in herpes zoster (HZ). Clinically, it presents as a unilateral vesicular rash accompanied by burning sensation and neuralgia. Approximately 9%-34% of patients develop postherpetic neuralgia (PHN), with pain ratings reaching 7 or higher. In some patients, the pain can last for 1-10 years or even longer. Currently, there is no specific drug for treating shingles and postherpetic neuralgia (PHN). Clinical treatment often involves antiviral drugs such as acyclovir or combined analgesics to relieve the severe neuralgia caused by shingles. However, limitations remain, including a high recurrence rate and the difficulty in treating PHN. Therefore, VZV vaccination is undoubtedly a key strategy for effectively preventing shingles and PHN.

[0004] Respiratory syncytial virus (RSV) is an enveloped, non-segmented, single-stranded, negative-sense RNA virus whose genome encodes 11 proteins, including three transmembrane surface glycoproteins (G, F, and SH), six internal proteins, and two non-structural proteins. The virus exists in two subtypes, A and B, differing primarily in the G glycoprotein, while the F glycoprotein sequence is more conserved between the two subtypes. The RSV fusion protein (F protein) consists of 574 amino acid residues. Initially, it is the F0 precursor, which is hydrolyzed by furin to form F1 and F2 subunits, linked by disulfide bonds to form heterodimers. These three heterodimers assemble into a mature F protein trimer. The RSV F protein trimer is unstable, exhibiting two conformations: pre-F and post-F. While the pre-F conformation is unstable, it is a key antigen in viral-mediated membrane fusion and immune responses. RSV is a major pathogen causing lower respiratory tract infections in infants and young children. Transmitted through the air, it can cause bronchitis, pneumonia, asthma, and other diseases, posing a particularly serious threat to premature infants, immunocompromised children, and the elderly, potentially leading to severe complications and even death. Statistics show that in 2019, there were 33 million RSV-related acute lower respiratory tract infections globally, resulting in 3.6 million hospitalizations and 26,300 deaths. Among these, 6.6 million were infants aged 0-6 months, with 1.4 million hospitalizations and 13,300 deaths. Of the children under 5 years old hospitalized globally due to RSV infection, 99% of deaths occurred in developing countries. The RSV infection rate in adults increases with age, with the highest in-hospital mortality rate among those over 65 years of age (approximately 72,000 deaths per 100,000 people annually due to RSV infection). There is currently no specific treatment for RSV infection, which has a high infection and mortality rate. After more than 60 years of research, only two RSV vaccines (GSK's Arexvy and Pfizer's Abrysvo) received FDA approval in 2023. However, these vaccines are primarily intended for specific age groups such as the elderly and do not cover the entire population, including infants and young children. As of now, there are no domestically produced RSV vaccines approved for marketing in China, making the acceleration of RSV vaccine development particularly urgent.

[0005] In the field of biomedicine, vaccination, as a core means of preventing and controlling infectious diseases, is becoming increasingly important due to the growing diversity and complexity of pathogens. Traditional vaccine designs often focus on single pathogens, making it difficult to address the challenges posed by multi-pathogen infections. For example, diseases such as pneumococcal pneumonia, RSV bronchitis, and varicella / shingles are caused by bacteria and viruses, respectively, with significantly different pathological mechanisms and immune responses. Existing vaccine products typically aim to prevent single-pathogen infection. To achieve protection against multiple diseases with a single vaccine, combination vaccines are mainly used, such as diphtheria-pertussis-tetanus trivalent, pentavalent, and hexavalent vaccines. These vaccines are prepared by mixing single products, but clinical evaluations show that some single-drug vaccines are not very effective. Therefore, developing multivalent vaccines to replace single-drug vaccines and achieving full coverage of the protected population is undoubtedly a primary task in future vaccine research and development.

[0006] However, developing multivalent conjugate vaccines faces numerous challenges, one key consideration being the carrier inhibition between different serotypes. The choice of carrier protein significantly impacts the immunogenicity of the conjugate vaccine. The production process for conjugating pneumococcal polysaccharides to carrier proteins and solutions to carrier inhibition between different serotypes are crucial technical hurdles that must be overcome in developing multivalent conjugate vaccines. Especially with increasing vaccine valence, the selection and design of polysaccharides and proteins must, on the one hand, avoid carrier inhibition effects while enhancing the immunogenicity of polysaccharides for different serotypes, and on the other hand, ensure the immunogenicity of the carrier protein itself, thereby producing a vaccine with multiple immunogenicities. These two aspects are the primary key challenges to be addressed in the development of multivalent vaccines. This invention, through screening for dominant serotype combinations and designing and selecting different carrier proteins, achieves highly efficient covalent binding of multiple antigens in a single immunizing agent, ultimately achieving the goal of one vaccine protecting against multiple diseases. Summary of the Invention

[0007] To address the issue that some serotype polysaccharides in multivalent pneumococcal conjugate vaccines exhibit weak immunogenicity due to carrier interference / inhibition, and the problem that the carrier protein design in existing pneumococcal conjugate vaccines does not consider autoimmunogenicity, this invention proposes a multi-pathogen conjugate vaccine based on polysaccharide-protein conjugation technology. Through an innovative dual-carrier system design, bacterial pneumococcal capsular polysaccharide antigens are screened and conjugated to viral RSV and VZV structural protein antigens, respectively. This avoids the seroinhibition phenomenon in multivalent conjugate vaccines, effectively enhancing the immunogenicity of different serotype polysaccharides, while ensuring the immunogenicity of the carrier protein itself. This results in an immunogenic preparation containing multiple antigens with highly efficient covalent binding, achieving synergistic prevention against bacterial (pneumococcal) and viral (VZV, RSV) respiratory infections.

[0008] The technical solution of this invention to solve the technical problem is as follows: In a first aspect of the invention, a composition of a pneumococcal polysaccharide-protein conjugate is provided, the composition comprising at least one first carrier protein of capsular polysaccharide-bound from different pneumococcal serotypes and at least one second carrier protein of capsular polysaccharide-bound from different pneumococcal serotypes, wherein the first carrier protein is selected from respiratory syncytial virus Pre-F protein and the second carrier protein is selected from varicella-zoster virus gE protein.

[0009] Furthermore, the first carrier protein is a respiratory syncytial virus Pre-F recombinant protein, namely F-1 or F-2, and its amino acid sequence is shown in SEQ ID NO: 2 or SEQ ID NO: 3; the second carrier protein is a varicella-zoster virus gE recombinant protein, namely E-1, and its amino acid sequence is shown in SEQ ID NO: 5.

[0010] Further, the capsular polysaccharide is selected from one or more of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

[0011] Further, the capsular polysaccharide bound to the first carrier protein is selected from one or more of serotypes 1, 2, 3, 4, 8, 12F, 14, 15B, 17F, 19A, 20, 22F, and 33F, and the capsular polysaccharide bound to the second carrier protein is selected from one or more of serotypes 5, 6A, 6B, 7F, 9N, 9V, 10A, 11A, 18C, 19F, and 23F.

[0012] Furthermore, the first carrier protein is F-1, whose amino acid sequence is shown in SEQ ID NO: 2, and the second carrier protein is E-1, whose amino acid sequence is shown in SEQ ID NO: 5.

[0013] Furthermore, the first carrier protein is F-2, whose amino acid sequence is shown in SEQ ID NO: 3, and the second carrier protein is E-1, whose amino acid sequence is shown in SEQ ID NO: 5.

[0014] In a preferred embodiment of the present invention, the composition is a composition comprising a 24-valent pneumococcal polysaccharide dual-carrier protein conjugate, wherein the capsular polysaccharides of serotypes 1, 2, 3, 4, 8, 12F, 14, 15B, 17F, 19A, 20, 22F, and 33F bind to a first carrier protein F-1, and the capsular polysaccharides of serotypes 5, 6A, 6B, 7F, 9N, 9V, 10A, 11A, 18C, 19F, and 23F bind to a second carrier protein E-1.

[0015] In another preferred embodiment of the invention, the composition is a composition comprising a 24-valent pneumococcal polysaccharide dual-carrier protein conjugate, wherein the capsular polysaccharides of serotypes 1, 2, 3, 4, 8, 12F, 14, 15B, 17F, 19A, 20, 22F, and 33F bind to a first carrier protein F-2, and the capsular polysaccharides of serotypes 5, 6A, 6B, 7F, 9N, 9V, 10A, 11A, 18C, 19F, and 23F bind to a second carrier protein E-1.

[0016] Furthermore, the composition of the pneumococcal polysaccharide-protein conjugate contains an adjuvant.

[0017] Furthermore, the adjuvant includes one or more selected from aluminum salt adjuvants, CpG, QS21, monophosphoryl ester A, MF59, stearoyl tyrosine, Freund's adjuvant, and other mucosal adjuvants.

[0018] In a second aspect of the invention, a composition of pneumococcal polysaccharide-protein conjugate as described in the first aspect is provided. The preparation method includes the following steps: (1) Prepare at least one capsular polysaccharide from different pneumococcal serotypes and react it with a first carrier protein in a buffer or organic solvent to obtain a first binding stock solution, wherein the first carrier protein is selected from respiratory syncytial virus Pre-F protein; (2) Prepare at least one capsular polysaccharide from different pneumococcal serotypes and react it with a second carrier protein in a buffer or organic solvent to obtain a second binding stock solution, wherein the second carrier protein is selected from varicella-zoster virus gE protein; (3) Mix the first binding stock solution obtained in step (1) with the second binding stock solution obtained in step (2) to obtain the composition stock solution of pneumococcal polysaccharide-protein conjugate.

[0019] Furthermore, the chemical synthesis reaction is selected from one of the following: the reduced amine method, the CDAP method, the adipic dihydrazide method, or the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride method.

[0020] Furthermore, the organic solvent is selected from dimethyl sulfoxide or dimethylformamide.

[0021] Furthermore, the preparation method also includes pretreatment of pneumococcal capsular polysaccharide, expression and purification of carrier protein, and purification of the conjugate.

[0022] Furthermore, the pretreatment of the capsular polysaccharide includes degradation and activation, wherein the degradation method is selected from high-pressure homogenization degradation, acid hydrolysis or enzymatic digestion.

[0023] In a third aspect of the invention, the use of a composition of pneumococcal polysaccharide-protein conjugate as described in the first aspect in the preparation of a vaccine or medicament for the prevention or treatment of a disease, said disease being caused by Streptococcus pneumoniae infection, varicella-zoster virus infection, and / or respiratory syncytial virus infection.

[0024] The present invention has the following technical effects: (1) The innovative dual-carrier combination design effectively reduces immune interference and enhances the polysaccharide immune effect. The dual-vector pneumococcal polysaccharide conjugate vaccine prepared in this invention, compared to the single CRM197 vector vaccine, fundamentally reduces immune interference between multiple serotype vaccines, resulting in superior overall immunogenicity. As shown in Examples 7 and 8, different carrier proteins combined with different serotype polysaccharide antigens exhibit varying immunizing effects. The immunogenicity of some serotype polysaccharides shows little correlation with the carrier protein (e.g., Pn1, Pn11A, Pn20), but the immunogenicity of most serotype polysaccharides shows a significant correlation with the carrier protein. Overall, in groups 2-5, the dual-vector protein approach resulted in better immunization than the single-vector group 1. Among the 11 serotypes tested outside of the positive vaccine, group 2 (a specific serotype combined with F-1 and E-1) and group 4 (a specific serotype combined with F-2 and E-1) showed outstanding immunogenicity. Specifically, group 2 exhibited significantly higher immunogenicity than group 1 for polysaccharides containing 8 serotypes (2, 9N, 10A, 12F, 15B, 17F, 22F, and 33F). Group 4 showed immunogenicity for 10 serotypes (2, 8, 9N, 10A, 12F, 15B, 17F, 20, 22F, and 33F). The immunogenicity of the serotype polysaccharides was significantly higher than that of group 1, while only 6 and 4 serotype polysaccharides in groups 3 and 5, respectively, were higher than that of the single-vector group 1. This indicates that the combination of serotype 2, 8, 12F, 15B, 17F, 22F, and 33F polysaccharides with F-1 or F-2 vectors, serotype 9N and 10A polysaccharides with E-1 vectors, and 11A and 20 polysaccharides with F-1, F-2, or E-1 vectors ensured that the immune responses of each serotype polysaccharide did not inhibit each other, and the overall immune effect was further improved. In the 13 serotype tests identical to those for the positive vaccine, groups 2 and 4 showed superior immunogenicity. Specifically, group 2 exhibited significantly higher immunogenicity than group 1 for 10 serotypes (3, 4, 5, 6A, 6B, 7F, 9V, 14, 19F, 23F polysaccharides). Similarly, group 4 showed significantly higher immunogenicity than group 1 for 11 serotypes (3, 4, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F, 23F polysaccharides). Groups 3 and 5, however, showed significantly lower immunogenicity. The immunogenicity of the 8- and 5-serotype polysaccharide groups was superior to that of the single-vector group 1. This indicates that the combination of serotypes 3, 4, 14, and 19A with F-1 or F-2 vectors, serotypes 5, 6A, 6B, 7F, 9V, 19F, and 23F with E-1 vectors, and serotypes 1 and 18C with F-1, F-2, or E-1 vectors effectively avoids immune interference between different serotype polysaccharide antigens, resulting in overall immunogenicity far exceeding that of single-vector vaccines. Therefore, the dual-vector combination prepared in this invention, through differentiated screening and allocation of the binding relationship between serotypes and vectors, establishes independent immune pathways for different polysaccharide antigens, significantly reducing competitive inhibition and achieving a synergistic enhancement of overall immune efficacy.

[0025] (2) Construct a 24-valent dual-vector combination vaccine to achieve a dual breakthrough of "broad coverage + strong immunization". The 24-valent dual-vector pneumococcal polysaccharide conjugate vaccine of this invention, while covering 24 serotypes and significantly broadening the scope of protection, exhibits significantly superior immunogenicity compared to commercially available single-vector vaccines, providing a better option for the prevention and control of pneumococcal infection. As can be seen from Example 9, there are significant differences in the titers of corresponding serotype polysaccharide antibodies induced in animals by the 24-valent pneumococcal polysaccharide dual-vector conjugate vaccine (Formulation B, Formulation C) and PCV13 (Formulation A). Overall, Formulation B induces the highest serum antibody titer in animals, followed by Formulation C. Among the 13 serotypes identical to those in the PCV13 positive vaccine, especially serotypes 3, 5, 6A, 6B, 7F, 9V, 14, 19F, and 23F, the polysaccharide antibody titers of formulations B and C were significantly higher than those of formulation A, indicating that their immunization effect clearly reached that of the marketed vaccine. Simultaneously, for the 11 serotypes other than those in the positive vaccine, formulations B and C also showed good immunogenicity, particularly for serotypes 8, 9N, 10A, 12F, 15B, 17F, 22F, and 33F, where the polysaccharide antibody titers were significantly higher and significantly higher than the overall average of the 13 serotype polysaccharide antibody titers in the marketed positive vaccine. This indicates that the dual-vector vaccine group induced higher polysaccharide antibody titers and higher serum protective efficacy in animals immunized with animals compared to the single-vector vaccine. In other words, the 24-valent pneumococcal polysaccharide dual-vector conjugate vaccine prepared in this invention can effectively increase the level of IgG antibodies in animals, effectively avoiding the immunosuppressive effect caused by a single vector, and further enhancing the antigenicity against the polysaccharides of 24 serotypes of pneumococcus. This also demonstrates that, compared to existing single-vector vaccine products, the pneumococcal polysaccharide dual-vector conjugate vaccine prepared using this invention covers higher-valence pneumococcal serotypes, expanding the vaccine's protective range, while also ensuring superior immunogenicity of the polysaccharide antigens. Furthermore, while both formulations B and C, which are dual-vector vaccines, showed good promoting effects on the immunization of 24 polysaccharide antigens, differences in the carrier proteins F-1 and F-2 resulted in variations in polysaccharide antibody titers for some serotypes. Specifically, for serotypes 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 15B, 17F, 19F, 22F, 23F, and 33F polysaccharide antibody titers, formulation B showed significantly higher values. The results were higher than those of formulation C, indicating that the combination of polysaccharides from serotypes 5, 6A, 6B, 7F, 9N, 9V, 10A, 11A, 18C, 19F, and 23F with carrier protein E-1, and polysaccharides from serotypes 1, 2, 3, 4, 8, 12F, 14, 15B, 17F, 19A, 20, 22F, and 33F with carrier protein F-2, has a stronger synergistic effect in multivalent vaccines and can more effectively enhance the immunogenicity of polysaccharide antigens.

[0026] (3) Multifunctional carrier proteins enable a breakthrough innovation of multiple protections with a single vaccine. The carrier protein of this invention is not only an immunostimulant for pneumococcal polysaccharide antigen, but also possesses independent antigenicity, capable of simultaneously stimulating immune protection against varicella-zoster virus and respiratory syncytial virus infection, effectively achieving "one vaccine for multiple protections." On one hand, as the E-1 carrier for varicella-zoster virus antigen, as seen in Examples 10 and 11, the total IgG, IgG1, and IgG2a antibody titers induced by the pneumococcal polysaccharide dual-carrier conjugate vaccine (Formulation B and Formulation C) are all higher than those induced by the unconjugated gE protein (Formulation D). Furthermore, in the dual-carrier conjugate vaccine formulations, the IgG antibody titer of Formulation B is higher than that of Formulation C. This indicates that the immunogenicity of the carrier protein E-1 in the pneumococcal dual-carrier conjugate vaccine prepared by this invention is no less than that of the recombinant protein formulation D, which is only a single antigen. It also demonstrates that the gE recombinant protein prepared by this invention ensures both its enhancing and promoting effect on the immunogenicity of pneumococcal polysaccharide antigen as a carrier protein and its immunogenicity as an antigen protein itself, further enhancing the protective effect against varicella-zoster virus infection. Meanwhile, in mice immunized with the pneumococcal polysaccharide dual-vector conjugate vaccine (formulations B and C), sensitized immune cells secreted higher levels of IFN-γ upon stimulation with the gE protein antigen. Furthermore, the amount of IFN-γ produced by spleen cells sensitized with formulations B and C was significantly higher than that produced with formulation D (gE protein). This indicates that the gE recombinant protein prepared in this invention, in addition to its inherently good cellular immune effect, exhibits stronger cellular immune response induced by the dual-vector conjugate vaccine prepared using the carrier protein E-1, further enhancing its ability to regulate immune responses and strengthen defense against pathogens. On the other hand, as shown in Example 12, the F-1 and F-2 vectors used as respiratory syncytial virus antigens significantly increased the IgG antibody titers against Pre-F protein in the serum of animals immunized with the pneumococcal polysaccharide dual-vector conjugate vaccine (formulations B and C) and the Pre-F recombinant protein (formulations G and H), with both immunizations showing a significant increase in antibody levels. In both the first and second immunizations, the antibody titers in the serum of patients immunized with the pneumococcal polysaccharide dual-vector conjugate vaccine (Formulation B and Formulation C) were higher than those of the unconjugated Pre-F recombinant protein (Formulation G and Formulation H). Furthermore, in the dual-vector conjugate vaccine formulations, the IgG antibody titer of Formulation B was higher than that of Formulation C. This indicates that the immunogenicity of the carrier proteins F-1 and F-2 in the pneumococcal polysaccharide dual-vector conjugate vaccine prepared in this invention is no less than that of recombinant protein formulations containing only a single antigen, and the combined immunogenicity of the combination of carriers F-2 and E-1 is superior to that of the combination of carriers F-1 and E-1. This also demonstrates that the Pre-F recombinant protein prepared in this invention not only ensures its enhancing and promoting effect on the immunogenicity of pneumococcal polysaccharide antigen as a carrier protein, but also ensures its immunogenicity as an antigen protein itself, greatly enhancing the preventive and protective effect against respiratory syncytial virus infection.

[0027] (4) Novel carrier protein precisely adapts, and immunogenicity is specifically enhanced. The F-1, F-2, and E-1 carrier proteins prepared in this invention exhibit serotype-specific compatibility with pneumococcal polysaccharides, allowing for precise selection of carriers based on different serotype characteristics. This enables targeted enhancement of the immunogenicity of polysaccharides from different serotypes compared to the traditional CRM197 carrier. As shown in Example 6, after both the first and second immunizations, the polysaccharides of serotypes 5, 6B, 9V, and 19F exhibited the strongest immunogenicity upon binding with carrier protein E-1, significantly higher than F-1, F-2, and CRM197. The polysaccharide of serotype 14 showed the best immunogenicity upon binding with carrier protein F-2, far exceeding F-1, E-1, and CRM197. Carrier proteins F-1 and F-2 showed similar immunomodulatory effects on most serotype polysaccharides, while the immunomodulatory effects of polysaccharides of serotypes 3, 6B, 9V, 14, and 19F were superior to CRM197. This invention breaks through the limitations of traditional "universal" carriers by innovatively adapting carrier proteins to specific polysaccharides of different serum types, and further customizes better immune enhancement schemes for polysaccharides of different serum types. Attached Figure Description

[0028] Figure 1 The results of the comparison of immunogenicity of conjugates obtained by binding type 3 pneumococcal polysaccharide to different carrier proteins; Figure 2 The results of the comparison of immunogenicity of conjugates obtained by binding pneumococcal polysaccharide to different carrier proteins; Figure 3 The immunogenicity of conjugates obtained by binding pneumococcal polysaccharide to different carrier proteins was compared. Figure 4 The immunogenicity of conjugates obtained by binding pneumococcal polysaccharide of type 9V to different carrier proteins was compared. Figure 5 The immunogenicity of conjugates obtained by binding polysaccharides from type 14 pneumococcus with different carrier proteins was compared. Figure 6 The immunogenicity of conjugates obtained by binding polysaccharides from type 19F pneumococcus with different carrier proteins was compared. Figure 7 The results show the comparison of immunogenicity of single- and dual-vector pneumococcal polysaccharide conjugate vaccines (11 serotypes other than the 13 serotypes in the positive vaccine). In the figure, each serotype is in the following order from left to right: group 1, group 2, group 3, group 4, and group 5. Figure 8The results show the comparison of immunogenicity of single- and dual-vector pneumococcal polysaccharide conjugate vaccines (13 serotypes identical to the positive vaccine). In the figure, each serotype is arranged from left to right as group 1, group 2, group 3, group 4, and group 5. Figure 9 The results show the comparison of polysaccharide antibody titers after immunization with the 24-valent dual-vector pneumococcal polysaccharide conjugate vaccine and the positive vaccine. Detailed Implementation

[0029] To more concisely and clearly demonstrate the technical solution, purpose, and advantages of the present invention, the technical solution of the present invention is described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the instruments, equipment, reagents, materials, etc., used can all be obtained through conventional commercial means unless otherwise specified.

[0030] Example 1: Preparation of carrier protein (RSV) (I) Protein Construction The term "wild-type" as used in this invention refers to a product that exists in nature and has not undergone any artificial modification or processing. Those skilled in the art will understand that wild-type RSV F protein can have multiple sequences, which may have minor differences but essentially the same biological activity. The wild-type full-length F protein mentioned in this invention refers to the sequence provided in GenBank, specifically as shown in SEQ ID NO. 1 (Fusion glycoprotein F0 OS=Human respiratory syncytialvirus A (strain A2) OX=11259 GN=F PE=1 SV=1).

[0031] (1) Amino acid sequence design This invention relates to a carrier protein, RSV Pre-F recombinant protein, whose amino acid sequence is designed as follows: The transmembrane and intracellular regions of the wild-type RSV pre-F protein full-length sequence were deleted, and the fibritin / Throm / 6his / Stretaq sequence was ligated to its C-terminus. Then, amino acid point mutations were performed on this basis. Specifically, the T at position 324 of the wild-type pre-fusion F protein amino acid sequence was mutated to C, and the N at position 437 was mutated to C, resulting in the full-length mutant F protein. Its amino acid sequence is shown in SEQ ID NO. 2, which is the carrier protein F-1.

[0032] This invention also relates to another carrier protein, RSV Pre-F recombinant protein, whose amino acid sequence is designed as follows: The transmembrane and intracellular regions of the wild-type RSV pre-F protein full-length sequence were deleted, and the fibritin / Throm / 6his / Stretaq sequence was ligated to its C-terminus. Then, amino acid point mutations were performed on this basis. Specifically, the T at position 318 of the wild-type pre-fusion F protein amino acid sequence was mutated to C, and the R at position 339 was mutated to C, resulting in the full-length mutant F protein. Its amino acid sequence is shown in SEQ ID NO. 3, which is the carrier protein F-2.

[0033] The sequences SEQ ID NO.1 through SEQ ID NO.3 are shown below: SEQ ID NO. 1: >sp|P03420|FUS_HRSVA Fusion glycoprotein F0 OS=Human respiratory syncytial virus A (strain A2) OX=11259 GN=F PE=1 SV=1 .

[0034] SEQ ID NO .2: >RSV F T324C N437C MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTCNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSCGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK。

[0035] SEQ ID NO .3: >RSV F T318C R339C .

[0036] (2) Synthesis of Pre-F target genes and vector construction Based on the designed RSV protein amino acid sequences SEQ ID NO.2 and SEQ ID NO.3 and the codon bias of the host cell, the corresponding gene coding sequence was determined. A restriction endonuclease XbaI sequence was added to the 5' end of this gene segment, and a restriction endonuclease EcoRI sequence was added to the 3' end, chemically synthesizing the designed nucleotide sequence. The synthesized target gene fragment was ligated into the pUC19 vector (containing the Amp resistance gene), transformed into DH5α competent cells, and positive clones were screened using LB (Amp+) plates. Single clones were picked and inoculated into LB (Amp+) liquid medium, cultured at 37°C and 200 rpm for 12 hours, and plasmids were extracted using a plasmid medium preparation kit (Sigma-Aldrich GenElute). The plasmids were digested with XbaI and EcoRI, and the target gene fragment was recovered by agarose gel electrophoresis and purified for later use.

[0037] The mammalian expression vector pGN (containing the CMV promoter and DHFR gene) was digested with XbaI and EcoRI. The vector DNA fragment was recovered using a kit (TaKaRa 9761). The target gene fragment was then ligated to the vector fragment, transformed into DH5α cells, and positive clones were screened for amplification culture. Finally, the amplified plasmid was extracted using an endotoxin-free plasmid DNA medium-quantity purification kit (TaKaRa9783).

[0038] (II) Cell transfection and screening of high-expression clones (1) Cell culture and transfection CHO cells (ATCC) were resuscitated and cultured in DMEM medium (Sigma-Aldrich) containing 10% newborn calf serum. The cells were passaged every 3 days for two passages, ensuring cell viability >90%. CHO cells were then seeded into 96-well plates (0.75 × 10⁶ cells / well). 6 Cells / well), the well plates were filled with Iscove's optimized DMEM medium (Sigma-Aldrich) and 10% fetal bovine serum (IMEM+FBS) (Gibco).

[0039] Cells were placed in a humidity-saturated incubator at 37°C with 5% CO2. Each well contained 4 μg of pcDNA RSVPre-F vector. DNA and Lipofectamine 2000 (Sigma-Aldrich) transfection reagent were mixed and added to two wells. A negative control was also set up (only transfection reagent was added).

[0040] (2) Stable expression and clone screening Forty-eight hours after transfection, the culture medium was removed, and the supernatant was collected by centrifugation and stored at -20°C. IMDM+FBS culture medium and 10 μg / mL Blastidin-HCl (Invitrogen) were added to one well of the transfected cells. The other well was washed with PBS, and then the cells were lysed with a mixture of 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% (v / v) Triton X-100 containing complete, and EDA-free protease inhibitor. The lysate was centrifuged at 16000 × g for 10 minutes at 4°C, and stored at -20°C. RSV pre-F protein expression was detected by Western blot.

[0041] After 5 days of culture, cells were eluted with trypsin (Invitrogen) and then seeded onto 9 cm Petri dishes for serial dilution to isolate monoclonal cells. During 7-11 days of continued culture, 42 monoclonal cells were selected and transferred to 96-well plates. The culture supernatant was analyzed by Western blot to screen for high-expression clones. Clones that secreted the highest amount of protein were selected for the next round of screening. Finally, the cells were expanded, and 30 new clones were selected and preserved.

[0042] (3) Adaptation to serum-free suspension culture High-expression clones were amplified into cell culture flasks, digested with trypsin, washed with PBS, and then resuspended in 250 mL rotary flasks containing 100 mL of ProCHO4 (Lonza), 1×ProHT, 4 mL of glutamine, and 2% FBS (Lonza). Cells were cultured in a humidified incubator at 37°C with 5% CO2 and a stirring speed of 90 rpm. Cell viability was monitored daily (trypan blue staining), and cells were passaged every 3-5 days. Cells were cultured when the viable cell concentration was >0.3 × 10⁻⁶ cells / mL. 6 Large-scale suspension culture was performed at a cell / mL ratio.

[0043] (III) Perfusion culture and protein purification (1) Bioreactor perfusion culture A perfusion system was configured in a bioreactor (1.5 L), and cells were separated from the culture medium using a rotary filter (10 μm). Culture parameters were controlled as follows: temperature 37 °C, pH 6.9 (adjusted by CO2 / NaOH), dissolved oxygen saturation 40% (N2 / O2 mixed gas), and stirring speed 200–300 rpm.

[0044] The perfusion rate was 0.3–0.8 VVD. Cell density and protein expression were monitored daily. 12.5 L of cell-free culture medium was collected. The medium was then centrifuged at 8000×g for 30 minutes at 4°C, filtered through a 0.45 μm membrane, and then concentrated by ultrafiltration through a 10 kDa membrane. The sample was washed with buffer and concentrated to 0.5 L. 0.5 L of PBS was added and then concentrated to 0.5 L.

[0045] (2) Purification of RSV protein The sample solution was loaded onto a Q-Sepharose FF column (GE Bioscience), equilibrated with 20 mM Tris-HCl (pH 7.5), and washed with 200 mM NaCl to further remove adsorbed protein impurities. The pre-F protein was eluted with NaCl solution at a concentration increased to 300 mM. The eluents were combined, and ammonium sulfate was added to a concentration of 800 mM. The eluent was then loaded onto a Butyl-Sepharose column (GE Bioscience), washed with PBS containing 400 mM ammonium sulfate, and eluted with pure water. Finally, the sample was loaded onto a Sephacryl S-400HR column (GE Bioscience), eluted with PBS, and lyophilized at -70°C. The final purity was >95%.

[0046] The carrier proteins F-1 and F-2 of the present invention were both prepared using the methods described above.

[0047] Example 2: Preparation of carrier protein (VZV) Varicella-zoster virus (VZV) is a member of the subfamily Alphaherpesvirus of the genus Herpesviridae, specifically Human herpesvirus 3. gE (glycoprotein E) is encoded by the ORF68 gene, located in a short segment of the VZV genome. The encoded gE contains 623 amino acids. The gE protein molecule is mainly composed of a hydrophilic extracellular region (containing a signal peptide) consisting of amino acids 1-546, a hydrophobic transmembrane region consisting of amino acids 547-623, and an intracellular tail. Its full-length sequence is referenced in NCBI Reference Sequence: NP_040190.1, and the specific sequence is shown in SEQ ID NO. 4 (envelope glycoprotein E [Human alphaherpesvirus 3] / strain="Dumas" / 623aa).

[0048] (I) Protein Construction (1) Amino acid sequence design The present invention relates to a carrier protein, namely VZV gE protein, which is a VZV gE protein fragment extracted from the original full-length protein (SEQ ID NO.4), with the hydrophobic transmembrane region and intracellular tail removed, and the N-terminal 546 amino acids retained. Its specific sequence is shown in SEQ ID NO.5, which is the carrier protein E-1.

[0049] The sequences SEQ ID NO. 4 through SEQ ID NO. 5 are shown below: SEQ ID NO. 4: > VZV gE 1-623 (NP_040190.1 / 623aa / envelope glycoprotein E [Humanalphaherpesvirus 3] / strain="Dumas") 1 MGTVNKPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHT DEDKLDTNSV YEPYYHSDHA 61ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG VYNQGRGIDS GERLMQPTQM 121 SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD QRQYGDVFKG DLNPKPQGQR LIEVSVEENH 181 PFTLRAPIQR IYGVRYTETW SFLPSLTCTG DAAPAIQHIC LKHTTCFQDV VVDVDCAENT 241 KEDQLAEISY RFQGKKEADQ PWIVVNTSTL FDELELDPPE IEPGVLKVLR TEKQYLGVYI 301 WNMRGSDGTS TYATFLVTWK GDEKTRNPTP AVTPQPRGAE FHMWNYHSHV FSVGDTFSLA 361 MHLQYKIHEA PFDLLLEWLY VPIDPTCQPM RLYSTCLYHP NAPQCLSHMN SGCTFTSPHL 421 AQRVASTVYQ NCEHADNYTA YCLGISHMEP SFGLILHDGG TTLKFVDTPE SLSGLYVFVV 481 YFNGHVEAVA YTVVSTVDHF VNAIEERGFP PTAGQPPATT KPKEITPVNP GTSPLLRYAA 541 WTGGLAAVVL LCLVIFLICT AKRMRVKAYR VDKSPYNQSM YYAGLPVDDF EDSESTDTEE 601 EFGNAIGGSH GGSSYTVYID KTR。

[0050] SEQ ID NO. 5: > VZV gE 1-546 MGTVNKPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHT DEDKLDTNSV YEPYYHSDHA ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG VYNQGRGIDS GERLMQPTQM SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD QRQYGDVFKG DLNPKPQGQR LIEVSVEENH PFTLRAPIQR IYGVRYTETW SFLPSLTCTG DAAPAIQHIC LKHTTCFQDV VVDVDCAENT KEDQLAEISY RFQGKKEADQ PWIVVNTSTL FDELELDPPE IEPGVLKVLR TEKQYLGVYI WNMRGSDGTS TYATFLVTWK GDEKTRNPTP AVTPQPRGAE FHMWNYHSHV FSVGDTFSLA MHLQYKIHEA PFDLLLEWLY VPIDPTCQPM RLYSTCLYHP NAPQCLSHMN SGCTFTSPHL AQRVASTVYQ NCEHADNYTA YCLGISHMEP SFGLILHDGG TTLKFVDTPE SLSGLYVFVV YFNGHVEAVA YTVVSTVDHF VNAIEERGFP PTAGQPPATT KPKEITPVNP GTSPLLRYAA WTGGLA.

[0051] (2) Synthesis of the VZV gE target gene Based on the amino acid sequence SEQ ID NO.5 of the aforementioned carrier protein VZV gE and the codon preference of the host cell, the corresponding gene coding sequence was determined, and the restriction endonuclease EcoRI sequence was added to the C-terminus of the gene segment, and the restriction endonuclease XbaI sequence was added to the N-terminus, resulting in a chemically synthesized nucleotide sequence.

[0052] (3) Plasmid amplification and target gene extraction The plasmid vector pUC19 was digested with EcoRI and XbaI restriction enzymes and ligated to the synthesized gene. This ligation was then introduced into the host DH5α for amplification. Single clones were screened using LB (Amp+) agar solid medium. Single clones containing the target gene were inoculated into LB (Amp+) liquid medium and amplified at 37°C and 200 rpm. The plasmid pUC19-gE was extracted using the Sigma-Aldrich GenElute™ HP plasmid medium-scale preparation kit. The target gene fragment was recovered from the plasmid extracted by double digestion with EcoRI and XbaI restriction enzymes using the TaKaRaMiniBest Agarose Gel Extraction Kit.

[0053] (4) Construction of eukaryotic expression vectors The mammalian cell expression plasmid pGN-M, containing the CMV promoter and dihydrofolate reductase (DHFR) gene, was digested with EcoRI and XbaI restriction enzymes. The vector DNA fragment was recovered using the TaKaRa MiniBEST DNA Fragment Purification Kit Ver. 4.0. The vector DNA fragment and the target gene fragment were ligated at sticky ends and introduced into the DH5α amplification host. Single clones containing the eukaryotic expression plasmid pGN-M_gE were obtained by screening. The clones were inoculated into LB (Amp+) cells for amplification culture, and the amplified plasmid was extracted using the TaKaRa MidiBEST Endo-free Plasmid Purification Kit.

[0054] (II) Expression and Cloning Screening of gE Protein in CHO Cells CHO K1 (ATCC) cells were used as host cells. After cell resuscitation, the cells were cultured in DMEM medium (Sigma-Aldrich) containing 10% newborn calf serum and passaged every 3 days. After two passages, the cells showed good growth. Then, CHO K1 cells were cultured at a rate of 0.75 × 10⁻⁶ cells / year. 6 Three 9.6cm cells / wells 2The cells were placed in wells containing Iscove's optimized DMEM medium (Sigma-Aldrich) and 10% fetal bovine serum (IMEM+FBS) (Gibco). Cells were incubated in a humidity-saturated incubator at 37°C and 5% CO2. Each well contained 4 μg of pcDNA VZVgE vector. DNA was mixed with Lipofectamine 2000 (Sigma-Aldrich) and added to two wells. Lipofectamine 2000 was added separately to a third well as a negative control. After 48 hours, the medium was removed, and the cells were centrifuged at 200×g for 5 minutes. The supernatant was stored at -20°C. Add IMDM+FBS culture medium and 10 μg / mL Blastidin-HCl (Invitrogen) to one well of transfected cells. Wash the other well with PBS, then lyse the cells with a mixture of 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% (v / v) Triton X-100 containing complete, and EDA-free protease inhibitor (Roche Diagnostics). Centrifuge at 16000×g for 10 min at 4°C, and store the lysate at -20°C. Use Western blot to detect the presence of recombinant protein in the supernatant and lysate. After culturing in selective medium for 5 days, wash the cells with trypsin (Invitrogen) and then seed them onto 9 cm Petri dishes for serial dilutions to isolate single clones. Over subsequent 7-11 days, select 42 single clones and transfer them to wells of a 96-well plate. Use Western blot to analyze the culture supernatant and screen for high expression of the protein gE. The clone that secretes the highest amount of protein is selected for the next round of screening, and the cells are eventually expanded. Thirty new clones are then selected and preserved.

[0055] The selected clones were amplified into three T175 flasks (NETS). After trypsin digestion and washing with PBS, the clones were resuspended in 250 mL rotating flasks containing 100 mL of ProCHO4 (Lonza), 1×ProHT, 4 mM L-glutamine, and 2% FBS (Lonza). The flasks were incubated at 37°C in a humidified incubator with 5% CO2 and a stirring speed of 90 rpm, with the lid slightly ajar to ensure air diffusion. Samples were taken daily, stained with trypan blue (Sigma-Aldrich), and cells were counted. Cells were passaged every 3-5 days until the viable cell concentration exceeded 0.3 × 10⁻⁶. 6 After the plateau phase, when the cell count exceeds 90% and the viable cell count is above 90%, and the cells have adapted and are growing well, FBS is gradually removed. At this point, the cells are considered fully adapted for serum-free suspension growth.

[0056] (iii) Production of gE protein in a bioreactor A 1.5 L perfusion culture was configured in a bioreactor, equipped with a rotary filtration (10 μm) separator. Culture parameters were set as follows: temperature controlled at 37 °C using a heating blanket; pH adjusted to 6.9 using CO2 or 0.3 M sodium hydroxide; stirring speed at 200-300 RPM; dissolved oxygen (dO2) adjusted to 40% of saturated air using a N2 and O2 mixture at a maximum flow rate of 200 mL / min. The perfusion rate was 0.3 to 0.8 V dilutions / day, and cell counting was performed daily by sampling the culture medium. Trypan blue staining was used, and glucose and lactate concentrations in the supernatant were detected offline. A total of 12.5 L of cell-free culture medium was collected, centrifuged at 8000 × g for 30 min at 4 °C, filtered through a 0.45 μm membrane, and then concentrated by ultrafiltration using a 10 kDa membrane. The ultrafiltration solution was washed with buffer, and the sample solution volume was concentrated to 0.5 L. After adding 0.5 L of PBS, it was further concentrated to 0.5 L. Repeat the above steps 5 times.

[0057] (iv) Purification of gE protein The sample solution was loaded onto a Q-Sepharose fast flow (GE Bioscience) column and washed with 20 mM Tris-HCl pH 7.5. Then, the column was washed with 20 mM Tris-HCl pH 7.5 containing 200 mM sodium chloride to further remove adsorbed protein impurities. gE protein was eluted with a solution containing 300 mM sodium chloride. Ammonium sulfate was added to the combined solution to a concentration of 800 mM, and the solution was loaded onto a Butyl-Sepharose (GE Bioscience) column. The column was washed with phosphate-buffered saline (PBS, 6 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 0.15 M sodium chloride pH 6.8) containing 800 mM ammonium sulfate, followed by washing with PBS containing 400 mM ammonium sulfate. Finally, gE protein was eluted with purified water. Finally, the sample was loaded onto a Sephacryl S-400HR (GEBioscience), the column was washed with PBS, the protein peak was collected, a co-solvent was added, and the sample was freeze-dried in a vacuum freeze dryer and stored at -70°C for later use. This is the carrier protein E-1.

[0058] This invention also relates to a carrier protein, CRM197, which is a non-toxic diphtheria toxin mutant with a single mutation replacing glycine at position 52 with glutamic acid. This alters the active site of the diphtheria toxin enzyme, thus preventing it from exerting cytotoxic effects on cells, while maintaining the same antigenicity and immunogenicity as the natural diphtheria toxin. CRM197 is a well-established and widely used carrier protein, currently used in many approved vaccines, such as polysaccharide conjugate vaccines for meningococcal, Haemophilus influenzae type b, and pneumococcal infections.

[0059] The CRM197 protein used in this invention was prepared using conventional methods in this technical field.

[0060] Example 3: Preparation of Pneumococcal Capsular Polysaccharide (1) Preparation of master seeds and working seeds The specific method for purifying capsular polysaccharides from the fermentation broth of pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F is as follows: Pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F were obtained from the American Type Culture Collection. The cultures from the lyophilized seed tubes were inoculated into 5 ml of yeast-acid-hydrolyzed casein culture medium and cultured at 36℃±2℃ for 18 hours. When the bacterial growth reached an OD600 reading of 1.0, the culture was transferred to 150 ml of fresh yeast-acid-hydrolyzed casein culture medium and cultured at 36℃±2℃ for 5-10 hours until the exponential growth phase. The culture was then stopped, aliquoted, lyophilized, and stored as the primary seed culture at 2-8℃.

[0061] The bacterial cultures of pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F were inoculated into 5 mL of yeast-acid-hydrolyzed casein culture medium and cultured at 36℃±2℃ for 18 hours. When the bacterial growth reached an OD600 reading of 1.0, the culture was transferred to 150 mL of fresh yeast-acid-hydrolyzed casein culture medium and cultured at 36℃±2℃ for 8 hours until the exponential growth phase. The culture was then stopped, aliquoted, freeze-dried, and stored at 4℃ as working seed cultures for the serotypes.

[0062] (2) Bacterial fermentation Seed tubes were taken from the working seed bank and inoculated into 5 mL of yeast-acid-hydrolyzed casein culture medium. The culture was incubated at 36℃±2℃ until the mid-exponential growth phase. The culture was then transferred to 150 mL of fresh yeast-acid-hydrolyzed casein culture medium and incubated at 36℃±2℃ for 5-10 hours until the exponential growth phase. 50 mL of the culture was then transferred to 2 L of yeast-acid-hydrolyzed casein culture medium and incubated at 36℃±2℃ until the mid-exponential growth phase to prepare the fermentation seed culture. This seed culture was then inoculated into a 50 L fermenter containing 30 L of yeast-acid-hydrolyzed casein culture medium. The pH of the fermentation broth was maintained at 6.8±0.2 using sodium hydroxide until the bacteria reached the late exponential growth phase.

[0063] (3) Purification of capsular polysaccharides 1. Add phosphoric acid to adjust the pH of the fermentation broth to around 4, and stir for 1 hour; 2. Centrifuge using a disc centrifuge at a speed of 9600 rpm, collect the supernatant, and discard the residue. 3. Use a microfiltration membrane to microfilter the centrifuged liquid to remove residual cell debris and insoluble small particulate matter. Use a 0.22μm membrane to microfilter the fermentation centrifuged supernatant and collect the filtrate. 4. The microfiltrate was concentrated and washed using a 100kD membrane to obtain a crude bacterial capsular polysaccharide solution. Then, 15 sample volumes were washed with buffer and ultrafiltration using a 30kD membrane. 5. Use a 50kD membrane to further wash and filter the polysaccharide solution for 10 sample volumes to concentrate the polysaccharide sample solution; 6. Collect the purified polysaccharide solution into a freeze-drying bottle, freeze-dry it in a vacuum freeze dryer, and store it at -70℃.

[0064] Example 4: Preparation of polysaccharide-protein conjugates (CDAP) of pneumococcal serotypes Pn1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. The preparation method includes the following steps: 1) Weigh 20 mg of the corresponding serum-type purified capsular polysaccharide and dissolve it in 4 mL of sodium phosphate buffer; 2) Add 15 mg of 1-cyano-4-dimethylammonium pyridine tetrafluoroborate (CDAP) (Sigma-Aldrich) to the polysaccharide solution, stir, and react at room temperature for 1 hour; 3) Add 3 Eqm of cystamine and react at room temperature for 1 hour; 4) Add 0.3 mL of 1 M lysine (Sigma-Aldrich) solution to quench the reaction, and react at room temperature for 1-2 hours; 5) Add 8 Eqm of 3(2-chloroethyl) phosphate to the polysaccharide solution to reduce the disulfide bonds in the polysaccharide; 6) Transfer the activated polysaccharide solution to a dialysis bag and dialyze against phosphate buffer at 4°C, changing the buffer four times; 7) Weigh 30 mg of carrier protein (CRM197 / F-1 / F-2 / E-1) and dissolve it in phosphate buffer to achieve a protein concentration of 10 mg / mL; 8) Add 8 mg of N-hydroxysuccinimide bromoacetate (BAANS) (Sigma-Aldrich) to the carrier protein solution and react at room temperature for 2 hours. Transfer the activated protein solution to a dialysis bag (Thermo Scientific) and dialyze against phosphate buffer at 4°C, changing the buffer four times. 9) Mix 4 mL of activated polysaccharide solution with 4 mL of activated protein solution and react at room temperature for 4 hours; 10) Add 4 Eqm of N-acetyl-L-cysteine ​​(Sigma-Aldrich), react at 2-8℃ for 4 hours, then add 12 Eqm of iodoacetamide (Sigma-Aldrich), and react at 2-8℃ for 4 hours. 11) Transfer the polysaccharide conjugate reaction solution to a dialysis bag and dialyze it against phosphate buffer at 4°C; 12) Load the sample solution onto Sepharose CL-4B, purify it during the process, and collect the external water volume bound. 13) After filtration through a 0.22μm filter membrane, store at 2-8℃ until preparation is complete.

[0065] The method of this embodiment can be used to prepare monovalent polysaccharide-protein conjugates of various serotypes of pneumococcus, and the obtained monovalent polysaccharide-protein conjugates can be further used in the formulation of multivalent conjugate vaccines.

[0066] Example 5: Preparation of polysaccharide-protein conjugates of pneumococcal serotypes Pn1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F (reduced amine method) The preparation method includes the following steps: 1) Weigh 500 mg of the corresponding serum-type purified capsular polysaccharide and dissolve it in 500 mL of purified water; 2) Degradation was carried out using a high-pressure homogenizer at a pressure of 600 bar for three cycles, with the addition of 0.12 eqm sodium periodate (Sigma-Aldrich), and the reaction was carried out in the dark for 18 hours; 3) Use ultrafiltration to wash the purified water with a membrane with a molecular weight of 50Kd, then concentrate and freeze-dry it; 4) Weigh 18 mg of activated polysaccharide, add 4 mL of DMSO, and stir until completely dissolved; 5) Add 19 mg of carrier protein (CRM197 / F-1 / F-2 / E-1) to 4 mL of DMSO (Sigma-Aldrich) solution, add 2 Eqm of sodium cyanoborohydride (Sigma-Aldrich), and react at room temperature for 22 hours; 6) After quenching the reaction with 2 Eqm sodium borohydride (Sigma-Aldrich) for 4 hours, transfer the synthesis reaction solution to a dialysis bag, dialyze against the buffer solution, and change the solution four times. 7) Load the sample solution onto Sepharose CL4B and collect the bound portion of the external water volume; 8) After filtration through a 0.22μm filter membrane, store at 4℃ until preparation.

[0067] The method of this embodiment can be used to prepare monovalent polysaccharide-protein conjugates of various serotypes of pneumococcus, and the obtained monovalent polysaccharide-protein conjugates can be further used in the formulation of multivalent conjugate vaccines.

[0068] Example 6: Immunogenicity detection of different carrier protein monovalent conjugates To improve the immunization effect and further optimize the immunogenicity of polysaccharide antigens, this invention selected six serotypes (3, 5, 6B, 9V, 14, and 19F) and studied their conjugation to different carrier proteins. Specifically, the polysaccharides of serotypes Pn3 and Pn14 were conjugated to carrier proteins F-1 / F-2 / E-1 / CRM197 using the method described in Example 4, while the polysaccharides of serotypes Pn5, Pn6B, Pn9V, and Pn19F were conjugated to carrier proteins F-1 / F-2 / E-1 / CRM197 using the method described in Example 5. Subsequently, the purified monovalent conjugates were used to immunize experimental rabbits. Blood samples were collected on day 0 before immunization, day 14 after the first immunization, and day 28 after the second immunization to determine the immunogenicity of the polysaccharides in each monovalent conjugate. Information on the test samples is shown in Table 1.

[0069] Table 1: Summary Table of Test Sample Information

[0070] Table 1 shows the antibody titers in the serum of rabbits immunized with the six test samples. The results are shown in Table 1. Figures 1-6 .

[0071] The results showed that the immunogenicity of pneumococcal polysaccharides differed after binding to different carrier proteins. Firstly, overall, after both the first and second immunizations (D14 and D28), the immunogenicity of pneumococcal polysaccharides from serotypes 3, 5, 6B, 9V, 14, and 19F with CRM197 as the carrier protein was relatively weak, while the immunogenicity with F-1, F-2, and E-1 as carrier proteins was relatively strong, and the second immunization was significantly more effective than the first. Secondly, significant differences in immunogenicity were also observed when F-1, F-2, and E-1 were used as carrier proteins. Figure 2-6 As can be seen, the immunogenicity of serotypes 5, 6B, 9V, and 19F polysaccharides bound to carrier protein E-1 is the strongest, significantly higher than that of the conjugates of carrier proteins F-1 and F-2, while the immunogenicity of the F-1 and F-2 conjugates is not significantly different. However, serotype 14 polysaccharide shows the strongest immunogenicity bound to carrier protein F-2, significantly higher than that of the conjugates of carrier proteins F-1 and E-1. Therefore, compared to CRM197 as a carrier protein, the carrier proteins F-1, F-2, and E-1 prepared in this invention have a significant advantage in enhancing the immunogenicity of pneumococcal polysaccharide antigens. However, the specific immunizing effects will vary significantly depending on the polysaccharide type and the choice of carrier protein, thus requiring further in-depth experimental research.

[0072] Example 7: Immunogenicity detection of single-vector and dual-vector pneumococcal polysaccharide-protein conjugate vaccines (11 serotypes other than the 13 serotypes in the positive vaccine) Eleven serotypes other than the 13 serotypes in the positive vaccine—serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F—were selected and screened for binding with different carrier proteins. Further research was conducted on the immunogenicity of polysaccharides in single- and dual-carrier protein pneumococcal polysaccharide conjugate vaccines. Specific details of the binding of each polysaccharide with different carrier proteins are shown in Table 2. Serotypes 2, 8, 12F, 15B, 17F, 22F, and 33F were prepared using the method described in Example 4, while serotypes 9N, 10A, 11A, and 20 were prepared using the method described in Example 5. The polysaccharide-protein conjugate stock solutions prepared according to Table 2 were diluted with a buffer solution at pH 5.8, mixed, and then aluminum phosphate adjuvant was added and stirred to prepare immunoantigens (test samples). The polysaccharide content in each conjugate was 2.2 μg per milliliter, and the aluminum phosphate adjuvant content was 0.125 mg per milliliter.

[0073] Table 2: Summary Table of Carrier Protein Information Group 1 The carrier protein is CRM197 All serotypes were bound to the CRM197 vector. Group 2 The carrier proteins are F-1 and E-1. The serotypes that bind to the F-1 vector are: 2, 8, 12F, 15B, 17F, 22F, and 33F; the serotypes that bind to the E-1 vector are: 9N, 10A, 11A, and 20. Group 3 The carrier proteins are F-1 and E-1. The serotypes that bind to the F-1 vector are 9N, 10A, 11A, and 20, while the serotypes that bind to the E-1 vector are 2, 8, 12F, 15B, 17F, 22F, and 33F. Group 4 The carrier proteins are F-2 and E-1. The serotypes that bind to the F-2 vector are: 2, 8, 12F, 15B, 17F, 22F, and 33F. The serotypes that bind to the E-1 vector are: 9N, 10A, 11A, and 20. Group 5 The carrier proteins are F-2 and E-1. The serotypes that bind to the F-2 vector are: 9N, 10A, 11A, and 20; the serotypes that bind to the E-1 vector are: 2, 8, 12F, 15B, 17F, 22F, and 33F.

[0074] Immunization protocol: New Zealand white rabbits weighing 2.5-3.5 kg were selected and immunized in groups of 4. Each immunization dose was 0.5 ml, administered twice daily at week 0 and week 2. Blood samples were collected on day 28 to evaluate the immunogenicity of the polysaccharides. Immunization results are as follows: Figure 7 As shown.

[0075] The test results showed that different combinations of carrier proteins with 11 serotype polysaccharide antigens other than the positive vaccine resulted in varying immunization effects. For some serotypes, the immunogenicity was not significantly correlated with the carrier protein, such as Pn11A and Pn20. However, the immunogenicity of most serotype polysaccharides showed a strong correlation with the carrier protein. Overall, the immunization effect of using dual carrier proteins with related serotype polysaccharides in groups 2-5 was better than that of group 1, which used a single carrier protein (CRM197) as all carrier proteins. Among them, in group 2, the immunogenicity of polysaccharides from 8 serotypes (2, 9N, 10A, 12F, 15B, 17F, 22F, and 33F) was significantly higher than that in group 1; in group 3, the immunogenicity of polysaccharides from 6 serotypes (9N, 10A, 12F, 15B, 17F, and 22F) was significantly higher than that in group 1; and in group 4, the immunogenicity of polysaccharides from 10 serotypes (2, 8, 9N, 10A, 12F, 15B, 17F, 20, 22F, and 33F) was significantly higher than that in group 1. In groups 1 and 5, the immunogenicity of polysaccharides from serotypes 2, 9N, 15B, and 17F was significantly higher than that of group 1. This indicates that among the five groups, groups 2 and 4 showed better immunization effects. Specifically, the overall immunogenicity of the combinations using serotypes 2, 8, 12F, 15B, 17F, 22F, and 33F with F-1 or F-2 vectors, serotypes 9N and 10A with E-1 vectors, and serotypes 11A and 20 with F-1, F-2, or E-1 vectors was higher. This demonstrates that the conjugate vaccine using the dual-vector combination of this invention can effectively reduce immune interference between different serotype polysaccharide antigens, and its overall immunogenicity is superior to that of conjugate vaccines using a single vector.

[0076] Example 8: Immunogenicity detection of single-vector and dual-vector pneumococcal polysaccharide-protein conjugate vaccines (13 serotypes identical to the positive vaccine) Thirteen serotypes identical to those in the positive vaccine—serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F—were selected and screened for binding with different carrier proteins. The immunogenicity of polysaccharides in single- and dual-carrier protein pneumococcal polysaccharide conjugate vaccines was further investigated. The specific binding details of each polysaccharide with different carrier proteins are shown in Table 3. Serotypes 1, 3, 4, 14, and 19A were prepared using the method described in Example 4, while serotypes 5, 6A, 6B, 7F, 9V, 18C, 19F, and 23F were prepared using the method described in Example 5. The polysaccharide-protein conjugate stock solutions prepared according to Table 3 were diluted with a buffer solution of pH 5.8 and mixed. Then, aluminum phosphate adjuvant was added and stirred to prepare an immunoantigen (test sample). The polysaccharide content in each conjugate was 2.2 μg per milliliter (except for 6B polysaccharide, which was 4.4 μg), and the aluminum phosphate adjuvant content was 0.125 mg.

[0077] Table 3: Summary Table of Carrier Protein Information Group 1 The carrier protein is CRM197 All serotypes bound to the CRM197 vector. Group 2 The carrier proteins are F-1 and E-1. The serotypes that bind to the F-1 vector are: 1, 3, 4, 14, and 19A; the serotypes that bind to the E-1 vector are: 5, 6A, 6B, 7F, 9V, 18C, 19F, and 23F. Group 3 The carrier proteins are F-1 and E-1. The serotypes that bind to the F-1 vector are: 5, 6A, 6B, 7F, 9V, 18C, 19F, and 23F. The serotypes that bind to the E-1 vector are: 1, 3, 4, 14, and 19A. Group 4 The carrier proteins are F-2 and E-1. The serotypes that bind to the F-2 vector are: 1, 3, 4, 14, and 19A; the serotypes that bind to the E-1 vector are: 5, 6A, 6B, 7F, 9V, 18C, 19F, and 23F. Group 5 The carrier proteins are F-2 and E-1. The serotypes that bind to the F-2 vector are: 5, 6A, 6B, 7F, 9V, 18C, 19F, and 23F. The serotypes that bind to the E-1 vector are: 1, 3, 4, 14, and 19A. Immunization protocol: New Zealand white rabbits weighing 2.5-3.5 kg were selected and immunized in groups of 4. Each immunization dose was 0.5 ml, administered twice daily at week 0 and week 2. Blood samples were collected on day 28 to evaluate the immunogenicity of the polysaccharides. Immunization results are as follows: Figure 8 As shown.

[0078] The test results showed that different carrier proteins, combined with the same 13 serotype polysaccharide antigens as the positive vaccine, produced varying immune responses. For some serotypes, such as Pn1, the immunogenicity was not significantly correlated with the carrier protein, but for most serotype polysaccharides, the correlation with the carrier protein was quite significant. Overall, in groups 2-5, the use of dual carrier proteins for related serotype polysaccharides resulted in better immune responses than the use of a single carrier protein in group 1, where all carrier proteins were CRM197. In group 2, the immunogenicity of polysaccharides from 10 serotypes (3, 4, 5, 6A, 6B, 7F, 9V, 14, 19F, and 23F) was significantly higher than that in group 1; in group 3, the immunogenicity of polysaccharides from 8 serotypes (5, 6A, 6B, 7F, 9V, 14, 19F, and 23F) was significantly higher than that in group 1; and in group 4, the immunogenicity of polysaccharides from 11 serotypes (3, 4, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F, and 23F) was significantly higher than that in group 1. 1. In group 5, the immunogenicity of polysaccharides from serotypes 4, 6A, 7F, 18C, and 19F was significantly higher than that in group 1. This indicates that groups 2 and 4 showed better immunization effects among the five groups. Specifically, the overall immunogenicity of the combinations using serotypes 3, 4, 14, and 19A with F-1 or F-2 vectors, serotypes 5, 6A, 6B, 7F, 9V, 19F, and 23F with E-1 vectors, and serotypes 1 and 18C with F-1, F-2, or E-1 vectors was higher. This demonstrates that the conjugate vaccine using the dual-vector combination of this invention can effectively reduce immune interference between different serotype polysaccharide antigens, and its overall immunogenicity is superior to that of conjugate vaccines using a single vector.

[0079] Example 9: Detection of polysaccharide antibody titers after immunization with 24-valent dual-vector pneumococcal polysaccharide conjugate vaccine Different 24-valent pneumococcal capsular polysaccharides Pn1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F were formulated by combining and conjugating them with different carrier proteins to create various 24-valent pneumococcal polysaccharide dual-carrier immunomodulators. Specific information is shown in Table 4. Serotypes 5, 6A, 6B, 7F, 9V, 18C, 19F, and 23F were prepared using the method described in Example 5, while the remaining serotypes were prepared using the method described in Example 4.

[0080] Table 4: Summary of Information on 24-valent Conjugate Carrier Proteins Formulation A PCV13 (positive vaccine - control) The carrier protein is CRM197 PREVENAR 13 (Pfizer) Formulation B 24-valent pneumococcal conjugate vaccine (dual vector) The carrier proteins are F-2 and E-1. Serum types 5, 6A, 6B, 7F, 9N, 9V, 10A, 11A, 18C, 19F, and 23F bind to carrier protein E-1, while the remaining serum types bind to carrier protein F-2. Formulation C 24-valent pneumococcal conjugate vaccine (dual vector) The carrier proteins are F-1 and E-1. Serum types 5, 6A, 6B, 7F, 9N, 9V, 10A, 11A, 18C, 19F, and 23F bind to carrier protein E-1; the remaining serum types bind to carrier protein F-1. Preparation of formulations B and C: Take the polysaccharide-protein conjugate stock solutions prepared according to Table 4, and measure out the equivalent of 2.2 μg of polysaccharide (except for Pn6B, which has 4.4 μg of polysaccharide), including Pn1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, into sterile containers. Add CpG (Genscript) to a final volume of 0.1 mg, then add phosphate buffer (pH 5.8). Filter sterilely through a 0.22 μm membrane. Add sterile aluminum phosphate gel (Benetag) to a final aluminum ion content of 0.125 mg. Stir at 4°C for 1 hour, aseptically dispense 0.5 mL / bottle, and store at 4°C for use in immunization.

[0081] Immunization protocol: Six New Zealand white rabbits weighing 2.5-3.5 kg were used as a group. Each group was immunized with formulation A (Pfizer), formulation B, and formulation C, respectively. Each rabbit was injected with 0.5 mL once, and immunized once on D0, D14, and D28. Blood samples were collected one week after the immunization was completed and stored for testing.

[0082] Polysaccharide antibody titer detection: Different serotypes of pneumococcal polysaccharides (1×PBS solution) were prepared and stored at 4°C. The polysaccharide of the target serotype was diluted to 4 μg / mL, and 100 μL of coating solution was added to each well to coat the ELISA plate. The plate was incubated overnight at room temperature. After washing four times with wash buffer, 100 μL of blocking buffer was added, and the plate was incubated for 2 hours at room temperature. After washing four times with wash buffer, the plate could be stored at 4°C for one week. The corresponding test sera obtained from rabbit vaccine injection and control samples were diluted 1:10 to prepare working sample sera. The appropriate dilution was added to the first row of wells in the ELISA plate, with a total volume of 200 μL. Serial dilutions were performed twofold from the first row downwards, and the plates were incubated for 2 hours at room temperature. After washing four times with wash buffer, 100 μL of alkaline phosphatase-labeled goat anti-rabbit antibody (1:2000 dilution) was added, and the plate was incubated for 4 hours at room temperature. Wash four times with wash buffer, add 100 μL of 4-nitrobenzene phosphate disodium salt substrate (Sigma-Aldrich) solution, and read the plate at 405 nm. Immunoassay results are as follows. Figure 9 As shown.

[0083] The test results showed that there were significant differences in the serum polysaccharide antibody titers induced by the 24-valent pneumococcal polysaccharide dual-vector conjugate vaccine (Formulation B and Formulation C) and Pfizer PCV13 (Formulation A). Overall, Formulation B induced the highest serum antibody titers in animals, followed by Formulation C.

[0084] Among the 13 serotypes identical to those in the PCV13 positive vaccine, especially serotypes 3, 5, 6A, 6B, 7F, 9V, 14, 19F, and 23F, the test results of formulations B and C were significantly higher than those of formulation A, indicating that their immunization effect has clearly reached that of the marketed vaccine. At the same time, for the 11 serotypes other than the 13 serotypes in the positive vaccine, formulations B and C also showed good immunogenicity, especially for serotypes 8, 9N, 10A, 12F, 15B, 17F, 22F, and 33F, whose polysaccharide antibody titers were significantly higher and significantly higher than the overall average level of polysaccharide IgG antibody titers for the 13 serotypes in the marketed positive vaccine.

[0085] Therefore, it is evident that the dual-vector vaccine group induced higher polysaccharide antibody titers and higher serum protective efficacy in animals immunized with animals compared to the single-vector vaccine. This indicates that the 24-valent pneumococcal polysaccharide dual-vector conjugate vaccine prepared in this invention can effectively increase IgG antibody levels in animals, effectively avoiding the immunosuppressive effects caused by single vectors and further enhancing the antigenicity against polysaccharides from 24 serotypes of pneumococcus. This demonstrates that compared to commercially available single-vector vaccine products, the pneumococcal polysaccharide dual-vector conjugate vaccine prepared in this invention covers a higher valence of pneumococcal serotypes, expanding the vaccine's protective range while ensuring superior immunogenicity of the polysaccharide antigens for a more effective immunization.

[0086] Furthermore, while both formulations B and C, which are dual-carrier formulations, showed good promotion of the immunization effect against 24 polysaccharide antigens, the titers of some serum polysaccharide antibodies differed due to the different carrier proteins F-1 and F-2 used in the two formulations. Specifically, the titers of polysaccharide antibodies against serum types 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 15B, 17F, 19F, 22F, 23F, and 33F were significantly higher in formulation B than in formulation C. This indicates that the immunization effect of polysaccharide antigens using a combination of carrier proteins F-2 and E-1 is superior.

[0087] Example 10: Detection of antibody titers against the carrier protein (VZV-gE) in a dual-vector pneumococcal polysaccharide conjugate vaccine Immunization protocol: 4-6 week old female BALB / c mice were randomly divided into 4 groups of 8 mice each. Subcutaneous immunization was performed every two weeks. One group used PBS (phosphate buffer, pH 5.8: 8.34 g potassium dihydrogen phosphate and 0.87 g dipotassium hydrogen phosphate dissolved in water to make 1000 ml) as an immunization control. The other two groups were immunized with preparations B and C (prepared as in Example 9), respectively. The remaining group was immunized with preparation D. Preparation D preparation: 37 μg of the carrier protein E-1 obtained in Example 2 was sampled, CpG was added to a final volume of 0.1 mg, phosphate buffer (pH 5.8) was added, and the mixture was sterilely filtered through a 0.22 μm membrane. Sterile aluminum phosphate gel was then added to a final aluminum ion concentration of 0.125 mg. The mixture was stirred at 4°C for 1 hour and aseptically dispensed into 0.5 mL vials.

[0088] Each preparation was administered twice, with 0.1 mL administered every two weeks. Blood was collected after the second immunization. A portion of the blood was prepared into PBMCs, flash-frozen on dry ice, and stored at low temperature. The other portion of blood was left at room temperature for 4 hours, centrifuged at 10,000 RPM at room temperature, and the supernatant serum was collected and stored at -70°C for testing.

[0089] Protein antibody titer assay: Prepare a purified gE protein stock solution of 1 mg / mL (1×PBS solution) and store at 4°C. Dilute the gE protein stock solution to 4 μg / mL coating buffer, add 100 μL of coating solution to each well to coat the ELISA plate, and incubate overnight at room temperature. Wash four times with wash buffer, add 100 μL of blocking buffer, incubate at room temperature for 2 hours, wash four times with wash buffer, and store at 4°C until use.

[0090] Serum samples obtained from injecting mice with various vaccine formulations and control samples were diluted 1:10 to prepare working sample serum. The appropriate dilution was added to the first row of wells in an ELISA plate, with a total volume of 200 μL. Serial dilutions were performed twofold from the first row downwards, and the plates were incubated at room temperature for 2 hours. The plates were washed four times with wash buffer, and 100 μL of alkaline phosphatase-labeled goat anti-mouse antibody (1:2000 dilution) was added. The plates were incubated at room temperature for 4 hours. The plates were then washed four more times with wash buffer, and 100 μL of disodium 4-nitrobenzene phosphate substrate solution was added. The plates were read at 405 nm. The immunoassay results are shown in Table 5.

[0091] Table 5: Geometric mean (Eu) titers of anti-gE IgG antibodies in different formulations of immunosuppressant serum

[0092] *CI: Confidence interval.

[0093] Table 5 shows that the titers of anti-gE protein IgG antibodies in the animal immune antiserum of pneumococcal polysaccharide dual-vector conjugate vaccines (Formulation B and Formulation C) and gE protein (Formulation D) were significantly enhanced, and the titers of total IgG, IgG1, and IgG2a antibodies were also significantly increased. Specifically, the titers of total IgG, IgG1, and IgG2a antibodies in the immune serum of the 24-valent pneumococcal polysaccharide dual-vector conjugate vaccines (Formulation B and Formulation C) were all higher than those of the unconjugated gE protein (Formulation D). Furthermore, among the dual-vector conjugate vaccine formulations, the IgG antibody titer of Formulation B was higher than that of Formulation C. Therefore, the immunogenicity of the carrier protein E-1 in the pneumococcal dual-vector conjugate vaccine prepared in this invention is no less than that of the recombinant protein formulation D, which is only a single antigen. This also demonstrates that the gE recombinant protein prepared in this invention, on the one hand, ensures its enhancing and promoting effect on the immunogenicity of pneumococcal polysaccharide antigen as a carrier protein, and on the other hand, ensures its immunogenicity as an antigen protein itself, further enhancing the protective effect against varicella-zoster virus infection.

[0094] Example 11: Detection of IFN-γ cytokine in mouse spleen cells after immunization with dual-vector pneumococcal polysaccharide conjugate vaccine After immunizing the mice with the formulation in Example 10, blood was collected from the mice, and their spleens were collected. The spleens were ground, added to PBS (pH 7.4), resuspended, and serially diluted. The resulting single-cell suspension was added to ELISpot plates pre-coated with capture antibodies. 5 μg / mL of gE protein and 1 μg / mL of concanavalin A (positive control) (Sigma-Aldrich) were added, and the plates were incubated at 37°C and 5% CO2 for 24 hours. IFN-γ secretory cells were detected using an ELISpot kit (MabTech), and positive spots were detected using a CTL ImmunoSpot S5UV Micro analyzer. The immunoassay results are shown in Table 6.

[0095] Table 6: IFN-γ SFC / 10 in spleen cells of mice immunized with different formulations 6 Cell count

[0096] *CI: Confidence interval.

[0097] Table 6 shows that, compared to PBS (control), both the pneumococcal polysaccharide dual-vector conjugate vaccine (formulations B and C) and gE protein (formulation D) significantly stimulated immune cells in the spleen of animals, such as T cells and natural killer cells (NK cells), to secrete IFN-γ cytokines. Specifically, the spleen cells of mice immunized with the 24-valent pneumococcal polysaccharide dual-vector conjugate vaccine (formulations B and C) showed higher IFN-γ secretion under stimulation by the gE protein antigen; and the amount of IFN-γ produced by spleen immune cells sensitized with the 24-valent pneumococcal polysaccharide dual-vector conjugate vaccine (formulations B and C) was significantly higher than that produced by formulation D (gE protein). This indicates that the recombinant gE protein prepared in this invention, in addition to its good cellular immune effect, demonstrates a stronger ability to induce cellular immune responses in mice when used as a carrier protein E-1 in the preparation of the pneumococcal polysaccharide dual-vector conjugate vaccine, further enhancing the regulation of immune responses and strengthening the defense against pathogens.

[0098] Example 12: Detection of antibody titer against carrier protein (RSV-preF) in dual-vector pneumococcal polysaccharide conjugate vaccine Immunization protocol: 4-6 week old female BALB / c mice were randomly divided into 4 groups of 6 mice each. Each group was subcutaneously immunized twice, with 0.1 mL administered each time. Two groups were immunized with preparations B and C (prepared as in Example 9), respectively, while the other two groups were immunized with preparations G and H, respectively. Preparation of preparations G and H: 20 μg of the carrier protein F-1 / F-2 obtained in Example 1 was sampled and added to CpG, bringing the final volume to 0.1 mg. Phosphate-buffered saline (pH 5.8) was added, and the mixture was sterilely filtered through a 0.22 μm membrane. Sterile aluminum phosphate gel was then added, bringing the final aluminum ion content to 0.125 mg. The mixture was stirred at 4°C for 1 hour and aseptically dispensed into 0.5 mL vials, i.e., preparation G (F-1) / preparation H (F-2).

[0099] Blood was collected one week after immunization, with collection times at three times: before week 0 immunization, two weeks after the first immunization, and four weeks after the second immunization. A portion of the blood was prepared into PBMCs, flash-frozen on dry ice, and stored at low temperature. The other portion of blood was left at room temperature for 4 hours, then centrifuged at 10,000 RPM at room temperature. The supernatant serum was collected and stored at -70°C for later testing.

[0100] Protein antibody titer detection: Prepare a purified pre-F protein stock solution of 1 mg / mL (1×PBS solution) and store it at 4°C; dilute the pre-F protein stock solution to 4 μg / mL coating buffer, add 100 μL of coating solution to each well to coat the ELISA plate, and incubate overnight at room temperature; wash 4 times with wash buffer, add 100 μL of blocking buffer, incubate at room temperature for 2 hours, wash 4 times with wash buffer again, and store at 4°C for later use.

[0101] Serum samples obtained after injecting mice with various vaccine formulations were diluted 1:10 to prepare working sample serum. The appropriate dilution was added to the first row of wells in an ELISA plate, with a total volume of 200 μL. Serial dilutions were performed twofold from the first row downwards, and the plates were incubated at room temperature for 2 hours. The plates were then washed four times with wash buffer, and 100 μL of alkaline phosphatase-labeled goat anti-mouse antibody (1:2000 dilution) was added. The plates were incubated at room temperature for 4 hours. The plates were then washed four more times with wash buffer, and 100 μL of disodium 4-nitrobenzene phosphate substrate solution was added. The plates were read at 405 nm. The immunoassay results are shown in Table 7.

[0102] Table 7: Geometric mean of anti-Pre-F IgG antibody titers in different formulations of immunosera

[0103] Table 7 shows that, compared to before immunization, the IgG antibody titers against Pre-F protein in animal serum were significantly increased after immunization with the pneumococcal polysaccharide dual-vector conjugate vaccine (Formulation B, Formulation C) and the Pre-F recombinant protein (Formulation G, Formulation H) formulations. The antibody levels were significantly improved after both immunizations. Specifically, after both the first and second immunizations, the protein antibody titers in the serum immunized with the 24-valent pneumococcal polysaccharide dual-vector conjugate vaccine (Formulation B, Formulation C) were higher than those with the unconjugated Pre-F recombinant protein (Formulation G, Formulation H). Furthermore, among the dual-vector conjugate vaccine formulations, the IgG antibody titer of Formulation B was higher than that of Formulation C. This indicates that the immunogenicity of the carrier proteins F-1 and F-2 in the pneumococcal polysaccharide dual-vector conjugate vaccine prepared in this invention is no less than that of the recombinant protein formulations (Formulation G, Formulation H) containing only a single antigen. Moreover, the combined immunogenicity of carriers F-2 and E-1 with the 24 pneumococcal polysaccharides is slightly better than the combined effect of carriers F-1 and E-1. Therefore, the Pre-F recombinant protein prepared by this invention not only ensures its role as a carrier protein in enhancing the immunogenicity of pneumococcal polysaccharide antigen, but also ensures its immunogenicity as an antigen protein itself, thereby increasing serum protective titer and further enhancing its preventive and protective effect against respiratory syncytial virus infection.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention pending approval.

Claims

1. A composition of pneumococcal polysaccharide-protein conjugate, characterized in that, The composition comprises at least one first carrier protein bound to capsular polysaccharides from different pneumococcal serotypes and at least one second carrier protein bound to capsular polysaccharides from different pneumococcal serotypes, wherein the first carrier protein is selected from respiratory syncytial virus Pre-F protein and the second carrier protein is selected from varicella-zoster virus gE protein.

2. The composition of a pneumococcal polysaccharide-protein conjugate according to claim 1, characterized in that, The first carrier protein is a respiratory syncytial virus Pre-F recombinant protein, namely F-1 or F-2, and its amino acid sequence is shown in SEQ ID NO: 2 or SEQ ID NO: 3; the second carrier protein is a varicella-zoster virus gE recombinant protein, namely E-1, and its amino acid sequence is shown in SEQ ID NO:

5.

3. The composition of a pneumococcal polysaccharide-protein conjugate according to claim 2, characterized in that, The capsular polysaccharide is selected from one or more of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

4. The composition of a pneumococcal polysaccharide-protein conjugate according to claim 3, characterized in that, The capsular polysaccharide that binds to the first carrier protein is selected from one or more of serotypes 1, 2, 3, 4, 8, 12F, 14, 15B, 17F, 19A, 20, 22F, and 33F, and the capsular polysaccharide that binds to the second carrier protein is selected from one or more of serotypes 5, 6A, 6B, 7F, 9N, 9V, 10A, 11A, 18C, 19F, and 23F.

5. The composition of a pneumococcal polysaccharide-protein conjugate according to claim 4, characterized in that, The first carrier protein is F-1, whose amino acid sequence is shown in SEQ ID NO: 2, and the second carrier protein is E-1, whose amino acid sequence is shown in SEQ ID NO:

5.

6. The composition of a pneumococcal polysaccharide-protein conjugate according to claim 4, characterized in that, The first carrier protein is F-2, and its amino acid sequence is shown in SEQ ID NO:

3. The second carrier protein is E-1, and its amino acid sequence is shown in SEQ ID NO:

5.

7. The composition of a pneumococcal polysaccharide-protein conjugate according to claim 5, characterized in that, The composition is a composition comprising a 24-valent pneumococcal polysaccharide dual-carrier protein conjugate, wherein the capsular polysaccharides of serotypes 1, 2, 3, 4, 8, 12F, 14, 15B, 17F, 19A, 20, 22F, and 33F bind to the first carrier protein F-1, and the capsular polysaccharides of serotypes 5, 6A, 6B, 7F, 9N, 9V, 10A, 11A, 18C, 19F, and 23F bind to the second carrier protein E-1.

8. The composition of a pneumococcal polysaccharide-protein conjugate according to claim 6, characterized in that, The composition is a composition comprising a 24-valent pneumococcal polysaccharide dual-carrier protein conjugate, wherein the capsular polysaccharides of serotypes 1, 2, 3, 4, 8, 12F, 14, 15B, 17F, 19A, 20, 22F, and 33F bind to the first carrier protein F-2, and the capsular polysaccharides of serotypes 5, 6A, 6B, 7F, 9N, 9V, 10A, 11A, 18C, 19F, and 23F bind to the second carrier protein E-1.

9. The composition of a pneumococcal polysaccharide-protein conjugate according to claim 1, characterized in that, The composition contains an adjuvant.

10. The composition of a pneumococcal polysaccharide-protein conjugate according to claim 9, characterized in that, The adjuvant includes one or more selected from aluminum salt adjuvants, CpG, QS21, monophosphoryl ester A, MF59, stearoyl tyrosine, Freund's adjuvant, and other mucosal adjuvants.

11. A method for preparing a composition of pneumococcal polysaccharide-protein conjugate as described in any one of claims 1-10, characterized in that, The preparation method includes the following steps: (1) Prepare at least one capsular polysaccharide from different pneumococcal serotypes and react it with a first carrier protein in a buffer or organic solvent to obtain a first binding stock solution, wherein the first carrier protein is selected from respiratory syncytial virus Pre-F protein; (2) Prepare at least one capsular polysaccharide from different pneumococcal serotypes and react it with a second carrier protein in a buffer or organic solvent to obtain a second binding stock solution, wherein the second carrier protein is selected from varicella-zoster virus gE protein; (3) Mix the first binding stock solution obtained in step (1) with the second binding stock solution obtained in step (2) to obtain the composition stock solution of pneumococcal polysaccharide-protein conjugate.

12. The preparation method according to claim 11, characterized in that, The chemical synthesis reaction is selected from one of the following: the reducing amine method, the CDAP method, the adipamide dihydrazine method, or the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride method.

13. The preparation method according to claim 11, characterized in that, The organic solvent is selected from dimethyl sulfoxide or dimethylformamide.

14. The preparation method according to claim 11, characterized in that, The preparation method also includes pretreatment of pneumococcal capsular polysaccharide, expression and purification of carrier protein, and purification of conjugate.

15. The preparation method according to claim 14, characterized in that, The pretreatment of the capsular polysaccharide includes degradation and activation, and the degradation method is selected from high pressure homogenizer degradation, acid hydrolysis or enzymatic digestion.

16. The use of a composition of a pneumococcal polysaccharide-protein conjugate as described in any one of claims 1-10 in the preparation of a vaccine or medicament for the prevention or treatment of a disease, characterized in that, The diseases mentioned are those caused by Streptococcus pneumoniae infection, varicella-zoster virus infection, and / or respiratory syncytial virus infection.

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