Multivalent pneumococcal polysaccharide-protein conjugate vaccine, preparation method therefor, and use thereof

Abstract

Provided in the present invention are a multivalent pneumococcal polysaccharide-protein conjugate vaccine for preventing pneumococcal infection and respiratory syncytial virus infection, a preparation method therefor, and use thereof. In the multivalent pneumococcal polysaccharide-protein conjugate vaccine of the present invention, a carrier protein thereof is an immunogenic RSV recombinant protein; the pneumococcal polysaccharide is linked to the RSV recombinant protein by means of a covalent bond; the RSV recombinant protein is selected from a Pre-F protein, and the amino acid sequence thereof is set forth in SEQ ID NO: 2 or SEQ ID NO: 3. The conjugate vaccine of the present invention can simultaneously prevent pneumonia and respiratory syncytial virus infection, thereby reducing the number of vaccinations required. According to the present invention, by means of the mutational modification of the carrier protein in the conjugate vaccine, the carrier protein can maintain the structural stability and antigenic cluster function under different environments.

Description

Multivalent pneumococcal polysaccharide-protein conjugate vaccine, its preparation method and application Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a multivalent pneumococcal polysaccharide-protein conjugate vaccine for the prevention of pneumococcal infection and respiratory syncytial virus infection, its preparation method, and its application. Background Technology

[0002] Streptococcus pneumoniae is a Gram-positive bacterium whose outer membrane is encapsulated by a layer of capsular polysaccharide. The capsule is its primary pathogenic factor. Based on the different chemical structures of the capsular polysaccharide and classification using specific antisera, 91 different serotypes of Streptococcus pneumoniae have been identified. Streptococcus pneumoniae typically colonizes the nasopharynx and generally does not cause clinical symptoms. However, when the colonization environment changes, such as a weakened immune system, infection with other respiratory viruses, malnutrition, or old age, Streptococcus pneumoniae may breach the mucosal defenses and easily cause invasive infections. Based on the site of infection, Streptococcus pneumoniae can be classified into invasive diseases, including meningitis, bacteremia, and bacteremic pneumonia, and non-invasive diseases, including acute otitis media, sinusitis, and non-bacteremic pneumonia.

[0003] Pneumococcal disease, caused by Streptococcus pneumoniae, is a global public health challenge posing a serious threat to human health, with an increasingly severe disease burden. Statistics show that in 2019, 33 bacterial infections caused 7.7 million deaths globally, with pneumococcus ranking third, accounting for 829,000 deaths. In terms of lost years of life, pneumococcus ranks first, and deaths from pneumococcal infection are primarily concentrated in children under 5 years old and the elderly over 60 years old. Infants, especially those under 2 years old, are highly susceptible to pneumococcal infection, and their mortality rate is extremely high; approximately 75% of invasive pneumococcal disease and 83% of pneumococcal meningitis occur in this age group (see WHO Position Paper 2019). Similarly, the morbidity and mortality rates of invasive pneumococcal disease in the elderly are also significant. According to reports, pneumonia is the fourth leading cause of death among the elderly in China, causing approximately 125,000 deaths annually. The infection rate varies by region, ranging from 28.0% to 71.5%, and the incidence of pneumonia increases dramatically with age. Meanwhile, in an era of increasingly severe global population aging, the UN's "World Society 2023" report indicates that the global population aged 65 and over was 761 million in 2021, and this number is projected to increase to 1.6 billion by 2050, with the population aged 80 and over growing at an even faster rate. Therefore, the elderly are a high-risk group for pneumococcal infection, and the aging population will inevitably lead to an increasingly heavy burden of pneumococcal disease.

[0004] Pneumococcal vaccines are the most effective means of preventing pneumococcal infection. Currently available vaccines include pneumococcal polysaccharide vaccines (PPV) and pneumococcal conjugate vaccines (PCV). In the 1980s, Wyeth's (acquired by Pfizer in 2009) 23-valent pneumococcal polysaccharide vaccine (PPV23) achieved approximately 90% global immunization coverage for the dominant serotypes. However, limitations remained. Studies found that bacterial polysaccharides are non-T-cell dependent antigens, inducing short-term immune responses in children and adults, but ineffective in infants, especially in children under 2 years old. Through development, the polysaccharide was covalently bound to a carrier protein, converting the non-T-cell dependent antigen into a T-cell dependent antigen. Pneumococcal polysaccharide-protein conjugate vaccine (PCV) prepared using this technology can be administered as early as 2 months of age, promoting a stronger immune response and memory response, making it a beneficial immunization method for faster and earlier prevention of pneumococcal infection. The first PCV vaccine to receive FDA approval globally in 2000 was PCV7 (Prevnar 7) manufactured by Wyeth. In 2010, Pfizer launched PCV13 (Prevnar 13), which covers more types, replacing PCV7 (Prevnar 7) for routine childhood vaccinations. Currently, three PCV vaccines have received WHO prequalification: GSK's PCV10 (Synflorix), Pfizer's PCV13 (Prevenar 13), and the Serum Institute of India's PCV10 (PNEUMOSIL). In 2021, the FDA approved even higher-value PCV vaccines, including Pfizer's PCV20 (Prevnar 20) and Merck's PCV15 (VAXNEUVANCE). In China, pneumococcal polysaccharide conjugate vaccines are PCV13 from Watson Bio and PCV13 from Minhai Biotechnology. Studies have found that conjugate vaccines can be used for people of any age, and their effects are longer-lasting and more potent than those of polysaccharide vaccines. Therefore, polysaccharide-protein conjugate vaccines will become the main development trend of pneumococcal vaccines.

[0005] With the widespread use of pneumococcal conjugate vaccines both domestically and internationally, the prevalent serotypes in different regions have changed significantly, and the incidence of some non-vaccine-covered serotypes has increased markedly. Therefore, the development of higher-valent pneumococcal conjugate vaccines is particularly urgent. However, a common drawback of current conjugate vaccines is that the carrier protein is not endowed with immunogenic protective function; that is, although the conjugate vaccine carrier can stimulate the body to produce antibodies, vaccine designers have not been able to utilize the antibodies produced by the carrier protein to prevent disease. The main reason why tetanus toxoid, diphtheria toxoid, and diphtheria avirulence variant toxoid are widely used as carrier proteins is not because the antibodies they produce are protective, but because of their safety and ability to enhance the immunogenicity of polysaccharides in the conjugate. Furthermore, tetanus toxoid and diphtheria toxoid are already two components of the DPT vaccine and are used routinely, so whether the carrier protein in the pneumococcal conjugate vaccine can stimulate the body to produce protective antibodies is not important. Therefore, while increasing the titer of pneumococcal vaccines and expanding coverage, the selection of carrier proteins is also a crucial consideration.

[0006] Respiratory syncytial virus (RSV) is a common, infectious virus primarily transmitted through the air and is a major pathogen causing lower respiratory tract infections in infants and young children. RSV infection can lead to mild to severe respiratory illnesses, including bronchitis, pneumonia, and asthma attacks. RSV infection can cause serious complications and even death, especially in premature infants, low birth weight infants, children with weakened immune systems, and the elderly.

[0007] According to statistics, in 2019, there were as many as 33 million cases of RSV-related acute lower respiratory tract infections globally, with 3.6 million hospitalizations and 26,300 deaths caused by RSV. Among infants aged 0-6 months, the number of RSV-related acute lower respiratory tract infections, hospitalizations, and hospital deaths were approximately 6.6 million, 1.4 million, and 13,300, respectively. China has one of the highest numbers of children with RSV-related acute lower respiratory tract infections, and globally, 99% of deaths among children under 5 years old hospitalized with RSV-related acute lower respiratory tract infections occur in developing countries. The RSV infection rate in adults increases with age, with the elderly being a particularly vulnerable group. Elderly patients with RSV infections often have poorer prognoses and face a heavier economic burden, with the highest in-hospital mortality rate among those over 65 years of age.

[0008] RSV is an enveloped, non-segmented, single-stranded, negative-sense RNA virus belonging to the order Cytovirales, family Pneumoviridae, and genus Orthopneumovirus. Its genome consists of single-stranded negative-sense RNA molecules encoding 11 proteins (including 9 structural proteins (3 glycoproteins and 6 internal proteins) and 2 non-structural proteins). The structural proteins include three transmembrane surface glycoproteins: attachment protein G, fusion protein F, and small hydrophobic SH protein. Two RSV subtypes exist: A and B, differing primarily in the G glycoprotein, while the F glycoprotein sequence is more conserved between the two subtypes.

[0009] The RSV fusion protein (F protein) is a class I transmembrane protein composed of 574 amino acid residues. Initially, it is generated in the host cell as the precursor F0. F0 protein undergoes glycosylation at the Golgi apparatus and is subsequently hydrolyzed by intracellular furin protease, releasing a 27-amino acid polypeptide, pep27. At the N- and C-terminal cleavage sites of this polypeptide, two subunits, F1 and F2, are generated. The F2 subunit consists of the signal peptide SP and the heptapeptide repeat sequence HRC. The F1 subunit consists of the fusion peptide FP, the heptapeptide repeat regions HRA and HRB, the transmembrane regions TM (Domain I and Domain II), and the cytoplasmic domain CP. F1 and F2 are linked by disulfide bonds, forming a heterodimer. The three heterodimers assemble into a mature F protein trimer. The RSV F protein trimer is unstable, exhibiting both prefusion and postfusion conformations. The F protein on the viral envelope initially exists in a metastable pre-F conformation. Upon viral adsorption to the cell membrane, the pre-F protein undergoes a conformational change triggered by factors such as cell receptors, temperature, and ion concentration, generating a highly stable post-F trimer. This process releases energy and mediates the fusion of the viral envelope with the cell membrane. Due to the high conformational instability of pre-F, the protein obtained through in vitro isolation and purification is usually post-F. However, research indicates that the metastable pre-F conformation is essential for viral-mediated membrane fusion and is also an important antigen inducing an immune response in humans.

[0010] The pre-F protein, a major antigen of respiratory syncytial virus (RSV), possesses good immunogenicity. However, the natural RSV pre-F protein is easily destroyed under conditions such as high temperature, high pressure, and extreme pH, resulting in poor protein structural stability and difficulty in ensuring its antigenicity. Therefore, further research is needed on the structure and function of the RSV pre-F protein. However, designing a highly stable and effective recombinant RSV pre-F protein remains a challenge, and currently, there are no domestically produced RSV vaccines approved for marketing in China. Therefore, an immunogen derived from the RSV F protein, possessing improved properties compared to the corresponding natural RSV F protein, is needed to lay the foundation for further vaccine development.

[0011] For vaccines, a key characteristic is that one vaccine can prevent infection by a specific pathogen. For example, pneumococcal polysaccharide conjugate vaccine can prevent pneumonia, meningitis, and otitis media caused by pneumococcal infection, while RSV vaccine can prevent respiratory diseases caused by respiratory syncytial virus infection. To achieve a single formulation that can prevent infection by multiple pathogens, the current approach is to use combination vaccines, such as the diphtheria-pertussis-tetanus trivalent vaccine for children, the diphtheria-pertussis-tetanus-tetanus pentavalent vaccine for children, the diphtheria-pertussis-tetanus-tetanus-poliomyelitis-Haemophilus influenzae pentavalent vaccine for children, and the diphtheria-pertussis-tetanus-tetanus-poliomyelitis-Haemophilus influenzae-hexavalent vaccine for children. These vaccines are prepared by mixing single-product vaccines. Current clinical evaluations show that the preventive efficacy of some single-product vaccines within combination vaccines is not ideal.

[0012] Currently, children of appropriate age in my country need to receive at least 15 types of vaccines, far exceeding the number of vaccine types. Furthermore, with the aging population, by the end of 2024, my country's population aged 60 and above had reached 310 million, accounting for 22% of the total population. Therefore, protecting the health of the elderly and developing vaccine products specifically for them is indispensable. It is evident that developing multivalent conjugate vaccines to replace single-drug vaccines and achieving full coverage of vaccine protection will inevitably be the primary task of future vaccine research and development. However, with the increasing valence of vaccines, key challenges in the development of polysaccharide-protein conjugate vaccines include: how to select the carrier protein to avoid carrier inhibition effects; how to design stable and highly expressed carrier proteins; and how to combine immunogenic carrier proteins with other immunogenic substances (such as pneumococcal capsular polysaccharide) to prepare vaccines with dual immunogenicity, achieving the goal of one vaccine protecting against two diseases. Summary of the Invention

[0013] To overcome the shortcomings of existing technologies, this invention utilizes polysaccharide-protein conjugation technology, using RSV Pre-F protein, modified with amino acid mutations to enhance stability, as a carrier protein to prepare a novel multivalent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine. This conjugate vaccine possesses dual immunogenicity, enabling simultaneous and effective prevention of diseases caused by 30 serotypes of pneumococcus and respiratory syncytial virus infection. This significantly reduces the number of vaccinations required, alleviates the suffering of infants and young children and the psychological burden on parents, lowers immunization costs, and increases immunization coverage, providing a new means of preventing respiratory infections in infants and the elderly.

[0014] The technical solution of this invention to solve the technical problem is as follows:

[0015] In a first aspect of the present invention, a multivalent pneumococcal polysaccharide-protein conjugate vaccine is provided, wherein the carrier protein in the conjugate vaccine is an immunogenic RSV recombinant protein; the pneumococcal polysaccharide is covalently linked to the RSV recombinant protein; the RSV recombinant protein is selected from Pre-F protein, and its amino acid sequence is shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0016] Furthermore, the pneumococcal polysaccharide in the conjugated vaccine is selected from one or more serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34, and 35B.

[0017] In a preferred embodiment of the present invention, the conjugate vaccine is a 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine, wherein the amino acid sequence of the RSV recombinant protein is shown in SEQ ID NO: 2, and the pneumococcal polysaccharide includes 30 serotypes, namely serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34, and 35B.

[0018] In a preferred embodiment of the present invention, the conjugate vaccine is a 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine, wherein the amino acid sequence of the RSV recombinant protein is shown in SEQ ID NO: 3, and the pneumococcal polysaccharide includes 30 serotypes, namely serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34, and 35B.

[0019] Furthermore, the conjugate vaccine is formulated as an aqueous solution or a lyophilized solution.

[0020] Furthermore, the conjugate vaccine contains an adjuvant.

[0021] Furthermore, the adjuvant is any one of CpG, QS21, aluminum phosphate, a mixture of CpG and aluminum phosphate, or a mixture of QS21 and aluminum phosphate.

[0022] In a second aspect of the present invention, a method for preparing a multivalent pneumococcal polysaccharide-protein conjugate vaccine as described in the first aspect is provided. The preparation method includes the following steps: chemically synthesizing pneumococcal polysaccharide and RSV recombinant protein in a buffer solution or organic solvent, thereby covalently binding the pneumococcal polysaccharide to the RSV recombinant protein through the reaction to obtain a conjugate.

[0023] Furthermore, the preparation method further includes pretreatment of pneumococcal polysaccharide, expression and purification of RSV recombinant protein, and purification of the conjugate. More preferably, the pretreatment of the polysaccharide includes degradation and activation, wherein the degradation method is selected from high-pressure homogenization, acid hydrolysis, or enzymatic digestion.

[0024] Furthermore, the organic solvent is selected from dimethyl sulfoxide or dimethylformamide.

[0025] Furthermore, the chemical synthesis reaction is selected from any one of the following: the amine reduction method, the 1-cyano-4-dimethylamino pyridinium tetrafluoroborate method, the adipicohydrazide method, or the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride method.

[0026] In a third aspect of the invention, a nucleic acid molecule is provided that encodes an RSV Pre-F recombinant protein with an amino acid sequence as described in the first aspect, such as SEQ ID NO: 2 or SEQ ID NO: 3.

[0027] In a fourth aspect of the invention, a recombinant expression vector containing nucleic acid molecules as described in the third aspect is provided.

[0028] In a fifth aspect of the invention, an expression method for preparing recombinant RSV protein using a recombinant expression vector as described in the fourth aspect is provided, wherein the expression method employs a CHO cell expression system or an insect baculovirus expression system.

[0029] The present invention has the following technical effects:

[0030] (1) This invention provides a multivalent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine, which is an innovative vaccine that can prevent two common diseases, namely pneumonia and respiratory syncytial virus infection, with one vaccine product. This greatly reduces the number of vaccinations, lowers the cost of immunization, and greatly improves the immunization coverage rate.

[0031] (2) This invention modifies the carrier protein, namely RSV Pre-F protein, in the conjugate vaccine by amino acid mutation, so that the carrier protein can maintain its structural stability and antigen cluster function under different environments, including high temperature, acidity and high osmotic pressure. Even after undergoing chemical reactions, it can still maintain its antigenicity as a carrier protein.

[0032] (3) The present invention also provides a 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine and its preparation method. The RSV recombinant protein is an RSV Pre-F protein carrier with enhanced stability after the amino acid sequence is innovatively designed by gene recombination technology and the amino acid is modified by mutation. After the conjugate polysaccharide antigen part is combined with a novel adjuvant preparation and animals are immunized, the conjugate polysaccharide antigen part can stimulate the body to produce specific serum polysaccharide antibodies, while the carrier protein part can stimulate the body to produce protective antibodies against RSV virus. Thus, after immunization with a vaccine, infection of 30 serotypes of pneumococcus and RSV can be prevented at the same time.

[0033] (4) The 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine prepared by the present invention has a synergistic effect: On the one hand, as can be seen from Example 10, compared with the Pfizer PCV20 conjugate vaccine with CRM197 as the carrier protein, the conjugate vaccine prepared by the present invention uses the RSV recombinant protein modified by amino acid mutation as the carrier protein, which has a better effect on enhancing the antigenicity of 30 serotype polysaccharides. In the second immunization, the titers of IgG antibodies against the corresponding serotypes produced by animals stimulated by the 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine of this invention (Formulation A) and PCV20 Pfizer (Formulation B) varied depending on the serotype. Data for serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 23F, and 33F showed that Formulation A was significantly higher than Formulation B, accounting for 90% of the components. Only for two serotypes, 12F and 22F, the test results showed that Formulation B was higher than Formulation A, accounting for only 10% of the components.

[0034] On the other hand, as can be seen from Example 11, the IgG antibody titer in the mouse immune serum of the 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine prepared in this invention is significantly higher than that of the RSV Pre-F recombinant protein. Specifically, the IgG antibody titers in the immune serum of the 30-valent conjugate (Pn-RSV Pre-F) after the first and second immunizations were approximately 1.6 and 2.4 times higher than those of RSV Pre-F (formulation C), respectively. Meanwhile, the results of the RSV A2 virus neutralization titer test also showed that the mean neutralization titer in the mouse serum of the 30-valent conjugate (Pn-RSV Pre-F) was 12600, which is approximately 1.9 times higher than that in the RSV Pre-F mouse serum. This demonstrates that the 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine prepared in this invention can obtain serum with high protective titers and can produce high neutralizing antibody titers against the main circulating RSV strain A.

[0035] (5) The 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine prepared by this invention covers more valences of pneumococcal serotypes, expanding the vaccine's protective range while also achieving better immunization effects. As can be seen from Example 10, compared to PCV20 (20-valent pneumococcal conjugate vaccine), the antiserum IgG antibody titers (GM) of the 10 newly added serotypes Pn2, Pn6C, Pn9N, Pn15A, Pn17F, Pn20, Pn22A, Pn24F, Pn34, and Pn35B in the conjugate vaccine of this invention are all significantly higher than the average value (168.06) of the polysaccharide IgG antibody titers of the 20 serotypes in PCV20. Attached Figure Description

[0036] Figure 1 shows the temperature stability test results of the Pre-F protein before and after modification.

[0037] Figure 2 shows the pH stability test results of Pre-F protein before and after modification.

[0038] Figure 3 shows the osmotic stability test results of Pre-F protein before and after modification. Detailed Implementation

[0039] To more concisely and clearly demonstrate the technical solution, objectives, 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.

[0040] Example 1: Preparation of carrier protein

[0041] (I) Protein Construction

[0042] 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 syncytial virus A(strain A2)OX=11259GN=F PE=1SV=1).

[0043] (1) Amino acid sequence design

[0044] This invention relates to a recombinant RSV Pre-F protein with enhanced stability obtained through amino acid mutation modification, the amino acid sequence of which is designed as follows:

[0045] 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 G at position 242 of the wild-type pre-fusion F protein amino acid sequence was mutated to C, and the M at position 289 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 RSV-modified Pre-F protein sequence.

[0046] This invention also relates to a pre-F recombinant protein before RSV modification, the amino acid sequence of which is designed as follows:

[0047] The transmembrane and intracellular regions of the full-length wild-type RSV pre-F protein sequence were deleted, and the fibritin / Throm / 6his / Stretaq sequence was added to its C-terminus to obtain the variant sequence shown in SEQ ID NO.2. This sequence is the RSV pre-modified Pre-F protein sequence.

[0048] The sequences SEQ ID NO.1 through SEQ ID NO.3 are shown below:

[0049] SEQ ID NO.1:

[0050] >sp|P03420|FUS_HRSVA Fusion glycoprotein F0 OS=Human respiratory syncytial virus A(strain A2)OX=11259GN=F PE=1SV=1

[0051] SEQ ID NO.2:

[0052] >RSV F

[0053] SEQ ID NO.3:

[0054] >RSV F G242C M289C

[0055] (2) Synthesis of RSV Pre-F target gene

[0056] Based on the designed RSV protein amino acid sequences SEQ ID NO.2 and SEQ ID NO.3 and the codon preference of the host cell, the corresponding gene coding sequence was determined, and the restriction endonuclease XbaI sequence was added to the 5' end of the gene segment and the restriction endonuclease EcoRI sequence was added to the 3' end, and the designed nucleotide sequence was chemically synthesized.

[0057] (3) Plasmid amplification and target gene extraction

[0058] The pUC19 plasmid vector was digested with XbaI and EcoRI restriction enzymes, ligated to the synthesized gene, and 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-preF 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 XbaI and EcoRI using the TaKaRa MiniBest Agarose Gel Extraction Kit.

[0059] (4) Construction of eukaryotic expression vectors

[0060] The mammalian cell expression plasmid pGN-M, containing the CMV promoter and dihydrofolate reductase (DHFR) gene, was digested with XbaI and EcoRI 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_preF were screened and amplified. The amplified plasmid was extracted using the TaKaRa MidiBEST Endo-free Plasmid Purification Kit.

[0061] (II) Protein expression and clone screening

[0062] 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 2Cells 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. 4 μg of pcDNA vector was added to each well. The 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 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 proteins in the supernatant and lysate. After culturing in selective medium for 5 days, wash cells with trypsin (Invitrogen) and then seed them onto 9 cm Petri dishes for serial dilutions to isolate single clones. Over subsequent days 7-11, 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 proteins. Clones that secrete the highest amount of RSV pre-F protein are selected for the next round of screening, and the cells are finally expanded. Thirty new clones are then selected and preserved.

[0063] The selected clones were amplified into three T175 flasks (NETS). After trypsin digestion and washing with PBS, the clones were resuspended in 250 mL rotational 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, the BFS (Bio-free suspension) is gradually removed. At this point, the cells are considered fully adapted for serum-free suspension growth.

[0064] (III) Production of RSV protein in a bioreactor

[0065] A 1.5-liter perfusion culture was configured in a bioreactor, equipped with a rotary filter (10 μm). Culture parameters were set as follows: temperature controlled at 37°C using a heating blanket; pH adjusted to 6.9 using CO2 or 0.3M 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.

[0066] A total of 12.5 liters of cell-free culture medium was collected, centrifuged at 8000×g for 30 minutes at 4°C, filtered through a 0.45 μm membrane, and then concentrated by ultrafiltration using a 10 kDa membrane. The sample was washed with buffer and concentrated to 0.5 liters. 0.5 liters of PBS were added, and the concentration was further reduced to 0.5 liters. This process was repeated five times.

[0067] (iv) Purification of RSV protein

[0068] 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. The pre-F protein was eluted with a solution containing 300 mM sodium chloride. Ammonium sulfate was added to the combined buffer 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, the pre-F protein was eluted with purified water. Finally, the sample was loaded onto a Sephacryl S-400HR (GE Bioscience), the column was washed with PBS, the protein peak was collected, a co-solvent was added, and the sample was lyophilized in a vacuum freeze dryer and stored at -70°C for later use.

[0069] Both the pre-F protein before RSV modification and the pre-F protein after RSV modification were prepared using the methods described above.

[0070] Example 2: Temperature stability test of the Pre-F protein obtained in Example 1 before and after modification.

[0071] 1) Dilute the test proteins (the pre-modified Pre-F protein prepared in Example 1 and the post-modified Pre-F protein prepared in Example 1) to 20ug / ml with 1*PBS pH7.4 buffer and place them in a 1.5mL centrifuge tube with a total volume of 1mL.

[0072] 2) Incubate at different temperatures according to the table below:

[0073] 3) After incubation, each sample was temporarily stored at 4℃.

[0074] 4) Perform ELISA testing according to the protocol:

[0075] Dilute the sample stored at 4℃ to 1ug / mL with 1*PBS pH 7.4, add 100µL / well to a microplate (NUNC 442404), and coat overnight at 4℃. Shake the plate dry, add 150µL / well of 1% BSA-PBS, and incubate at 37℃ for 2 hours. Wash the plate 3 times according to the programmed procedure, add 100µL / well of the detection antibody PA1 (Medimmuneorigion), and incubate at 37℃ for 2 hours. Wash the plate 3 times according to the programmed procedure, add 100µL / well of AP-labeled goat anti-human secondary antibody at a 1:2000 dilution, and incubate at 37℃ for 1 hour. Wash the plate 3 times according to the programmed procedure, add 100µL / well of pNPP substrate solution, set the microplate reader to 405nm, and read the value.

[0076] The test results are shown in Figure 1. The results show that the antigen-binding activity of the Pre-F protein before and after modification is significantly different after treatment at different temperatures. The detection values ​​of the modified Pre-F protein at 4℃, 50℃ and 70℃ are significantly higher than those of the unmodified protein. This indicates that the modified Pre-F protein prepared in this invention can still maintain high antigen-binding activity after treatment at different temperatures. That is, compared with the unmodified Pre-F protein, the temperature stability of the modified Pre-F protein prepared in this invention is significantly enhanced.

[0077] Example 3: pH stability test of the Pre-F protein obtained in Example 1 before and after modification.

[0078] 1) Solution preparation

[0079] ① 25mM acetate buffer, pH 3.5.

[0080] Preparation method (based on a 100mL volume):

[0081] 1. Accurately weigh 98.953 mg of sodium acetate and add it to a 200 mL beaker.

[0082] 2. Accurately weigh 1.429g of acetic acid and add it to a 5mL centrifuge tube.

[0083] 3. Add about 80 mL of purified water to sodium acetate and dissolve it completely, then add acetic acid.

[0084] 4. Adjust the pH of the solution to 3.5.

[0085] 5. Bring the volume to 100 mL, store at room temperature, shelf life 3 months.

[0086] ② 25mM acetate buffer, pH 5.0.

[0087] Preparation method (based on a 100mL volume):

[0088] 1. Accurately weigh 1.381g of sodium acetate and add it to a 200mL beaker.

[0089] 2. Accurately weigh 490.3 mg of acetic acid and add it to a 5 mL centrifuge tube.

[0090] 3. Add about 80 mL of purified water to sodium acetate and dissolve it completely, then add acetic acid.

[0091] 4. Adjust the pH of the solution to 5.0.

[0092] 5. Bring the volume to 100 mL, store at room temperature, shelf life 3 months.

[0093] ③ 25mM Tris-HCl buffer, pH 8.0.

[0094] Preparation method (based on a 100mL volume):

[0095] 1. Add 80 mL of purified water to a 200 mL beaker.

[0096] 2. Accurately measure 302.85 mg Tris and add it.

[0097] 3. Add 1 mL of 1N hydrochloric acid (theoretical value 1.42 mL), then add a small amount to adjust the pH of the solution to 8.0.

[0098] 4. Bring the volume to 100 mL, store at room temperature, shelf life 3 months.

[0099] ④ 25mM Tris-HCl buffer, pH 10.0.

[0100] Preparation method (based on a 100mL volume):

[0101] 1. Add 80 mL of purified water to a 200 mL beaker.

[0102] 2. Accurately measure 302.85 mg Tris and add it.

[0103] 3. Add 20 μL of 1N hydrochloric acid (theoretical value 33 μL), then add a small amount to adjust the pH of the solution to 10.0.

[0104] 4. Bring the volume to 100 mL, store at room temperature, shelf life 3 months.

[0105] ⑤ 25mM PBS buffer control, pH 7.5.

[0106] Preparation method (based on a 100mL volume):

[0107] 1. Add 80 mL of purified water to a 200 mL beaker.

[0108] 2. Accurately measure and add 142 mg of Na2HPO4, 27 mg of KH2PO4, 800 mg of NaCl, and 20 mg of KCl.

[0109] 3. Add a small amount of HCl to adjust the pH of the solution to 7.5.

[0110] 4. Bring the volume to 100 mL, store at room temperature, shelf life 3 months.

[0111] 2) Dilute the test proteins (the pre-modified Pre-F protein prepared in Example 1 and the post-modified Pre-F protein prepared in Example 1) to 20 ug / mL using buffers with different pH values, and place them in 1.5 mL centrifuge tubes with a total volume of 1 mL.

[0112] 3) Incubate the samples at different pH values ​​according to the table below:

[0113] 4) After incubation, neutralize the pH of each sample to 7.5 with acid or alkali (measured with pH test paper) and temporarily store at 4℃. Perform ELISA testing according to the protocol (operation is the same as temperature stability test).

[0114] The test results are shown in Figure 2. The results show that the antigen-binding activity of the Pre-F protein before and after modification is significantly different after treatment at different pH values. The detection values ​​of the modified Pre-F protein at pH 5.0, pH 7.5, pH 8.0 and pH 10 are significantly higher than those of the unmodified protein. This indicates that the modified Pre-F protein prepared in this invention can still maintain high antigen-binding activity after treatment at different pH values. That is, compared with the unmodified Pre-F protein, the pH stability of the modified Pre-F protein prepared in this invention is significantly enhanced.

[0115] Example 4: Osmotic stability test of the Pre-F protein obtained in Example 1 before and after modification.

[0116] 1) Solution preparation

[0117] ①10mM Tris-HCl buffer, pH 7.5.

[0118] Preparation method (based on a 25mL volume):

[0119] 1. Add 10 mL of 25 mM Tris-HCl buffer (pH 8.0) to a 50 mL centrifuge tube.

[0120] 2. Add purified water to a volume of 25 mL.

[0121] 3. Add 6.0 mL of 1 N hydrochloric acid (theoretical value 6.45 mL), then add a small amount to adjust the pH of the solution to 7.5.

[0122] 4. Make up to 25 mL, store at room temperature, shelf life 3 months.

[0123] ②80mM Tris-HCl buffer, pH 7.5.

[0124] Preparation method (based on a 100mL volume):

[0125] 1. Add 80 mL of purified water to a 200 mL beaker.

[0126] 2. Accurately measure 969.12 mg of Tris and add it.

[0127] 3. Add 6 mL of 1N hydrochloric acid (theoretical value 6.45 mL), then add a small amount to adjust the pH of the solution to 7.5.

[0128] 4. Bring the volume to 100 mL, store at room temperature, shelf life 3 months.

[0129] ③ 1.5M MgCl2 solution

[0130] Preparation method (based on a 100mL volume):

[0131] 1. Accurately measure 20 mL of 3M MgCl2 solution and add it to a 50 mL centrifuge tube.

[0132] 2. Add purified water to 20 mL and mix thoroughly.

[0133] 3. Store at room temperature. Shelf life is 3 months.

[0134] ④ 3M MgCl2 solution

[0135] Preparation method (based on a 100mL volume):

[0136] 1. Accurately measure 60.99g of magnesium chloride hexahydrate and add it to a 200mL beaker.

[0137] 2. Add purified water to about 80mL and stir until fully dissolved.

[0138] 3. Bring the volume to 100 mL, store at room temperature, shelf life 3 months.

[0139] ⑤ 150mM Tris-HCl buffer, pH 7.5.

[0140] Preparation method (based on a 100mL volume):

[0141] 1. Add 80 mL of purified water to a 200 mL beaker.

[0142] 2. Accurately measure 1.815 mg of Tris and add it.

[0143] 3. Add 6 mL of 1N hydrochloric acid (theoretical value 6.45 mL), then add a small amount to adjust the pH of the solution to 7.5.

[0144] 4. Bring the volume to 100 mL, store at room temperature, shelf life 3 months.

[0145] ⑥ 4M NaCl solution

[0146] Preparation method (based on a 125mL volume):

[0147] 1. Accurately weigh 29.22g of sodium chloride and add it to a 200mL beaker.

[0148] 2. Add 100mL of purified water and stir thoroughly to dissolve.

[0149] 3. Bring the volume to 125 mL, store at room temperature, shelf life 3 months.

[0150] 2) Dilute the test proteins (the pre-modified Pre-F protein prepared in Example 1 and the post-modified Pre-F protein prepared in Example 1) to 20 ug / mL with buffers of different osmotic pressure values, and place them in 1.5 mL centrifuge tubes with a total volume of 1 mL.

[0151] 3) Incubate the samples at different osmotic pressures according to the table below:

[0152] 4) The 10mM and 80mM groups were diluted with Tris buffer, and the 1500mM and 3000mM groups were diluted with MgCl2 buffer respectively.

[0153] 5) After incubation, the 10mM and 80mM groups were adjusted to 150mM with 4M NaCl buffer, and the 1500mM and 3000mM groups were diluted 10 times and 20 times with purified water, respectively, and stored at 4℃.

[0154] Perform the ELISA test according to the protocol. (The procedure is the same as the temperature stability test).

[0155] The results are shown in Figure 3. The results show that the antigen-binding activity of the Pre-F protein before and after modification is significantly different after incubation at different osmotic pressures. The detection values ​​of the modified Pre-F protein at 10mM, 80mM, 150mM, 1500mM and 3000mM are significantly higher than those of the unmodified protein. This indicates that the modified Pre-F protein prepared in this invention can still maintain high antigen-binding activity after incubation at different osmotic pressures. That is, compared with the unmodified Pre-F protein, the stability of the modified Pre-F protein prepared in this invention is significantly enhanced under different osmotic pressures.

[0156] Therefore, compared with the unmodified Pre-F protein, the modified Pre-F protein prepared by this invention still maintains its structural stability and antigen cluster function under different environments, including high temperature, acidity and high osmotic pressure. Even after undergoing chemical reactions, it can still retain its good antigenicity.

[0157] Example 5: Preparation of capsular polysaccharides from 30 serotypes of Streptococcus pneumoniae (1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34 and 35B)

[0158] (1) Preparation of master seed and working seed

[0159] The specific method for purifying capsular polysaccharides from the fermentation broth of pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34, and 35B is as follows:

[0160] Pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34, and 35B were obtained from the American Type Culture Collection. The inoculum in the lyophilized seed tubes was inoculated into 5 ml of yeast-acid-hydrolyzed casein culture medium and cultured at 36℃±2℃ for 18 hours. When the bacteria 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℃.

[0161] The bacterial cultures of pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34, and 35B were inoculated into 5 mL of yeast-acid-hydrolyzed casein culture medium and cultured at 36℃±2℃ for 18 hours. When the bacteria 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.

[0162] (2) Bacterial fermentation

[0163] 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.

[0164] (3) Purification of capsular polysaccharides

[0165] 1. Add phosphoric acid to adjust the pH of the fermentation broth to about 4, and stir for 1 hour;

[0166] 2. Centrifuge using a disc centrifuge at a speed of 9600 rpm, collect the supernatant, and discard the residue.

[0167] 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.

[0168] 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.

[0169] 5. Use a 50kD membrane to further wash and filter the polysaccharide solution for 10 sample volumes to concentrate the polysaccharide sample solution.

[0170] 6. Collect the purified polysaccharide solution into a freeze-drying bottle, freeze-dry it in a vacuum freeze dryer, and store it at -70℃.

[0171] Example 6: Preparation of polysaccharide-RSV recombinant protein conjugates of 30 serotypes of Streptococcus pneumoniae (Pn1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34 and 35B) (CDAP method)

[0172] 1) Weigh 17.5 mg of the corresponding serum-type purified capsular polysaccharide and dissolve it in 4 mL of sodium phosphate buffer;

[0173] 2) Add 12 mg of 1-cyano-4-dimethylammonium pyridine tetrafluoroborate (CDAP) (Sigma-Aldrich) to the polysaccharide solution, stir, and react at room temperature for 1 hour;

[0174] 3) Add 3 Eqm of cystamine and react at room temperature for 1 hour;

[0175] 4) Add 0.3 mL of 1M lysine (Sigma-Aldrich) solution to quench the reaction, and react at room temperature for 1-2 hours;

[0176] 5) Add 8 Eqm of 3(2-chloroethyl) phosphate to the polysaccharide solution to reduce the disulfide bonds in the polysaccharide;

[0177] 6) Transfer the activated polysaccharide solution to a dialysis bag and dialyze against phosphate buffer at 4°C, changing the buffer four times;

[0178] 7) Weigh 30 mg of carrier protein and dissolve it in phosphate buffer to achieve a protein concentration of 10 mg / mL;

[0179] 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.

[0180] 9) Mix 4 mL of activated polysaccharide solution with 4 mL of activated protein solution and react at room temperature for 4 hours;

[0181] 10) Add 4 Eqm of N-acetyl-L-cysteine ​​(Sigma-Aldrich), react at 2-8℃ for 4 hours, add 12 Eqm of iodoacetamide (Sigma-Aldrich), and react at 2-8℃ for 4 hours;

[0182] 11) Transfer the polysaccharide conjugate reaction solution to a dialysis bag and dialyze it against phosphate buffer at 4°C;

[0183] 12) Load the sample solution onto Sepharose CL-4B, purify it, and collect the external water volume bound.

[0184] 13) After filtration through a 0.22μm filter membrane, store at 2-8℃ until preparation is complete.

[0185] The carrier protein used in step 7) is the modified Pre-F protein, or the unmodified Pre-F protein can also be used as the carrier protein.

[0186] Example 7: Preparation of polysaccharide-RSV recombinant protein conjugates of 30 serotypes of Streptococcus pneumoniae (Pn1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34 and 35B) (reduced amine method)

[0187] 1) Weigh 500 mg of the corresponding serum-type purified capsular polysaccharide and dissolve it in 500 mL of purified water;

[0188] 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.

[0189] 3) Use ultrafiltration to wash the purified water with a membrane with a molecular weight of 50Kd, then concentrate and freeze-dry it;

[0190] 4) Weigh 18 mg of activated polysaccharide, add 4 mL of DMSO, and stir until completely dissolved;

[0191] 5) Add 19 mg of carrier protein to 4 mL of DMSO (Sigma-Aldrich) solution, add 2 Eqm of sodium cyanoborohydride (Sigma-Aldrich), and react at room temperature for 22 hours;

[0192] 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.

[0193] 7) Load the sample solution onto Sepharose CL4B and collect the bound portion of the external water volume.

[0194] 8) After filtration through a 0.22μm filter membrane, store at 4℃ until preparation.

[0195] 9) Sample and test the molecular weight of the conjugate, as well as the concentration and ratio of polysaccharides and proteins.

[0196] The carrier protein used in step 5) is the modified Pre-F protein, or the unmodified Pre-F protein can also be used as the carrier protein.

[0197] Example 8: Preparation of Immunotherapy Agents A, B, and C

[0198] (1) Formulation A (30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine):

[0199] The polysaccharide concentration in the monovalent conjugates was determined by measuring 2.2 μg of RSV recombinant protein conjugate solution (the conjugate prepared in Example 6 was used in this example, and the carrier protein in the conjugate was the modified Pre-F protein), including Pn1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, and 23F. Samples of 24F, 33F, 34, and 35B were transferred to sterile containers. Samples were taken and the modified Pre-F protein content was measured to be approximately 88.6 μg. CpG (Genscript) 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 (Benetag) was added to a final aluminum ion content of 0.125 mg. The mixture was stirred at 4°C for 1 hour, aseptically dispensed into 0.5 mL vials, and stored at 4°C for use in immunization.

[0200] (2) Formulation B (Pfizer PCV20):

[0201] In June 2021, the U.S. Food and Drug Administration (FDA) approved Pfizer's Prevnar 20 (pneumococcal 20-valent conjugate vaccine) for use in adults 18 years of age and older to prevent invasive disease and pneumonia caused by the 20 pneumococcal serotypes contained in the vaccine. Pfizer Prevnar 20 (PCV20) contains, in addition to the ingredients already listed in PREVENAR... The 13-valent pneumococcal conjugate vaccine [Diphtheria CRM 197 protein] contains capsular polysaccharide conjugates of 13 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F), as well as capsular polysaccharide conjugates of seven other serotypes (8, 10A, 11A, 12F, 15B, 22F, and 33F) that account for 40% of pneumococcal disease cases and deaths in the United States. These conjugates are associated with high mortality, antibiotic resistance, and / or meningitis.

[0202] Among them, CRM197 used in PCV20 is a carrier protein used in the preparation of traditional polysaccharide-protein conjugates. This carrier protein can enhance the immunogenicity of polysaccharides in the conjugate, but it does not take into account that the carrier protein itself stimulates the body to produce protective antibodies to prevent disease.

[0203] This invention uses Pfizer Prevnar 20 (PCV20) conjugate vaccine as a control for detecting polysaccharide antibody titers after immunization with formulation A (30-valent pneumococcal capsular polysaccharide-RSV recombinant protein conjugate vaccine) of this invention.

[0204] (3) Formulation C (RSV Pre-F):

[0205] The modified Pre-F protein obtained in Example 1 was sampled at a concentration of 25.0 μg. CpG (Genscript) was added to a final amount of 0.1 mg. Phosphate buffer (pH 5.8) was added, and the sample was sterilized and filtered through a 0.22 μm membrane. Sterile aluminum phosphate gel (Benetag) was added to a final aluminum ion concentration of 0.125 mg. The sample was stirred at 4°C for 1 hour, aseptically dispensed into 0.5 mL / bottle, and stored at 4°C for use in immunization.

[0206] Example 9: Immunization of rabbits with the formulation and blood collection

[0207] Ten New Zealand white rabbits weighing 2.5-3.5 kg were selected and divided into groups of five. One group received immunizing agent A (prepared in Example 8), and the other group received immunizing agent B (Pfizer PCV20). Each rabbit was injected with 0.5 mL of the agent once, twice a day at week 0 and week 2. Blood samples were collected three times: before week 0 immunization, one week after the first immunization, and one week 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 the 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 analysis.

[0208] Example 10: ELISA detection of polysaccharide antibody titers in rabbit serum immunized with 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine

[0209] Prepare different serotypes of pneumococcal polysaccharides (1×PBS solution) and store at 4°C. Dilute the polysaccharide of the desired serotype of pneumococcal polysaccharide to 4 μg / mL, 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 for 2 hours at room temperature, wash four times with wash buffer, and store at 4°C for one week.

[0210] The corresponding test serum obtained from rabbit vaccination and control samples was diluted 1:10 to prepare working sample serum. The appropriate dilution factor was added to the first row of wells in an ELISA plate, with a total volume of 200 μL. Serial dilutions of 2-fold were performed 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-rabbit antibody (1:2000 dilution) was added. The plates were incubated at room temperature for 4 hours. The plates were washed four times with wash buffer, and 100 μL of disodium 4-nitrobenzene phosphate substrate (Sigma-Aldrich) solution was added. The plates were read at 405 nm.

[0211] Table 1: Detection results of IgG antibody titers against 30 types of pneumococcal polysaccharides in rabbit antiserum of formulations A and B

[0212] The main difference between formulation A and formulation B lies in the different carrier proteins. Results of IgG antibody titer detection in rabbit immunized antiserum after the first (D14) and second (D28) doses of the pneumococcal capsular polysaccharide conjugate vaccine showed a significant upward trend in the titers of IgG antibodies against the corresponding 30 serotypes stimulated by the 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine (formulation A). The immunization effect of the second dose was significantly better than that of the first dose; that is, with the increase of immunization frequency, the antibody titer significantly increased. Furthermore, after the second immunization (D28), the titers of IgG antibodies against the corresponding serotypes of pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine (Formulation A) and PCV20 Pfizer (Formulation B) varied depending on the serotype. Specifically, for serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 23F, and 33F, the titers of IgG antibodies produced by Formulation A were significantly higher than those of Formulation B, accounting for 90% of the total titers. Only for two serotypes, 12F and 22F, were higher in Formulation B than in Formulation A, accounting for 10% of the total titers. This indicates that Formulation A, using RSV Pre-F recombinant protein as the carrier protein, has a better immunizing effect than Formulation B, which uses CRM197 as the carrier protein. This demonstrates that the RSV Pre-F recombinant protein carrier in the conjugate vaccine prepared in this invention has a good enhancing effect on the antigenicity of pneumococcal polysaccharides from 30 serotypes.

[0213] Meanwhile, compared to formulation B (PCV20), the antiserum IgG antibody titers (GM) of the 10 newly added serotypes Pn2, Pn6C, Pn9N, Pn15A, Pn17F, Pn20, Pn22A, Pn24F, Pn34, and Pn35B in the conjugate vaccine formulation A of this invention are all significantly higher than the average value (168.06) of the 20 polysaccharide IgG antibody titers in formulation B. This also demonstrates that the 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine prepared by this invention, while covering more valences of pneumococcal serotypes and expanding the vaccine's protective range, can also achieve better immunization effects.

[0214] Example 11: Detection of pre-F protein antibody titer in mouse immune serum from RSV recombinant protein and 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine

[0215] (1) ELISA detection of IgG antibody titer

[0216] Female BALB / c mice aged 4-6 weeks were randomly divided into two groups of 8 mice each. Each group was subcutaneously immunized every two weeks. One group was immunized with immunizing agent A (prepared in Example 8), and the other group was immunized with immunizing agent C (prepared in Example 8). Each immunization was 0.1 mL, and a total of two immunizations were performed.

[0217] 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.

[0218] Prepare a purified pre-F protein stock solution of 1 mg / mL (1×PBS solution) and store at 4°C. Dilute the 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 for 2 hours at room temperature, wash four times with wash buffer, and it can be stored at 4°C for one week.

[0219] Serum samples obtained from mouse vaccination and control samples were diluted 1:10 to prepare working sample serum. The appropriate dilution factor 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 washed four times with wash buffer, and 100 μL of disodium 4-nitrobenzene phosphate substrate solution was added. The plates were read at 405 nm.

[0220] (2) Detection of RSV A2 virus neutralizing titer

[0221] RSV type A virus was cultured in DMEM medium containing 10% bovine serum using Hep-2 cells. Hep-2 cells were seeded at a density of 20,000 cells / well and 100 μL of complete medium / well in 96-well plates. The cells were incubated at 37°C and 5% CO2 for 24 h until the cell density reached 60-80%. The medium was then replaced, the medium in the 96-well plates was aspirated, and maintenance medium was added at 100 μL / well. The serum was heat-inactivated and incubated at 56°C for 30 minutes. Serum was aliquoted into at least four tubes at 15 μL / tube and stored at -80°C. Antiserum was diluted in 96-well V-plates to an appropriate dilution ratio, resulting in 70 μL per well, with two replicates for each dilution. A mixture of virus and different dilutions of antiserum was prepared by diluting the virus to 100 TCID50 with DMEM and adding 70 μL / well to the aforementioned 96-well V-plates. Incubate at 37℃ for 1 hour; transfer 100 μL / well to the above Hep2 cell plate; culture at 37℃ and 5% CO2 for about 3-5 days; observe CPE status daily, and stain cells with 5% glutaraldehyde containing 0.25% crystal violet.

[0222] Table 2: Geometric mean (Eu) of IgG antibody titers in mouse serum immunized with RSV Pre-F protein and 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine

[0223] Table 2 shows that the geometric mean (Eu) of IgG antibody titers in mouse immune serum from the 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine was significantly higher than that from the RSV Pre-F recombinant protein. Specifically, the IgG antibody titers in the immune serum from the 30-valent conjugate (Pn-RSV Pre-F) after the first and second immunizations were approximately 1.6 and 2.4 times higher than those from RSV Pre-F (formulation C), respectively.

[0224] The results of the RSV A2 virus neutralizing titer assay showed that the mean neutralizing titer of the 30-valent conjugate (Pn-RSV Pre-F) mouse serum was 12600, significantly higher than that of RSV Pre-F mouse serum by approximately 1.9 times. This demonstrates that the 30-valent pneumococcal polysaccharide RSV recombinant protein conjugate vaccine prepared in this invention can produce serum with high protective titers after immunization of mice, and the mouse serum can generate high neutralizing antibody titers against the main circulating RSV strain A.

[0225] 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 protection scope of the claims of the present invention.

Claims

1. A multivalent pneumococcal polysaccharide-protein conjugate vaccine, characterized in that, The carrier protein in the conjugate vaccine is an immunogenic RSV recombinant protein; the pneumococcal polysaccharide is covalently linked to the RSV recombinant protein; the RSV recombinant protein is selected from Pre-F protein, and its amino acid sequence is shown in SEQ ID NO: 3; the pneumococcal polysaccharide is selected from at least two of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34, and 35B.

2. The multivalent pneumococcal polysaccharide-protein conjugate vaccine according to claim 1, characterized in that, The conjugate vaccine is a 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine, wherein the amino acid sequence of the RSV recombinant protein is shown in SEQ ID NO: 3, and the pneumococcal polysaccharide includes 30 serotypes, namely serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34 and 35B.

3. The multivalent pneumococcal polysaccharide-protein conjugate vaccine according to claim 1, characterized in that, The conjugate vaccine is formulated as an aqueous solution or a lyophilized solution.

4. The multivalent pneumococcal polysaccharide-protein conjugate vaccine according to claim 3, characterized in that, The conjugate vaccine contains an adjuvant.

5. The multivalent pneumococcal polysaccharide-protein conjugate vaccine according to claim 4, characterized in that, The adjuvant is any one of CpG, QS21, aluminum phosphate, a mixture of CpG and aluminum phosphate, or a mixture of QS21 and aluminum phosphate.

6. The method for preparing the multivalent pneumococcal polysaccharide-protein conjugate vaccine according to any one of claims 1-5, characterized in that, The preparation method includes the following steps: chemically synthesizing pneumococcal polysaccharide and RSV recombinant protein in a buffer or organic solvent, thereby covalently binding pneumococcal polysaccharide to RSV recombinant protein to obtain a conjugate.

7. The preparation method according to claim 6, characterized in that, The preparation method also includes pretreatment of pneumococcal polysaccharide, expression and purification of RSV recombinant protein, and purification of the conjugate.

8. The preparation method according to claim 7, characterized in that, The pretreatment of the pneumococcal polysaccharide includes degradation and activation, and the degradation method is selected from high pressure homogenization, acid hydrolysis or enzymatic digestion.

9. The preparation method according to claim 6, characterized in that, The organic solvent is selected from dimethyl sulfoxide or dimethylformamide.

10. The preparation method according to claim 6, characterized in that, The chemical synthesis reaction is selected from any one of the following: the amine reduction method, the 1-cyano-4-dimethylamino pyridinium tetrafluoroborate method, the adipic dihydrazide method, or the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride method.

11. A nucleic acid molecule encoding the RSV recombinant protein with the amino acid sequence shown in SEQ ID NO:

3.

12. A recombinant expression vector containing the nucleic acid molecule as described in claim 11.

13. A method for preparing RSV recombinant protein expression using the recombinant expression vector as described in claim 12, characterized in that, The expression method described uses a CHO cell expression system or an insect baculovirus expression system.

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