Pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine and preparation method therefor

Abstract

A pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine for preventing pneumococcal infection and RSV infection and a preparation method therefor. The pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine has an RSV recombinant protein that is an RSV Pre-F recombinant protein with enhanced stability due to amino acid mutation modification. The RSV recombinant protein has immunogenicity and is used as a carrier protein of pneumococcal polysaccharide. In the pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine, by means of the amino acid mutation modification on an RSV PreF protein carrier, the protein can still maintain the structural stability and an antigen cluster function in different environments, including high temperature, acidity, and high osmotic pressure. The conjugate vaccine has dual immunogenicity and can simultaneously effectively prevent diseases caused by streptococcus pneumoniae and RSV.

Description

Pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine and preparation method thereof Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine for preventing pneumococcal infection and RSV infection and a preparation method thereof. Background Art

[0002] Pneumococcus (Streptococcus pneumoniae) is a Gram-positive bacterium with an outer membrane encapsulated by a capsular polysaccharide. There are currently 91 distinct serotypes of pneumococcus, classified based on the chemical structure of the capsular polysaccharide and the availability of specific antisera. This bacterium can cause a variety of diseases, posing a serious threat to human health. Pneumonia, febrile bacteremia, and meningitis are the most common manifestations of invasive pneumococcal disease, while dissemination of the bacteria within the respiratory tract can lead to otitis media, sinusitis, or recurrent bronchitis. Compared to invasive disease, noninvasive disease is generally less severe but more common.

[0003] In Europe and the United States, pneumococcal pneumonia is the most common community-acquired bacterial pneumonia, affecting approximately 100 per 100,000 adults annually. Febrile bacteremia and meningitis affect approximately 15-19 per 100,000 adults and 1-2 per 100,000, respectively. The risk of developing one or more of these illnesses is much higher in infants, the elderly, and immunocompromised individuals of any age. Even in economically developed regions, the mortality rate from invasive pneumococcal disease is high; the mortality rate for adults with pneumococcal pneumonia is approximately 10-20%, while in high-risk groups, the mortality rate may exceed 50%. According to the World Health Organization (WHO), approximately 1.5 million people die each year from pneumococcal infection worldwide, including millions of children. Pneumonia is by far the most common cause of death among people infected with pneumococcus worldwide.

[0004] In China, the pneumonia incidence rate among people over 65 is 1.6% (approximately 1,600 per 100,000 adults). The incidence rate rises sharply with age, reaching 11.6% among those over 75. Not only is pneumonia more common in the elderly, but pneumonia infections often last a long time, are difficult to recover from, and can even lead to death in severe cases. Data show that 46%-76% of community-acquired pneumonia is caused by pneumococci.

[0005] Currently, preventive measures for pneumococcal infection primarily include vaccination and antibiotics. However, due to the widespread use of antibiotics, pneumococcal resistance to antibiotics has been increasing year by year, seriously compromising the effectiveness of antibiotics in treating pneumococcal infections. Therefore, the development of new vaccines has become an important approach to prevent pneumococcal infection.

[0006] There are two main types of pneumococcal vaccines currently on the market: pneumococcal polysaccharide vaccines and polysaccharide conjugate vaccines, such as the 23-valent pneumococcal polysaccharide vaccine (PPSV23) and the 13-valent pneumococcal conjugate vaccine (PCV13). Although current vaccine products for preventing pneumococcal infection have been developed in the direction of multivalent polysaccharide conjugate vaccines, PCV13 produced by different manufacturers also uses different carrier proteins. For example, the carrier protein used in Prevnar 13 produced by Pfizer in the United States is a non-toxic diphtheria toxoid mutant (CRM197), while the carrier protein used in Woanxin produced by Yunnan Watson is tetanus toxoid (TT). In addition, Weimin Feibao produced by Beijing Minhai uses a dual carrier protein of tetanus toxoid and diphtheria toxoid mutant. However, these conjugate vaccines have a common shortcoming, which is that the carrier protein does not confer immunogenic protective function; that is, although the conjugate vaccine carrier can stimulate the body to produce antibodies, the vaccine designers have not been able to use the antibodies produced by the carrier protein to prevent the disease. At the same time, these vaccines cannot cover all pneumococcal serotypes and require multiple booster immunizations after vaccination. Their protective effect still needs to be improved.

[0007] Respiratory Syncytial Virus (RSV) is a common virus that is primarily transmitted through the air and is the leading cause of lower respiratory tract infections in infants and young children. RSV infection can cause mild to severe respiratory illnesses, including bronchitis, pneumonia, and asthma attacks. RSV infection can lead to serious complications and even death, especially in premature infants, low-birth-weight infants, and children with weakened immune systems.

[0008] According to statistics, in 2019, RSV-related acute lower respiratory tract infections (ALRIs) accounted for 33 million cases worldwide, 3.6 million hospitalizations, and 26,300 deaths. Among infants aged 0-6 months, RSV-related ALRIs accounted for approximately 6.6 million cases, 1.4 million hospitalizations, and 13,300 in-hospital deaths, respectively. China is one of the countries with the highest number of children suffering from ALRIs due to RSV. Globally, 99% of deaths among children under 5 years of age hospitalized for RSV-related ALRIs occur in developing countries. RSV infection rates in adults increase with age, making the elderly a particularly susceptible group. The elderly also face a poorer prognosis and a greater economic burden. The highest in-hospital mortality rate is among those aged 65 and older, with approximately 72,000 deaths per 100,000 population annually from RSV infection. The RSV mortality rate in patients from developing countries reaches 9.1%.

[0009] RSV is an enveloped, nonsegmented, single-stranded, negative-sense RNA virus belonging to the order Monovirales, family Pneumoviridae, and genus Orthopneumovirus. Its genome consists of a single-stranded, negative-sense RNA molecule encoding 11 proteins, including nine structural proteins (three glycoproteins and six internal proteins) and two nonstructural proteins. Structural proteins include three transmembrane surface glycoproteins: the G attachment protein, the F fusion protein, and the small hydrophobic SH protein. There are two RSV subtypes, A and B, which differ primarily in the G glycoprotein, while the F glycoprotein sequence is more conserved between the two subtypes.

[0010] The RSV fusion protein (F protein) is a class I transmembrane protein composed of 574 amino acid residues. It is initially produced within host cells as the F protein precursor, F0. F0 is glycosylated at the Golgi apparatus and subsequently hydrolyzed by intracellular furin to release the 27-amino acid polypeptide pep27. Cleavage sites at the N- and C-termini of this peptide, respectively, produce two subunits, F1 and F2. The F2 subunit consists of the signal peptide SP and the heptad repeat sequence HRC. The F1 subunit consists of the fusion peptide FP, the heptad repeat regions HRA and HRB, the transmembrane regions Domain I and II TM, and the cytoplasmic domain CP. F1 and F2 are linked by disulfide bonds to form a heterodimer. Three heterodimers assemble into a mature F protein trimer. The RSV F protein trimer is unstable and exists in two conformations: prefusion and postfusion. 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 shift triggered by factors such as cellular receptors, temperature, and ion concentration, transforming into a highly stable post-F trimer. This process releases energy, mediating fusion of the viral envelope with the cell membrane. Because the pre-F conformation is highly unstable, the protein isolated and purified in vitro is typically post-F. However, research indicates that the metastable pre-F conformation is essential for virus-mediated membrane fusion and is a key antigen in inducing an immune response in humans.

[0011] RSV infection is associated with high infection rates, severe morbidity, and mortality, and there are no specific treatments for it. A major obstacle to vaccine development is the legacy of vaccine-enhanced disease from clinical trials using formalin-inactivated (FI) RSV vaccines in the 1960s. Children vaccinated with FI-RSV were not protected from natural infection and suffered more severe disease than unvaccinated children, including two deaths. This phenomenon is known as "enhanced disease." Since trials with FI-RSV vaccines, various approaches to developing RSV vaccines have been explored. Attempts have included classical live-attenuated, cold-passaged, or temperature-sensitive mutants of RSV, (chimeric) protein subunit vaccines, brain vaccines, and RSV proteins expressed by recombinant viral vectors, including adenovirus vectors. Although some of these vaccines have shown promising preclinical data, they still have many deficiencies in terms of protective efficacy, safety, and stability. Therefore, the development of new RSV vaccines is of great importance.

[0012] As one of the primary RSV antigens, the PreF protein possesses strong immunogenicity. However, the natural PreF protein is easily destroyed by conditions such as high temperature, high pressure, heavy metal ions, oxidants, and extreme pH, resulting in poor structural stability and difficulty maintaining its antigenicity. Therefore, further research is needed to understand the structure and function of the PreF protein. Studies have shown that amino acid mutations in the PreF protein can improve its immunogenicity and stability. However, obtaining stable and highly expressed PreF protein has become a major challenge in the development of RSV antibody drugs and vaccines.

[0013] In addition, how to obtain stable and highly expressed pre-F protein, and at the same time combine the stable and highly expressed PreF protein with other immunogenic substances, such as pneumococcal polysaccharide, to prepare a vaccine with dual immunogenicity and improve the protective effect of the vaccine, also has very important research and development value.

[0014] Summary of the Invention

[0015] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine for preventing pneumococcal infection and RSV infection and a preparation method thereof.

[0016] The technical solution of the present invention to solve the technical problem is as follows:

[0017] In a first aspect of the present invention, a RSV recombinant protein is provided, wherein the RSV recombinant protein is an RSV Pre-F recombinant protein with enhanced stability after amino acid mutation modification, wherein the amino acid mutation modification is any one of the following two methods:

[0018] (1) amino acid point mutations were performed on the full-length sequence of the wild-type pre-F protein as shown in SEQ ID NO. 1, such that I at position 28 was mutated to C and G at position 464 was mutated to C;

[0019] (2) First, the transmembrane region / intracellular region of the wild-type pre-F protein full-length sequence as shown in SEQ ID NO.1 is deleted, and the fibritin / Throm / 6his / Stretaq sequence is connected to its C-terminus to obtain a mutant with a sequence as shown in SEQ ID NO.2. Then, amino acid point mutations are performed on the basis of SEQ ID NO.2, mutating the I at position 28 to C and the G at position 464 to C. In this way, a RSV recombinant protein with an amino acid sequence as shown in SEQ ID No: 3 can be obtained.

[0020] In the second aspect of the present invention, two vaccines containing the RSV recombinant protein as described in the first aspect are provided.

[0021] The first vaccine is a vaccine that uses RSV recombinant protein as the sole immunogen and can be used to prevent RSV infection.

[0022] Furthermore, in the vaccine using RSV recombinant protein as the sole immunogen, the amino acid sequence of the RSV recombinant protein is shown in SEQ ID No: 3.

[0023] The second vaccine is a pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine using RSV recombinant protein as a carrier protein. The conjugate vaccine uses RSV recombinant protein as a carrier protein and includes two immunogens, pneumococcal polysaccharide and RSV recombinant protein. In other words, the RSV recombinant protein in the conjugate vaccine not only serves as a carrier protein for the conjugate vaccine, but also has immunogenicity and can be used as an immunogen for the conjugate vaccine. Therefore, the conjugate vaccine has dual immunogenicity and can be used to prevent pneumococcal infection and RSV infection.

[0024] Furthermore, in the pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine, the amino acid sequence of the RSV recombinant protein is shown in SEQ ID No: 3, and the pneumococcal capsular polysaccharide is selected from one or more of 24 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.

[0025] In a preferred embodiment of the present invention, the conjugate vaccine is a 24-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 capsular polysaccharide includes 24 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.

[0026] Furthermore, the pneumococcal capsular polysaccharide is linked to the RSV recombinant protein via a covalent bond.

[0027] Furthermore, the combination vaccine is in the form of an aqueous solution or a lyophilized preparation.

[0028] Furthermore, the combined vaccine contains an adjuvant.

[0029] 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.

[0030] In the third aspect of the present invention, a nucleotide sequence encoding the RSV recombinant protein as described in the first aspect is provided.

[0031] In the fourth aspect of the present invention, a recombinant expression vector containing the nucleotide sequence as described in the third aspect is provided.

[0032] The construction method of the recombinant expression vector is as follows:

[0033] 1) Synthesis of RSV Pre-F Target Gene The corresponding coding sequence (i.e., DNA sequence) was determined based on the amino acid sequence SEQ ID NO. 3. The restriction endonuclease sequence EcoRI was added to the C-terminus and the restriction endonuclease sequence XbaI was added to the N-terminus of the gene segment, and the designed nucleotide sequence was chemically synthesized.

[0034] 2) Plasmid amplification and target gene extraction

[0035] The pUC19 plasmid vector was double-digested with EcoRI and XbaI restriction enzymes, ligated with the synthesized gene, and introduced into the amplification host DH5α. Single colonies were screened using LB (Amp+) agar solid medium. Single colonies 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 MidiPrep Kit. The extracted plasmid was double-digested with EcoRI and XbaI restriction enzymes, and the target gene fragment was recovered using the TaKaRa MiniBest Agarose Gel Extraction Kit.

[0036] 3) Construction of eukaryotic expression vector

[0037] The mammalian cell expression plasmid pGN-M, which contains the CMV promoter and dihydrofolate reductase (DHFR) gene, was double-digested with EcoRI and XbaI restriction enzymes, and 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 via sticky ends and introduced into the DH5α amplification host. A single clone containing the eukaryotic expression plasmid pGN-M_preF was screened and obtained. The plasmid was inoculated in LB (Amp+) for amplification and culture, and the amplified plasmid was extracted using the TaKaRa MidiBEST Endo-free Plasmid Purification Kit and named RSV pre-F.

[0038] In the fifth aspect of the present invention, a method for preparing an RSV recombinant protein using the recombinant expression vector as described in the fourth aspect is provided, wherein the expression method adopts a CHO cell expression system or an insect baculovirus expression system.

[0039] In the sixth aspect of the present invention, a method for preparing the pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine as described in the second aspect is provided, comprising the following steps: reacting the pneumococcal capsular polysaccharide and RSV recombinant protein in a buffer or an organic solvent to obtain a conjugate by chemical synthesis.

[0040] Furthermore, the organic solvent is selected from dimethyl sulfoxide or dimethylformamide.

[0041] Furthermore, the chemical synthesis reaction is selected from one of the reductive amine method, the 1-cyano-4-dimethylamino pyridinium tetrafluoroborate method, the adipic acid dihydrazide method or the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride method.

[0042] Furthermore, the preparation method also includes pre-treatment of pneumococcal capsular polysaccharide, expression and purification of RSV recombinant protein, and purification of the conjugate.

[0043] Furthermore, the polysaccharide pretreatment includes degradation and activation, and the degradation method is selected from high-pressure homogenizer degradation, acid hydrolysis or enzyme digestion.

[0044] The present invention has the following technical effects:

[0045] 1) The present invention utilizes RSV Pre-F protein with enhanced stability obtained by amino acid mutation modification as a carrier protein to prepare a novel pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine. By performing amino acid mutation modification on the RSV PreF protein carrier, the vaccine can make the protein maintain its structural stability and antigen cluster function under different environments, including high temperature, acidity, and high osmotic pressure, and can still preserve its antigenicity even after undergoing chemical reactions. The conjugate vaccine of the present invention has dual immunogenicity. One vaccine can effectively prevent diseases caused by pneumococcus and RSV at the same time, further improving the protective effect and safety of the vaccine, greatly reducing the number of vaccinations, alleviating the pain of infants and the mental burden of parents, reducing the cost of immunization, and increasing the immunization coverage, providing a new means for preventing respiratory infections in infants and the elderly. The advantage of the present invention is that it can provide more efficient, safer, broader and more stable protection, and is expected to become an effective means for preventing pneumococcal and RSV infections.

[0046] 2) Using the modified RSV Pre-F protein as a conjugate vaccine carrier protein, a new type of pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine can be prepared, which can realize the production of effective protective antibodies by the protein carrier, thereby achieving the purpose of preventing two diseases simultaneously with one vaccine. As can be seen from Example 11, the pneumonia polysaccharide conjugate vaccine prepared using the RSV pre-F recombinant protein of the present invention as a carrier protein has a better antigenicity enhancement effect than the existing pneumonia polysaccharide conjugate vaccine (such as Pfizer PCV13) using other carrier proteins. As can be seen from Example 12, when the 24-valent pneumococcal polysaccharide RSV Pre-F recombinant protein conjugate vaccine prepared by the present invention is injected into mice, high protective titer serum can be obtained, and the serum neutralization titer is significantly higher than that of the serum of mice injected with the RSV Pre-F recombinant protein alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is an SDS-PAGE gel image of the purity test of the modified Pre-F protein.

[0048] FIG2 is a graph showing the temperature stability of the modified Pre-F protein.

[0049] FIG3 is a graph showing the pH stability of the modified Pre-F protein.

[0050] FIG4 is a graph showing the osmotic pressure stability of the modified Pre-F protein.

[0051] FIG5 shows the affinity test of the conjugates of pneumococcal serotypes 6A, 23F, 1, and 4 polysaccharide Pre-F protein and PA1 antibody. DETAILED DESCRIPTION

[0052] The technical solution provided by the present invention is further described in detail below with reference to the embodiments, but it should not be understood as limiting the scope of protection of the claims of the pending application.

[0053] Example 1: Preparation of RSV recombinant protein

[0054] (1) Protein construction

[0055] Term used in the present invention " wild-type " represents to exist in nature, without any modification or processed product artificially.Those skilled in the art understand that wild RSV F albumen can be multiple sequences, and there may be slight differences in these sequences, but biological activity is basically the same.The wild-type full-length F albumen mentioned among the present invention is with reference to the sequence that GenBank provides, and concrete sequence is (Fusion glycoprotein FOOS=Human respiratory syncytial virus A (strain A2) OX=11259GN=F PE=1SV=1) shown in SEQ ID NO.1.

[0056] (1) Amino acid mutation modification

[0057] The RSV Pre-F protein provided by the present invention, which is modified by amino acid mutation to obtain enhanced stability, can be mutated in any of the following two ways:

[0058] (1) Amino acid point mutations were performed on the full-length sequence of the wild-type pre-F protein;

[0059] (2) The transmembrane region / intracellular region in the full-length sequence of the wild-type pre-F protein was deleted, and the fibritin / Throm / 6his / Stretaq sequence was connected to its C-terminus to obtain the mutant sequence shown in SEQ ID NO.2. Amino acid point mutations were further performed on the basis of the SEQ ID NO.2 sequence.

[0060] In this embodiment, the second mutation method is used, in which the I at position 28 in the mutant shown in SEQ ID NO.2 is mutated to C, and the G at position 464 is mutated to C, to obtain a pre-F recombinant protein with an amino acid sequence shown in SEQ ID NO.3.

[0061] The amino acid sequence of SEQ ID NO.1-3 is as follows:

[0062] SEQ ID NO.1:

[0063] >sp|P03420|FUS_HRSVA Fusion glycoprotein F0 OS=Human respiratory syncytial virus A(strain A2)OX=11259GN=F PE=1SV=1

[0064] SEQ ID NO.2:

[0065] >RSVF

[0066] SEQ ID NO.3:

[0067] >RSV F I28C G464C

[0068] (2) Synthesis of RSV Pre-F target gene

[0069] The corresponding coding sequence (ie, DNA sequence) was determined based on the amino acid sequence SEQ ID NO.3, and the restriction endonuclease EcoRI sequence was added to the C-terminus of the gene in this segment, and the restriction endonuclease XbaI sequence was added to the N-terminus, and the designed nucleotide sequence was chemically synthesized.

[0070] (3) Plasmid amplification and target gene extraction

[0071] The pUC19 plasmid vector was double-digested with EcoRI and XbaI restriction enzymes, ligated with the synthesized gene, and introduced into the amplification host DH5α. Single colonies were screened using LB (Amp+) agar solid medium. Single colonies 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 MidiPrep Kit. The extracted plasmid was double-digested with EcoRI and XbaI restriction enzymes, and the target gene fragment was recovered using the TaKaRa MiniBest Agarose Gel Extraction Kit.

[0072] (4) Construction of eukaryotic expression vector

[0073] The mammalian cell expression plasmid pGN-M, which contains the CMV promoter and dihydrofolate reductase (DHFR) gene, was double-digested with EcoRI and XbaI restriction enzymes, and 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 via sticky ends and introduced into the DH5α amplification host. A single clone containing the eukaryotic expression plasmid pGN-M_preF was screened and obtained. The plasmid was inoculated in LB (Amp+) for amplification and culture, and the amplified plasmid was extracted using the TaKaRa MidiBEST Endo-free Plasmid Purification Kit and named RSV pre-F.

[0074] (II) Protein expression and cloning screening

[0075] CHO K1 cells purchased from ATCC were used as host cells. After cell recovery, they were cultured in DMEM medium (Sigma-Aldrich) supplemented with 10% newborn calf serum and passaged every 3 days. After passage 2, the cells were observed to be growing well, and then CHO K1 cells were plated at 0.75×10 6 Three 9.6cm cells / well 2Wells were filled with Iscove's optimized DMEM medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (IMEM+FBS) (Gibco). Cells were incubated in a humidified incubator with 5% CO₂ and 37°C. 4 μg of pcDNAVZVE vector was added to each well. DNA was mixed with Lipofectamine 2000 (Sigma-Aldrich) and added to two wells. Lipofectamine 2000 alone was added to the third well as a negative control. After 48 hours, the medium was removed, the cells were centrifuged at 200 × g for 5 minutes, and the supernatant was stored at -20°C. IMDM+FBS medium and 10 μg / mL blasticidin-HCl (Invitrogen) were added to one well of transfected cells. The other well was washed with PBS, and the cells were lysed with 50 mM Tris-HCl, pH 8, 150 mM NaCl, and 1% (v / v) Triton X-100 containing complete, EDA-free protease inhibitor cocktail (Roche Diagnostics). The cells were centrifuged at 16,000 × g for 10 minutes at 4°C, and the lysate was stored at -20°C. The supernatant and lysate were assayed for the presence of recombinant protein by Western blot. After 5 days of culture in selective medium, the cells were eluted with trypsin (Invitrogen) and seeded onto 9 cm Petri dishes. Serial dilutions were performed to isolate single clones. Over the next 7–11 days, 42 single clones were selected and transferred to wells of a 96-well plate. The culture supernatant was analyzed by Western blot to screen for highly expressed proteins. The clones secreting the highest amount of RSV pre-F protein were selected for the next round of screening, and the cells were finally expanded, and 30 new clones were selected and preserved.

[0076] The selected clones were expanded into three T175 flasks (NETS). Trypsin was added for digestion, washed with PBS, and resuspended in 100mL of ProCHO4 (Lonza) in a 250mL spinner flask, with 1×ProHT, 4mM L-glutamine and 2% FBS (Lonza). Culture was carried out in a humidified incubator at 37°C, 5% CO2, with a stirring speed of 90rpm and the lid slightly open to ensure air diffusion. Samples were taken daily, stained with trypan blue (Sigma-Aldrich), the cells were counted, and passaged every 3-5 days, when the viable cell concentration was higher than 0.3×106 cells / mL and the viable cell count exceeded 90% after the plateau period. When the cells adapted and grew well, BFS was gradually removed, and the cells were considered to be completely suitable for serum-free suspension growth.

[0077] (III) Production of RSV pre-F protein in bioreactors

[0078] A 1.5-liter perfusion culture was set up in a 3-liter bioreactor equipped with a 10 μm spin filter. Culture parameters were as follows: temperature maintained at 37°C using a heating blanket, pH adjusted to 6.9 with CO₂ or 0.3 M sodium hydroxide, agitation at 200-300 rpm, and dissolved oxygen (dO₂) maintained at 40% of saturated air using a mixture of N₂ and O₂ at a maximum flow rate of 200 mL / min. The perfusion rate was 0.3 to 0.8 dilutions / day, and culture fluid was sampled daily for cell counts. Supernatants were stained with trypan blue, and glucose and lactate concentrations were measured offline.

[0079] A total of 12.5 liters of cell-free culture fluid was collected, centrifuged at 8000×g for 30 minutes at 4°C, filtered through a 0.45 μm membrane, and then ultrafiltered and concentrated using a 10 kDa membrane pack. Ultrafiltration and filtration were performed with buffer and the sample solution volume was concentrated to 0.5 liters.

[0080] After adding 0.5 L of PBS, concentrate to 0.5 L. Repeat the above steps 5 times.

[0081] (IV) RSV pre-F protein purification

[0082] The sample solution was loaded onto a Q-Sepharose fast flow (GE Bioscience) column and washed with 20mM Tris-HCl, pH 7.5. The column was then washed with 20mM Tris-HCl, pH 7.5, supplemented with 200mM sodium chloride to further remove adsorbed protein impurities. The pre-F protein was eluted with a solution in which the sodium chloride concentration was increased to 300mM. Ammonium sulfate was added to the combined solution to a concentration of 800mM and loaded onto a Butyl-Sepharose (GE Bioscience) column. The column was then washed with phosphate buffered saline (PBS, 6mM Na2HPO4, 1.5mM KH2PO4, 0.15M sodium chloride, pH 6.8) supplemented with 800mM ammonium sulfate. Finally, the column was washed with PBS containing 400mM ammonium sulfate, and the pre-F protein was eluted with purified water. Finally, Sephacryl S-400HR (GE Bioscience) was loaded, the column was washed with PBS, the protein peak was collected, a cosolvent was added, the protein was lyophilized in a vacuum freeze dryer, and stored at -70°C until use.

[0083] The test results in FIG1 show that the purity of the RSV preF recombinant protein prepared by the present invention reaches 87.27% (SEC-280nm), and its reduction purity (SDS-PAGE) is above 95%, indicating that the RSV preF recombinant protein in the vaccine prepared by the present invention has high purity.

[0084] Example 2: Temperature stability test of the modified Pre-F protein obtained in Example 1

[0085] 1) Dilute the test protein (pre-modified Pre-F protein prepared in Reference Example 1 and post-modified Pre-F protein prepared in Example 1) to 20 μg / mL with 1*PBS pH 7.4 buffer and place in a 1.5 mL centrifuge tube to a total volume of 1 mL.

[0086] 2) Incubate at different temperatures according to the table below.

[0087]

[0088] 3) After incubation, each sample was temporarily stored at 4°C.

[0089] 4) Perform ELISA test according to the protocol:

[0090] Samples stored at 4°C were diluted to 1 μg / mL with 1% PBS, pH 7.4, and 100 μL / well of the plate was added to an ELISA plate (NUNC 442404). Coat the plate overnight at 4°C. Shake the plate dry and add 150 μL / well of 1% BSA-PBS. Incubate at 37°C for 2 hours. Wash the plate three times using a plate washer according to the protocol. Add the detection antibody (PA1) at 100 μL / well and incubate at 37°C for 2 hours. Wash the plate three times using a plate washer according to the protocol. Add AP-conjugated goat anti-human secondary antibody at a 1:2000 dilution at 100 μL / well and incubate at 37°C for 1 hour. Wash the plate three times using a plate washer according to the protocol. Add 100 μL / well of pNPP substrate solution. Read the plate on a microplate reader set to a wavelength of 405 nm.

[0091] The test results are shown in Figure 2, which show that the antigen-binding activity of the pre- and post-modification Pre-F proteins after treatment at different temperatures is significantly different. The OD values ​​of the modified Pre-F protein at 4°C, 50°C, and 70°C are significantly higher than those of the pre-modification protein, indicating that the modified Pre-F protein prepared by the present invention can still maintain a high antigen-binding activity after treatment at different temperatures, that is, the modified protein has good temperature stability.

[0092] Example 3: pH stability test of the modified Pre-F protein obtained in Example 1

[0093] 1) Liquid preparation

[0094] ①25 mM acetate buffer, pH 3.5.

[0095] Preparation method (calculated based on 100mL volume):

[0096] 1. Accurately weigh 98.953 mg of sodium acetate and add it to a 200 mL beaker.

[0097] 2. Accurately weigh 1.429 g of acetic acid and add it to a 5 mL centrifuge tube.

[0098] 3. Add about 80 mL of purified water to the sodium acetate to fully dissolve it, and then add acetic acid.

[0099] 4. Adjust the pH of the solution to 3.5.

[0100] 5. Dilute to 100 mL and store at room temperature. The shelf life is 3 months.

[0101] ②25mM acetate buffer, pH 5.0.

[0102] Preparation method (calculated based on 100mL volume):

[0103] 1. Accurately weigh 1.381 g of sodium acetate and add it to a 200 mL beaker.

[0104] 2. Accurately weigh 490.3 mg of acetic acid and add it to a 5 mL centrifuge tube.

[0105] 3. Add about 80 mL of purified water to the sodium acetate to fully dissolve it, and then add acetic acid.

[0106] 4. Adjust the pH of the solution to 5.0.

[0107] 5. Dilute to 100 mL and store at room temperature. The shelf life is 3 months.

[0108] ③25mM Tris-HCl buffer, pH8.0.

[0109] Preparation method (calculated based on 100mL volume):

[0110] 1. Add 80 mL of purified water to a 200 mL beaker.

[0111] 2. Accurately measure 302.85 mg of Tris and add.

[0112] 3. Take 1mL of 1N hydrochloric acid and add it (theoretical value 1.42mL), then add it in small amounts to adjust the pH of the solution to 8.0.

[0113] 4. Dilute to 100 mL and store at room temperature. The shelf life is 3 months.

[0114] ④25 mM Tris-HCl buffer, pH 10.0.

[0115] Preparation method (calculated based on 100mL volume):

[0116] 1. Add 80 mL of purified water to a 200 mL beaker.

[0117] 2. Accurately measure 302.85 mg of Tris and add.

[0118] 3. Take 20uL of 1N hydrochloric acid and add it (theoretical value 33uL), then add it in small amounts to adjust the pH value of the solution to 10.0.

[0119] 4. Dilute to 100 mL and store at room temperature. The shelf life is 3 months.

[0120] ⑤25mM PBS buffer control, pH 7.5.

[0121] Preparation method (calculated based on 100mL volume):

[0122] 1. Add 80 mL of purified water to a 200 mL beaker.

[0123] 2. Accurately measure 142 mg of Na2HPO4, 27 mg of KH2PO4, 800 mg of NaCl, and 20 mg of KCl and add them.

[0124] 3. Add a small amount of HCl to adjust the pH of the solution to 7.5.

[0125] 4. Dilute to 100 mL and store at room temperature. The shelf life is 3 months.

[0126] 2) The test proteins (pre-modified Pre-F protein prepared in Reference Example 1 and post-modified Pre-F protein prepared in Example 1, respectively) were diluted to 20 μg / mL using buffers of different pH values ​​and placed in 1.5 mL centrifuge tubes with a total volume of 1 mL.

[0127] 3) Incubate the samples at different pH values ​​according to the table below.

[0128]

[0129] 4) After incubation, neutralize the pH of each sample to 7.5 with acid or base (measured with pH paper) and store at 4°C.

[0130] Perform ELISA test according to the protocol (operation is the same as temperature stability test).

[0131] The test results are shown in Figure 3. The results show that the antigen-binding activity of the pre- and post-modification Pre-F proteins after treatment at different pH values ​​is significantly different. The OD values ​​of the modified Pre-F protein at pH 3.5, pH 5.0, pH 7.5, pH 8.0, and pH 10 are significantly higher than those of the pre-modification protein, indicating that the modified Pre-F protein prepared by the present invention can still maintain a high antigen-binding activity after treatment at different pH values, that is, the modified Pre-F protein has good pH stability.

[0132] Example 4: Osmotic Stability Test of the Modified Pre-F Protein Obtained in Example 1 1) Liquid Preparation

[0133] ①10mM Tris-HCl buffer, pH7.5.

[0134] Preparation method (calculated based on 25mL volume):

[0135] 1. Add 10 mL of 25 mM Tris-HCl buffer (pH 8.0) to a 50 mL centrifuge tube.

[0136] 2. Add purified water to a volume of 25 mL.

[0137] 3. Add 1N hydrochloric acid to adjust the pH of the solution to 7.5.

[0138] 4. Dilute to 25 mL and store at room temperature. The shelf life is 3 months.

[0139] ②80mM Tris-HCl buffer, pH7.5.

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

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

[0142] 2. Accurately measure 969.12 mg of Tris and add.

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

[0144] 4. Dilute to 100 mL and store at room temperature. The shelf life is 3 months.

[0145] ③1.5M MgCl2 solution preparation method (calculated based on 100mL volume):

[0146] 1. Accurately measure 20 mL of 3 M MgCl2 solution and add it to a 50 mL centrifuge tube.

[0147] 2. Add purified water to 20 mL and mix thoroughly.

[0148] 3. Store at room temperature, valid for 3 months.

[0149] ④3M MgCl2 solution

[0150] Preparation method (calculated based on 100mL volume):

[0151] 1. Accurately measure 60.99 g of magnesium chloride hexahydrate and add it to a 200 mL beaker.

[0152] 2. Add purified water to about 80 mL and stir to fully dissolve.

[0153] 3. Dilute to 100 mL and store at room temperature. The shelf life is 3 months.

[0154] ⑤150mM Tris-HCl buffer, pH7.5.

[0155] Preparation method (calculated based on 100mL volume):

[0156] 1. Add 80 mL of purified water to a 200 mL beaker.

[0157] 2. Accurately measure 1.815 mg of Tris and add.

[0158] 3. Add 1N hydrochloric acid to adjust the pH of the solution to 7.5.

[0159] 4. Dilute to 100 mL and store at room temperature. The shelf life is 3 months.

[0160] ⑥4M NaCl solution

[0161] Preparation method (calculated based on 125mL volume):

[0162] 1. Accurately weigh 29.22 g of sodium chloride and add it to a 200 mL beaker.

[0163] 2. Add 100 mL of purified water and stir thoroughly to dissolve.

[0164] 3. Dilute to 125 mL and store at room temperature. The shelf life is 3 months.

[0165] 2) Dilute the test proteins (pre-modified Pre-F protein prepared in Reference Example 1 and post-modified Pre-F protein prepared in Example 1) to 20 μg / mL using buffers with different osmotic pressure values ​​and place them in 1.5 mL centrifuge tubes with a total volume of 1 mL.

[0166] 3) Incubate the samples at different osmotic pressures according to the table below.

[0167]

[0168] 4) The 10mM and 80mM groups were diluted with Tris buffer, and the 1500mM and 3000mM groups were diluted with MgCl2 buffer.

[0169] 5) After incubation, the 10 mM and 80 mM groups were adjusted to 150 mM with 4 M NaCl buffer, and the 1500 mM and 3000 mM groups were diluted 10-fold and 20-fold with purified water, respectively, and stored at 4°C.

[0170] Perform ELISA test according to the protocol (the operation is the same as the temperature stability test).

[0171] The results are shown in Figure 4, which show that the antigen-binding activity of the Pre-F protein before and after modification after incubation at different osmotic pressures was significantly different. The OD values ​​of the modified Pre-F protein at 10 mM, 80 mM, 150 mM, 1500 mM, and 3000 mM were significantly higher than those of the unmodified protein, indicating that the modified Pre-F protein prepared by the present invention can still maintain a high antigen-binding activity after incubation at different osmotic pressures, that is, the modified protein has good stability to different osmotic pressures.

[0172] Example 5: Preparation 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 Capsular Polysaccharides

[0173] (1) Preparation of master seeds and working seeds

[0174] Capsular polysaccharide was purified 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 by the following method:

[0175] 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 strains from freeze-dried seed tubes were inoculated into 5 ml of yeast-acid hydrolyzed casein medium and cultured at 36°C ± 2°C for 18 h. When the bacteria grew to an OD600 reading of 1.0, the culture was transferred to 150 ml of fresh yeast-acid hydrolyzed casein medium and cultured at 36°C ± 2°C for 5-10 h until the exponential growth phase. The culture was then stopped, the cells were aliquoted and freeze-dried, and stored at 2-8°C as master seeds.

[0176] The strains of the master seed freeze-dried tubes 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°C ± 2°C for 18 hours. When the bacteria grew to an OD600 reading of 1.0, the culture solution was transferred to 150 mL of fresh yeast-acid hydrolyzed casein culture medium and cultured at 36°C ± 2°C for 8 hours until the exponential growth phase. The culture was stopped, the cells were aliquoted and freeze-dried, and stored at 4°C as working seeds of the serotype.

[0177] (2) Bacterial fermentation

[0178] Remove a seed tube from the working seed bank and inoculate it into 5 mL of yeast-acid hydrolyzed casein culture medium. Cultivate at 36°C ± 2°C until the bacterial growth phase reaches the mid-exponential phase. Transfer the bacterial solution into 150 mL of fresh yeast-acid hydrolyzed casein culture medium and incubate at 36°C ± 2°C for 5-10 hours until the exponential growth phase. Transfer 50 mL of the bacterial solution into 2 L of yeast-acid hydrolyzed casein culture medium and incubate at 36°C ± 2°C until the mid-exponential growth phase to prepare the fermentation seed solution. This fermentation seed solution is inoculated into a 50 L fermentor containing 30 L of yeast-acid hydrolyzed casein culture medium. Maintain the fermentation solution pH at 6.8 ± 0.2 with sodium hydroxide until the bacteria reach the late exponential phase.

[0179] (3) Capsular polysaccharide purification

[0180] 1. Centrifuge in a disc centrifuge at a speed of 9600 rpm, collect the supernatant, and discard the residue;

[0181] 2. Microfilter the centrifuge liquid with a microfiltration membrane to remove residual cell debris and insoluble small particles, and microfilter the fermentation centrifuge supernatant with a 0.22 μm membrane to collect the filtrate;

[0182] 3. Concentrate and diafiltration the microfiltrate to obtain a crude bacterial capsular polysaccharide solution, and then ultrafilter 15 sample volumes using a 30Kd membrane with buffer;

[0183] 4. Use a 50kD membrane package to further filter the polysaccharide solution for 10 sample volumes to concentrate the polysaccharide sample solution;

[0184] 5. Collect the purified polysaccharide solution into a freeze-drying bottle, freeze-dry it in a vacuum freeze dryer, and store it at -70°C.

[0185] Example 6: Preparation of Pneumococcal Serotype 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 Polysaccharide-RSV Recombinant Protein Conjugates (CDAP-CYS Method)

[0186] 1) Weigh 17.5 mg of purified capsular polysaccharide of the corresponding serotype and dissolve it in 4 mL of sodium phosphate buffer;

[0187] 2) Add 12 mg of 1-cyano-4-dimethylammonium pyridinium tetrafluoroborate (CDAP) (Sigma-Aldrich) to the polysaccharide solution, stir, and react at room temperature for 1 hour;

[0188] 3) Add 3Eqm cystamine and react at room temperature for 1 hour;

[0189] 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;

[0190] 5) adding 8 Eqm of 3(2-chloroethyl) phosphate to the polysaccharide solution to reduce the disulfide bonds in the polysaccharide;

[0191] 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 and dissolve it in phosphate buffer to a protein concentration of 10 mg / mL;

[0192] 8) Add 8 mg of bromoacetic acid N-hydroxysuccinimide ester (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.

[0193] 9) Mix 4 mL of the activated polysaccharide solution with 4 mL of the activated protein solution and react at room temperature for 4 hours;

[0194] 10) Add 4 Eq m of N-acetyl-L-cysteine ​​(Sigma-Aldrich) and react at 2-8° C. for 4 hours. Add 12 Eq m of iodoacetamide (Sigma-Aldrich) and react at 2-8° C. for 4 hours.

[0195] 11) Transfer the polysaccharide conjugate reaction solution to a dialysis bag and dialyze against phosphate buffer at 4°C;

[0196] 12) The sample solution was loaded onto Sepharose CL-4B, purified, and the bound product in the external water volume was collected;

[0197] 13) After filtering with a 0.22 μm filter membrane, store the product at 2-8°C.

[0198] Example 7: Preparation of Pneumococcal Serotype 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 Polysaccharide-RSV Recombinant Protein Conjugates (Reductive Amine Method (Aqueous Phase))

[0199] 1) Weigh 500 mg of purified capsular polysaccharide of the corresponding serotype and dissolve it in 500 mL of purified water;

[0200] 2) Degrade the product by high-pressure homogenization at 600 bar for three cycles, add 0.12 eqm sodium periodate (Sigma-Aldrich), protect from light, and react for 18 hours;

[0201] 3) Using an ultrafiltration filter with a molecular weight of 50Kd membrane, the purified water was ultrafiltered, concentrated, and freeze-dried;

[0202] 4) Weigh 18 mg of activated polysaccharide, add 4 mL of DMSO, and stir to dissolve completely;

[0203] 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;

[0204] 6) After adding 2 Eqm sodium borohydride (Sigma-Aldrich) to quench the reaction for 4 hours, the synthesis reaction solution was transferred to a dialysis bag and dialyzed against the buffer solution, changing the buffer four times;

[0205] 7) Load the sample solution onto Sepharose CL4B and collect the bound fraction in the void volume;

[0206] 8) After filtering with a 0.22 μm filter membrane, store at 4°C for preparation;

[0207] 9) Take samples to detect the molecular weight of the conjugate, the concentration and ratio of polysaccharide and protein.

[0208] Example 8: Affinity Detection of Pneumococcal Serotype 6A, 23F, 1, 4 Polysaccharide Pre-F Protein Conjugates with PA1 Antibody

[0209] 1) Pre-treat the 96-well plate by adding wash buffer (PBS) and incubate at 4°C overnight.

[0210] 2) Discard the liquid in the wells and wash with PBS three times, 5 minutes each time.

[0211] 3) Pneumococcal serotypes 6A, 23F, 1, and 4 polysaccharide Pre-F protein conjugates (prepared as described in Example 6, and also as described in Example 7) were diluted to appropriate concentrations and added to 96-well plates, 100 μL per well, and incubated at 37°C for 1 hour.

[0212] 4) Wash three times with washing buffer (PBS), 5 minutes each time.

[0213] 5) Add PA1 (Medimmune origin) antibody, 100 μL per well, and incubate at 37°C for 1 hour.

[0214] 6) Wash three times with washing buffer (PBS), 5 minutes each time.

[0215] 7) Add secondary antibody (AP-labeled goat anti-human), 100 μL per well, and incubate at 37°C for 1 hour.

[0216] 8) Wash three times with washing buffer (PBS), 5 minutes each time.

[0217] 9) Add TMB substrate solution, 100 μL per well, and incubate at 37°C for 15 minutes.

[0218] 10) Add 50 μL of stop solution (2 mol / L sulfuric acid) to each well, react at room temperature for 5 minutes, and then immediately measure the OD value.

[0219] The results are shown in Figure 5. Compared to the affinity of RSV Pre-F protein antibodies, the affinity of the four polysaccharide Pre-F protein conjugates to PA1 antibodies reached over 75% of the affinity of the Pre-F protein antibodies. The 6A-PreF protein conjugate had an affinity of 90.7%, the 23F-PreF protein conjugate had an affinity of 86.8%, and the 1-PreF protein conjugate had an affinity of 88.3%. This indicates that the polysaccharide Pre-F protein conjugates have good antibody affinity.

[0220] Example 9: 24-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine + CpG + Alum (Preparation A) and RSV recombinant protein conjugate vaccine + CpG + Alum (Preparation C)

[0221] The preparation method of preparation A is as follows:

[0222] To detect the polysaccharide concentration in the monovalent conjugate, an RSV recombinant protein conjugate solution (see Example 6 or 7) equivalent to 2.2 μg of polysaccharide (excluding 4.4 μg of Pn6B polysaccharide) was measured, 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, was placed in a sterile container. A sample was taken for protein detection, and the protein content was 39.2 μg. CpG (Genscript) was added to a final amount of 0.1 mg. Phosphate buffer, pH 5.8, was added and sterilized by filtration using a 0.22 μm membrane; sterile aluminum phosphate gel (Benetag) was added to a final aluminum ion amount of 0.125 mg. The solution was stirred at 4°C for 1 hour, aseptically aliquoted into 0.5 mL / bottle, and stored at 4°C for immunization.

[0223] The preparation method of preparation C is as follows:

[0224] The modified Pre-F protein obtained in Example 1 was sampled at a content of 5 μg, CpG (Genscript) was added to a final amount of 0.1 mg, phosphate buffer pH 5.8 buffer was added, and sterile filtration was performed with a 0.22 μm membrane; sterile aluminum phosphate gel (Benetag) was added to a final aluminum ion amount of 0.125 mg, stirred at 4°C for 1 hour, aseptically divided into 0.5 mL / bottle, and stored at 4°C for immunization.

[0225] Example 10: Immunization of rabbits with preparations and blood collection

[0226] Ten 2.5-3.5 kg New Zealand white rabbits were collected, divided into groups of five. One group received Immunization Preparation A (prepared in Example 9) and the other received Immunization Preparation B (Pfizer PCV13). Each rabbit was injected with 0.5 mL of the vaccine once at week 0 and week 2. Blood was collected three times: before week 0 immunization, one week after immunization, and one week after the second immunization. PBMCs were prepared from one portion of the blood, quickly frozen on dry ice, and stored at low temperatures. The remaining portion of the blood was allowed to stand at room temperature for 4 hours and then centrifuged at 10,000 rpm. The supernatant serum was collected and stored at -70°C for testing.

[0227] Example 11: ELISA detection of polysaccharide antibody titers in rabbit immune serum of the 24-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine of Example 9

[0228] Prepare pneumococcal polysaccharides of different serotypes (in 1× PBS) and store in a refrigerator at 4°C. Dilute the pneumococcal polysaccharide of the serotype to be tested to 4 μg / mL and coat the ELISA plate with 100 μL of coating solution per well. Incubate overnight at room temperature. Wash four times with plate wash buffer, add 100 μL of blocking buffer, incubate at room temperature for 2 hours, and wash four times with plate wash buffer. Store at 4°C for one week.

[0229] Serum obtained from rabbits injected with the vaccine and control samples was diluted 1:10 to prepare working samples. Appropriate dilutions were added to the first row of ELISA plates in a total volume of 200 μL. Two-fold serial dilutions were performed starting from the first row and proceeding downwards. The plates were incubated for 2 hours at room temperature. The plates were washed four times with wash buffer, and 100 μL of alkaline phosphatase-conjugated goat anti-rabbit antibody (1:2000 dilution) was added. The plates were incubated for 4 hours at room temperature. The plates were washed four times with wash buffer, and 100 μL of 4-nitrophenyl phosphate disodium salt substrate (Sigma-Aldrich) was added. The plate was read at 405 nm.

[0230] Table 1: IgG titer test results of rabbit immune antiserum against 24-valent pneumococcal polysaccharide-RSV pre-F recombinant protein conjugate vaccine

[0231] The primary difference between Formulations A and B lies in the carrier protein. IgG testing for polysaccharide and carrier protein antibodies showed no significant differences in polysaccharide antibody titers across serotypes before immunization. However, after the first dose, polysaccharide antibody titers for Formulations A (24-valent pneumococcal polysaccharide protein conjugate vaccine) and B (PCV13 Pfizer) stimulated animals to produce polysaccharide IgG titers that varied by serotype. For example, Formulation A produced higher titers for serotypes 1, 3, 4, 9V, 14, 18C, 19A, 19F, and 23F, accounting for 69% of the components. For serotypes 5, 6A, 6B, and 7F, Formulation B produced slightly higher titers than Formulation A, accounting for 31%. This suggests that Formulation A, using the RSV pre-F recombinant protein as a carrier protein, is superior to Formulation B, using the PCV13 Pfizer CRM197 as a carrier protein. After the second immunization, antibody IgG testing revealed a more significant difference between preparations A and B. Serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, and 23F were significantly higher in preparation A than in preparation B, accounting for 77% of the components. Serotypes 3 and 19F showed no significant difference, accounting for 15.3% of the components. Serotype 6A testing showed a slight increase in preparation B, accounting for 7.7% of the components. This suggests that using the RSV pre-F recombinant protein as a carrier protein is more effective in enhancing the antigenicity of the polysaccharide.

[0232] According to the antibody titer test results of the 24-valent pneumococcal polysaccharide RSV pre-F protein conjugate vaccine, the antibody titer increased significantly with the increase in the number of immunizations, and the IgG titer after the second immunization was significantly higher than the IgG titer after the first immunization.

[0233] Example 12: Detection of pre-F protein antibody titers in mouse immune sera from the RSV recombinant protein vaccine of Example 9 and the 24-valent pneumonia polysaccharide-RSV recombinant protein combined vaccine

[0234] (1) ELISA detection of IgG antibody titer (Eu)

[0235] Female BALB / c mice aged 4-6 weeks were randomly divided into groups of 8 and immunized subcutaneously once every two weeks with 0.1 mL of immunization preparation A and 0.1 mL of immunization preparation C (RSV Pre-F protein vaccine + CpG + Alum) in one group.

[0236] Blood was collected one week after immunization, three times before immunization at week 0, two weeks after immunization, and four weeks after the second immunization. One portion of the blood was prepared into PBMCs, snap-frozen on dry ice, and stored at low temperature. Another portion of the blood was allowed to stand at room temperature for 4 hours, centrifuged at 10,000 RPM at room temperature, and the supernatant serum was aspirated and stored at -70°C until testing.

[0237] Prepare a 1 mg / mL stock solution of purified pre-F protein in 1× PBS and store in a 4°C refrigerator. Dilute the protein stock solution to 4 μg / mL in coating buffer and coat the ELISA plate with 100 μL of coating solution per well. Incubate overnight at room temperature. Wash four times with plate wash buffer, add 100 μL of blocking buffer, incubate at room temperature for 2 hours, and wash four times with plate wash buffer. Store at 4°C for one week.

[0238] Serum obtained from mice injected with the vaccine and control samples was diluted 1:10 to prepare working samples. Appropriate dilutions were added to the first row of wells of an ELISA plate in a total volume of 200 μL. Two-fold serial dilutions were performed starting from the first row and proceeding downwards. The plates were incubated for 2 hours at room temperature. The plates were washed four times with wash buffer, and 100 μL of alkaline phosphatase-conjugated goat anti-mouse antibody (1:2000 dilution) was added. The plates were incubated for 4 hours at room temperature. The plates were washed four times with wash buffer, and 100 μL of 4-nitrophenyl phosphate disodium salt substrate solution was added. The plate was read at 405 nm.

[0239] (2) RSV A2 virus neutralization titer

[0240] RSV type A was cultured in Hep-2 cells in DMEM medium supplemented with 10% bovine serum. Eight aliquots of mouse sera from each group were diluted in DMEM medium supplemented with 2% bovine serum. Starting with a 40-fold dilution, the serum was diluted in a 3-fold series to 29-160 fold. The serum was then mixed with an equal volume of 200 TCID50 of virus solution and incubated at 37°C for 1 hour. 200 μl per well was plated onto Hep-2 cell plates, with three replicates per mouse serum. The serum was incubated at 37°C for 5-7 days and observed for cytopathic effects.

[0241] Table 2: Geometric mean (Eu) of IgG antibody titers in mice immunized with RSV Pre-F protein and 24-valent pneumococcal polysaccharide RSV Pre-F conjugate vaccine

[0242] Test results showed that the geometric mean (Eu) of IgG antibody titers in mouse sera immunized with the 24-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine was significantly higher than that of the RSV Pre-F recombinant protein vaccine. Specifically, IgG antibody titers in sera immunized with the 24-valent conjugate (Pn-RSV Pre-F) after both the first and second immunizations were significantly higher than those with the RSV Pre-F vaccine, by approximately 1.5 times.

[0243] The results of the RSV A2 virus neutralization titer test showed that the average neutralization titer of the 24-valent conjugate (Pn-RSV Pre-F) in mouse serum was 8303, which was significantly higher than the neutralization titer of RSV Pre-F mouse serum by about 1.3 times. This shows that the 24-valent pneumococcal polysaccharide RSV Pre-F recombinant protein conjugate vaccine prepared by the present invention can be injected into mice to obtain serum with high protective titers, and the mouse serum can produce high neutralizing antibody titers against the main prevalent RSV type A strain.

[0244] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than 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 the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be included within the scope of protection of the claims of the present invention to be approved.

Claims

1. An RSV recombinant protein, characterized in that, The RSV recombinant protein described above is an RSV Pre-F recombinant protein with enhanced stability through amino acid mutation modification, and the amino acid mutation modification is any one of the following two methods: (1) Based on the full-length wild-type pre-F protein sequence shown in SEQ ID NO.1, perform amino acid point mutations, mutate I at position 28 to C, and mutate G at position 464 to C; (2) First, delete the transmembrane region / intracellular region in the full-length wild-type pre-F protein sequence shown in SEQ ID NO.1, and connect the fibritin / Throm / 6his / Stretaq sequence to its C-terminus to obtain a mutant shown in SEQ ID NO.

2. Then, perform amino acid point mutations on the basis of the SEQ ID NO.2 sequence, mutate I at position 28 to C, and mutate G at position 464 to C.

2. A vaccine containing the RSV recombinant protein as described in claim 1, characterized in that, The vaccine described above is a vaccine with the RSV recombinant protein as the only immunogen.

3. The vaccine according to claim 2, wherein , The amino acid sequence of the RSV recombinant protein is shown in SEQ ID No:

3.

4. A pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine containing the RSV recombinant protein as described in claim 1, characterized in that, The conjugate vaccine described above uses the RSV recombinant protein as a carrier protein and includes two immunogens, pneumococcal polysaccharide and the RSV recombinant protein, and the RSV recombinant protein has immunogenicity.

5. The pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine according to claim 4, wherein , The amino acid sequence of the RSV recombinant protein is shown in SEQ ID No: 3, and the pneumococcal capsular polysaccharide is selected from one or more of 24 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.

6. The pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine according to claim 4, characterized in that , The conjugate vaccine is a 24-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine, in which the amino acid sequence of the RSV recombinant protein is shown in SEQ ID No: 3, and the pneumococcal capsular polysaccharide includes 24 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.

7. The pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine according to claim 4, wherein The pneumococcal capsular polysaccharide is covalently linked to the RSV recombinant protein.

8. The pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine according to claim 4, wherein The conjugate vaccine is in the form of an aqueous solution or a freeze-dried preparation.

9. The pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine according to claim 4, wherein, The conjugate vaccine contains an adjuvant.

10. The pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine according to claim 9, wherein 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.

11. The nucleotide sequence encoding the RSV recombinant protein as claimed in claim 1.

12. A recombinant expression vector containing the nucleotide sequence as claimed in claim 11.

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

14. The preparation method of the pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine according to any one of claims 4-10, characterized in that, It includes the following steps: The pneumococcal capsular polysaccharide and the RSV recombinant protein are conjugates obtained through a chemical synthesis reaction in a buffer solution or an organic solvent.

15. The preparation method according to claim 14, wherein, The organic solvent is selected from dimethyl sulfoxide or dimethylformamide.

16. The preparation method according to claim 14, wherein, The chemical synthesis reaction is selected from one of the reduction amination method, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate method, adipic dihydrazide method or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride method.

17. The preparation method according to claim 14, characterized in that, The preparation method further includes the pretreatment of pneumococcal capsular polysaccharide, the expression and purification of RSV recombinant protein, and the purification of the conjugate.

18. The preparation method according to claim 17, characterized in that, The polysaccharide pretreatment includes degradation and activation, and the degradation method is selected from high-pressure homogenizer degradation, acid hydrolysis method or enzymatic digestion method.

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