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

Abstract

The present invention relates to the field of biotechnology, and particularly relates to a multivalent pneumococcal polysaccharide-protein conjugate vaccine for preventing pneumococcal infection and respiratory syncytial virus (RSV) infection, and a preparation method and application thereof. In the multivalent pneumococcal polysaccharide-protein conjugate vaccine of the present invention, the carrier protein is an RSV recombinant protein with immunogenicity; the pneumococcal polysaccharide is covalently linked to the RSV recombinant protein; the RSV recombinant protein is selected from the Pre-F protein, and its amino acid sequence is as shown in SEQ ID NO: 2 or SEQ ID NO: 3. The conjugate vaccine of the present invention is an innovative vaccine, and can be used to simultaneously prevent two common diseases, namely pneumonia and RSV infection, by using one vaccine product, greatly reducing the number of vaccine inoculations. By mutating and modifying the carrier protein in the conjugate vaccine, the present invention can enable the carrier protein to maintain structural stability and antigenic cluster function under different environments.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a multivalent pneumococcal polysaccharide-protein conjugate vaccine for preventing pneumococcal infection and respiratory syncytial virus infection, and a preparation method and application thereof. Background Art

[0002] Streptococcus pneumoniae is a Gram-positive bacterium, and its outer membrane is wrapped by a layer of capsular polysaccharide. The capsule is its main pathogenic factor. According to the different chemical structures of different capsular polysaccharides and classified with specific antiserum, 91 different serotypes of Streptococcus pneumoniae have been discovered. Streptococcus pneumoniae is usually colonized in the nasopharynx and generally does not cause clinical symptoms. However, when the colonization environment changes, such as when the human immune system declines, suffering from other respiratory virus infections, malnutrition, or being old and weak, Streptococcus pneumoniae may break through the mucosal defense and is extremely likely to cause invasive infections. According to the infection site of Streptococcus pneumoniae, it can be divided 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 problem, posing a serious threat to human health, and the disease burden is becoming increasingly serious. According to statistics, in 2019, 33 bacterial infections globally caused 7.7 million deaths, among which Streptococcus pneumoniae ranked the 3rd, and Streptococcus pneumoniae infection caused 829,000 deaths. If converted into years of life lost, Streptococcus pneumoniae ranked the 1st, and the death cases caused by Streptococcus pneumoniae infection are mainly distributed among children under 5 years old and the elderly over 60 years old. For infants and young children, especially children under 2 years old, they are extremely susceptible to Streptococcus pneumoniae infection, and the fatality rate is extremely high. Approximately 75% of invasive pneumococcal diseases and 83% of pneumococcal meningitis occur in this age group (see the WHO position paper 2019 edition). Similarly, for the elderly, the incidence and mortality of diseases caused by invasive Streptococcus pneumoniae infection cannot be underestimated. It is reported that in China, pneumonia is the fourth leading cause of death among the elderly, with approximately 125,000 deaths per year. The pneumonia infection rate varies by region, ranging from 28.0% to 71.5%, and with the increase in age, the incidence of pneumonia in the elderly also rises sharply. At the same time, in the current era of the increasingly accelerating global population aging trend, the "World Social Report 2023" released by the United Nations pointed out that the global population aged 65 and above in 2021 was 761 million, and this figure will increase to 1.6 billion by 2050, and the population aged 80 and above is growing even faster. Thus, as the elderly are a high-risk group for Streptococcus pneumoniae infection, the aggravation of population aging will inevitably lead to an increasing burden of pneumococcal disease.

[0004] Pneumococcal vaccines are the most effective means of preventing pneumococcal infections. Currently, the vaccines available on the market are pneumococcal polysaccharide vaccine (PPV) and pneumococcal conjugate vaccine (PCV). In the 1980s, the 23-valent pneumococcal polysaccharide vaccine (PPV23) produced by Wyeth (acquired by Pfizer in 2009) achieved an immunization coverage rate of around 90% for the dominant serotypes globally. However, there are still limitations. Studies have found that bacterial polysaccharides are non-T cell-dependent antigens, which can induce short-term immune effects in children and adults, but are ineffective in infants, especially in children under 2 years old, where they cannot induce an effective immune response. Through development, covalently binding polysaccharides to carrier proteins can transform non-T cell-dependent antigens into T cell-dependent antigens. The pneumococcal polysaccharide-protein conjugate vaccine (PCV) prepared using this technology can be administered to infants starting at 2 months of age, promoting a stronger immune response and memory response, and is a favorable immunization method for preventing pneumococci faster and earlier. The world's first PCV approved by the FDA for market in 2000 was PCV7 (Prevnar 7) produced by Wyeth. In 2010, Pfizer launched PCV13 (Prevnar 13) that can cover more serotypes, replacing PCV7 (Prevnar 7) for routine childhood vaccination. Currently, there are 3 PCVs that have passed WHO prequalification, PCV10 (Synflorix) from GSK, PCV13 (Prevenar 13) from Pfizer, and PCV10 (PNEUMOSIL) from the Serum Institute of India. In 2021, the FDA successively approved the market launch of higher-valent PCVs, including PCV20 (Prevnar 20) from Pfizer and PCV15 (VAXNEUVANCE) from Merck. The pneumococcal polysaccharide conjugate vaccines available on the domestic market are PCV13 from Watson Bio and PCV13 from Minhai Bio. Studies have found that conjugate vaccines can be used in people of any age group, and the vaccination effect is also longer-lasting than that of polysaccharide vaccines. Therefore, polysaccharide-protein conjugate vaccines will become the main development trend of pneumococcal vaccines.

[0005] With the use of pneumococcal conjugate vaccines at home and abroad, the prevalent serotypes in different regions have changed significantly. The incidence of some non-vaccine-covered serotypes has increased markedly. Therefore, the development of higher-valent pneumococcal conjugate vaccines is particularly urgent. However, at the same time, there is a common drawback in the conjugate vaccines currently on the market, that is, the carrier protein is not endowed with the immunogenic protection function; that is to say, 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 diseases. The main reasons for widely using tetanus toxoid, diphtheria toxoid, and non-toxic mutant diphtheria toxin as carrier proteins are not because the antibodies they produce are protective, but rather considering their safety and ability to enhance the immunogenicity of polysaccharides in the conjugate. In addition, tetanus toxoid and diphtheria toxoid are already two components of the diphtheria, tetanus, and pertussis triple vaccine and have been used for routine vaccination. Therefore, whether the carrier protein in the pneumococcal conjugate vaccine can stimulate the body to produce protective antibodies is not important. Thus, it can be seen that while increasing the valency of pneumococcal vaccines and expanding the coverage range, the selection of carrier proteins is also a very important consideration factor.

[0006] Respiratory Syncytial Virus (RSV) is a common infectious virus, mainly transmitted through the air, and is the main pathogen of lower respiratory tract infections in infants and young children. RSV infection can cause mild to severe respiratory diseases, including bronchitis, pneumonia, asthma attacks, etc. Especially for premature infants, low birth weight infants, children with weakened immune systems, and the elderly, RSV infection may lead to serious complications and even death.

[0007] According to statistics, in 2019, the number of global RSV-related acute lower respiratory tract infections reached as high as 33 million person-times, and the number of hospitalizations and deaths caused by RSV-related acute lower respiratory tract infections were 3.6 million person-times and 26,300 cases respectively; among infants aged 0 - 6 months, the number of occurrences, hospitalizations, and in-hospital deaths caused by RSV-related acute lower respiratory tract infections were approximately 6.6 million person-times, 1.4 million person-times, and 13,300 cases respectively. Among them, China is one of the countries with a relatively large number of children suffering from acute lower respiratory tract infections caused by RSV, and 99% of the deaths among children under 5 years old hospitalized due to RSV acute lower respiratory tract infections globally come from developing countries. The RSV infection rate in adults increases with age, and the elderly are one of the susceptible populations. Moreover, the prognosis of elderly infected patients is poor and the economic burden is heavy, and the in-hospital fatality rate of those over 65 years old is the highest.

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

[0009] The RSV fusion protein (F protein) belongs to class I transmembrane proteins and is composed of 574 amino acid residues. It is initially generated as a precursor F0 of the F protein in host cells. The F0 protein is glycosylated in the Golgi apparatus and then proteolytically cleaved by intracellular furin protease to release a 27-amino acid polypeptide pep27. At the N- and C-terminal cleavage sites of this peptide segment, two subunits, F1 and F2, are generated. The F2 subunit consists of a signal peptide SP and a heptapeptide repeat sequence HRC. The F1 subunit consists of a fusion peptide FP, heptapeptide repeat regions HRA and HRB, Domain I, a transmembrane region TM of Domain II, and a 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 surface of the viral envelope is initially in a metastable pre-F conformation. When the virus adsorbs to the cell membrane surface, 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 can mediate the fusion of the viral envelope with the cell membrane. Since the conformation of pre-F is highly unstable, the protein isolated and purified in vitro is usually post-F. However, studies have shown that the metastable pre-F conformation is essential for the virus-mediated membrane fusion process and is also an important antigen that induces an immune response in the human body.

[0010] Before fusion, the F protein (i.e., pre-F protein), as one of the main antigens of respiratory syncytial virus, has good immunogenicity. However, due to the fact that the native respiratory syncytial virus pre-F protein is easily damaged under conditions such as high temperature, high pressure, and extreme pH, and its protein structure has poor stability, it is difficult to ensure its good antigenicity. Therefore, further research on the structure and function of the respiratory syncytial virus pre-F protein is needed. However, there are still many challenges in specifically designing and obtaining a highly stable and immunologically effective recombinant pre-F protein of respiratory syncytial virus, and currently there is no domestically produced RSV vaccine approved for marketing in China. Therefore, there is a need for an immunogen derived from the respiratory syncytial virus F protein, which has improved characteristics compared to the corresponding native respiratory syncytial virus F protein, to lay the foundation for the further development of related vaccines.

[0011] For products in the vaccine field, one of their characteristics is that a vaccine can prevent the infection of specific pathogens. For example, the pneumococcal polysaccharide conjugate vaccine can prevent pneumonia, meningitis, and otitis media caused by pneumococcal infection, and the RSV vaccine can prevent respiratory diseases caused by respiratory syncytial virus infection. In order to achieve the prevention of infections by multiple pathogens with one preparation, the current approach is to use combination vaccines, such as diphtheria, pertussis, and tetanus triple vaccine for children, diphtheria, pertussis, tetanus, poliomyelitis, and Haemophilus influenzae type b pentavalent vaccine for children, diphtheria, pertussis, tetanus, poliomyelitis, Haemophilus influenzae type b, and hepatitis B hexavalent vaccine for children, etc. These vaccines are prepared by mixing single-product vaccines. From the current clinical evaluation, the preventive effects of some single vaccines in the combination vaccines are not good.

[0012] Currently, school-age children in China need to be vaccinated with at least 15 vaccines, and the number of vaccination times far exceeds the number of vaccine types. And with the intensification of population aging, as of the end of 2024, the elderly population aged 60 and above in China has reached 310 million, accounting for 22% of the total national population. Therefore, protecting the health of the elderly and developing vaccine products for the elderly cannot be ignored. Thus, it is inevitable that developing multi-valent conjugate vaccines to replace single-vaccine products and achieving full coverage of the vaccinated population will be the primary task in the future vaccine research and development. However, with the increase in the number of vaccine valences, how to select the carrier protein in the polysaccharide-protein conjugate vaccine to avoid the carrier inhibition effect, how to design and obtain a stable and highly expressed carrier protein, and at the same time combine the immunogenic carrier protein with other immunogenic substances (such as pneumococcal capsular polysaccharide) to prepare a vaccine with dual immunogenicity and achieve the goal of preventing two diseases with one vaccine are all the key challenges faced in the development of polysaccharide-protein conjugate vaccines. Summary of the Invention

[0013] To overcome the deficiencies in the prior art, the present invention utilizes the polysaccharide-protein conjugation technology to prepare a novel multivalent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine by using the RSV Pre-F protein with enhanced stability obtained through amino acid mutation modification as the carrier protein. This conjugate vaccine has dual immunogenicity and can effectively prevent diseases caused by the infection of 30 serotypes of pneumococcus and respiratory syncytial virus with a single vaccine, greatly reducing the number of vaccine inoculations, alleviating the pain of infants and young children and the mental burden of parents, reducing the cost of immunization, increasing the immunization coverage rate, and providing a new means for preventing respiratory disease infections in infants and the elderly.

[0014] The technical solution for the present invention to solve the technical problems is as follows:

[0015] In the first aspect of the present invention, a multivalent pneumococcal polysaccharide-protein conjugate vaccine is provided. 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 the Pre-F protein, and its amino acid sequence is as shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0016] Furthermore, the pneumococcal polysaccharide in the conjugate vaccine is selected from one or more 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.

[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 as 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 as shown in SEQ ID NO: 3, and the pneumococcal polysaccharide includes 30 serotypes, namely serotype 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 in the form of an aqueous solution or a freeze-dried preparation.

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

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

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

[0025] Furthermore, the chemical synthesis reaction is any one of the reductive amination method, the 1-cyano-4-dimethylaminopyridinium tetrafluoroborate method, the adipic dihydrazide method, or the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride method.

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

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

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

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

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

[0031] (2) By mutating and modifying the amino acids of the carrier protein, i.e., the RSV Pre-F protein, in the conjugate vaccine of the present invention, the carrier protein can maintain its structural stability and antigenic cluster function under different environments, including high temperature, acidic conditions, 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 has an amino acid sequence innovatively designed by genetic recombination technology. After mutating and modifying the amino acids, the RSV Pre-F protein carrier with enhanced stability, in combination with a novel adjuvant formulation and immunizing animals, the polysaccharide antigen part of the conjugate can stimulate the body to produce specific serotype polysaccharide antibodies, while the carrier protein part can stimulate the body to produce protective antibodies against the RSV virus, thereby achieving the prevention of infections by 30 serotypes of pneumococcus and RSV after immunization with a single vaccine.

[0033] (4)The 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine prepared by the present invention has a synergistic technical effect: on the one hand, as can be seen from Example 10, compared with the conjugate vaccine with CRM197 as the carrier protein of Pfizer PCV20, the conjugate vaccine prepared by the present invention uses the RSV recombinant protein modified by amino acid mutation as the carrier protein, and has a better antigenicity enhancement effect on 30 serotype polysaccharides. Among them, after the second immunization, the IgG titers of the corresponding serotype polysaccharide antibodies induced by the 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine (Formulation A) and PCV20 Pfizer (Formulation B) of the present invention vary according to the serotype. The data of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 23F and 33F show that Formulation A is significantly higher than Formulation B, accounting for 90% of the components; only the test results of two serotypes 12F and 22F are that Formulation B is 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 value of the mouse immune serum of the 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine prepared by the present invention is significantly higher than that of the RSV Pre-F recombinant protein. Among them, the IgG antibody titers in the immune sera of the 30-valent conjugate (Pn-RSV Pre-F) were about 1.6 and 2.4 times higher than those of RSV Pre-F (Formulation C) respectively after the first and second immunizations. At the same time, the test results of the RSV A2 virus neutralization titer also show that the average value of the mouse serum neutralization titer of the 30-valent conjugate (Pn-RSV Pre-F) is 12600, which is about 1.9 times higher than the mouse serum neutralization titer of RSV Pre-F. This shows that the 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine prepared by the present invention can obtain a serum with a high protective titer, and can produce a relatively high neutralizing antibody titer against the main epidemic strain A of RSV.

[0035] (5)The 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine prepared by the present invention can cover more serotypes of pneumococcus, expand the protection range of the vaccine, and at the same time achieve better immune effects. As can be seen from Example 10, compared with PCV20 (20-valent pneumococcal conjugate vaccine), the GM test values of the IgG antibody titers of the antisera of the 10 newly added serotypes Pn2, Pn6C, Pn9N, Pn15A, Pn17F, Pn20, Pn22A, Pn24F, Pn34 and Pn35B in the conjugate vaccine of the present invention are all significantly higher than the average value (168.06) of the IgG antibody titers of the 20 serotype polysaccharides of PCV20. Description of the Drawings

[0036] Figure 1 Results of detecting the temperature stability of the Pre-F protein before and after modification.

[0037] Figure 2 Results of detecting the pH stability of the Pre-F protein before and after modification.

[0038] Figure 3 Results of detecting the osmotic pressure stability of the Pre-F protein before and after modification. Specific implementation manners

[0039] In order to more concisely and clearly demonstrate the technical solutions, objectives, and advantages of the present invention, the technical solutions of the present invention will be described in detail below in conjunction with specific embodiments and the accompanying drawings. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the instruments, equipment, reagents, materials, etc. used, unless otherwise specified, can all be obtained through conventional commercial means.

[0040] Example 1: Preparation of carrier protein

[0041] (1) Construction of protein

[0042] The term "wild type" used in the present invention refers to a product that exists in nature and has not been modified or processed artificially. Those skilled in the art understand that the wild RSV F protein can be of multiple sequences, and there may be slight differences in these sequences, but the biological activities are basically the same. The wild-type full-length F protein mentioned in the present invention refers to the sequence provided by GenBank, and the specific sequence is shown in SEQ ID NO.1 (Fusion glycoprotein F0 OS=Human respiratory syncytial virus A (strain A2) OX=11259 GN=F PE=1 SV=1).

[0043] (1) Amino acid sequence design

[0044] The present invention relates to an RSV Pre-F recombinant protein with enhanced stability obtained by amino acid mutation modification, and its amino acid sequence design method is as follows:

[0045] Delete the transmembrane region and intracellular region in the full-length sequence of the wild-type RSV pre-F protein, connect the fibritin / Throm / 6his / Stretaq sequence to its C-terminus, and then perform amino acid point mutations on this basis. The specific mutation method is: mutate the G at the 242nd position of the amino acid sequence of the wild-type pre-fusion F protein to C, and mutate the M at the 289th position to C to obtain a full-length mutant of the F protein, and its amino acid sequence is shown in SEQ ID NO.3, and this sequence is the RSV modified Pre-F protein sequence.

[0046] The present invention also relates to a pre-F recombinant protein before RSV modification, and its amino acid sequence is designed as follows:

[0047] Delete the transmembrane region and intracellular region in the full-length sequence of the wild-type RSV pre-F protein, and connect the fibritin / Throm / 6his / Stretaq sequence to its C-terminus to obtain a variant sequence as shown in SEQ ID NO. 2, and this sequence is the RSV pre-F protein sequence before modification.

[0048] The sequences of SEQ ID NO.1 - SEQ ID NO.3 are shown as follows:

[0049] SEQ ID NO.1:

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

[0051] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLS LIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN。

[0052] SEQ ID NO.2:

[0053] >RSV F

[0054] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK。

[0055] SEQ ID NO.3:

[0056] >RSV F G242C M289C

[0057] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNACVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSICSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK。

[0058] (2)Synthesis of the RSV Pre-F target gene

[0059] According to the RSV protein amino acid sequences SEQ ID NO.2, SEQ ID NO.3 designed above and the codon preference of the host cell, determine the corresponding gene coding sequence, add the restriction endonuclease XbaI sequence to the 5' end of the gene in this section, add the restriction endonuclease EcoRI sequence to the 3' end, and chemically synthesize the designed nucleotide sequence.

[0060] (3)Plasmid amplification and extraction of the target gene

[0061] The pUC19 plasmid vector was digested with XbaI and EcoRI restriction enzymes and ligated with the synthesized gene, then introduced into the amplification host DH5α, and monoclonal colonies were screened using LB (Amp+) solid agar medium; the monoclonal colonies containing the target gene were inoculated into LB (Amp+) liquid medium and cultured and amplified at 37°C with 200 rpm, and the plasmid pUC19-preF was extracted using the Sigma-Aldrich GenEluteTM HP Plasmid Midiprep Kit; the extracted plasmid was digested with XbaI and EcoRI restriction enzymes, and the target gene fragment was recovered using the TaKaRa MiniBest Agarose Gel Extraction Kit.

[0062] (4)Construction of eukaryotic expression vector

[0063] The mammalian cell expression plasmid pGN-M, which contains the CMV promoter and the dihydrofolate reductase (DHFR) gene, was digested with XbaI and EcoRI 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 by sticky ends and introduced into the DH5α amplification host, and monoclonal colonies containing the eukaryotic expression plasmid pGN-M_preF were screened; inoculated into LB (Amp+) for amplification culture, and the amplified plasmid was extracted using the endotoxin-free plasmid midiprep kit TaKaRa MidiBEST Endo-free Plasmid Purification Kit.

[0064] (II)Protein expression and clone screening

[0065] Using CHO K1 (ATCC) cells as host cells, after cell resuscitation, they were cultured in DMEM medium (Sigma-Aldrich) with 10% newborn bovine serum and passaged once every 3 days. After 2 passages, observing that the cell growth was good, then the CHO K1 cells were seeded at 0.75×10 6 cells / well in three 9.6 cm 2In the wells, Iscove's Modified DMEM Medium (Sigma-Aldrich) was added, along with 10% fetal bovine serum (IMEM+FBS) (Gibco). The cells were placed in a humidified incubator at 5% CO2 and 37°C. 4 μg of the pcDNA vector was added to each well. The DNA was mixed with Lipofectamine 2000 (Sigma-Aldrich) and added to two of the wells, while Lipofectamine 2000 alone was added to the 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 centrifuged supernatant was stored at -20°C. IMDM+FBS culture medium and 10 μg / mL of Blasticidin-HCl (Invitrogen) were added to one well of the transfected cells. The other well of transfected cells was washed with PBS, and then the cells were lysed with a mixture of 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% (v / v) Triton X-100 containing complete, EDA-free protease inhibitors (Roche Diagnostics). The lysate was centrifuged at 16,000×g for 10 minutes at 4°C and stored at -20°C. Western blot was used to detect the presence of recombinant protein in the supernatant and the lysate. After 5 days of culture in selective medium, the cells were detached with trypsin (Invitrogen) and then seeded onto a 9-cm Petri dish for serial dilution to isolate monoclonal cells. In the following 7 - 11 days, 42 single clones were picked and transferred to the wells of a 96-well plate. Western blot was used to detect the culture supernatant to screen for high-expressing proteins. The clone secreting the highest amount of RSV pre-F protein was subjected to the next round of screening. Finally, the cells were amplified and 30 new clones were screened and preserved.

[0066] The selected clones were amplified in three T175 flasks (NETS). Trypsin digestion was added, the cells were washed with PBS, and then resuspended in 100 mL of ProCHO4 (Lonza) in a 250-mL spinner flask, supplemented with 1×ProHT, 4 mM L-glutamine, and 2% FBS (Lonza). The cells were cultured in a humidified incubator at 37°C, 5% CO2, with a stirring speed of 90 rpm, and the lid was slightly opened to ensure air diffusion. Samples were taken daily, stained with trypan blue (Sigma-Aldrich), the cells were counted, and subcultured every 3 - 5 days when the viable cell concentration was higher than 0.3×10 6 cells / mL and after the plateau phase when the viable cell count exceeded 90%. When the cells were adapted and growing well, BFS was gradually removed, and at this point, the cells were considered fully suitable for serum-free suspension growth.

[0067] (III) Production of RSV Protein in a Bioreactor

[0068] Configure a 1.5-liter perfusion culture in a bioreactor and configure a rotary filter (10 μm) separator. The culture parameters are set as follows: control the temperature at 37 °C with a heating blanket, adjust the pH to 6.9 with CO2 or 0.3 M sodium hydroxide, the stirring speed is 200 - 300 RPM, and adjust the dissolved oxygen (dO2) to 40% of saturated air with a mixed gas of N2 and O2 with a maximum flow rate of 200 mL / min. The perfusion rate is 0.3 to 0.8 V dilution / day, and samples are taken from the culture medium every day for cell counting. Use trypan blue staining and offline detection of the glucose and lactate concentrations in the supernatant.

[0069] A total of 12.5 liters of cell-free culture medium is collected, centrifuged at 8000×g for 30 minutes at 4 °C, filtered through a 0.45 μm membrane, and then ultrafiltered and concentrated with a 10 kDa membrane package. Ultrafiltration washing is performed with a buffer, and the volume of the sample solution is concentrated to 0.5 liters. After adding 0.5 liters of PBS, it is further concentrated to 0.5 liters. The above steps are repeated 5 times.

[0070] (IV) Purification of RSV Protein

[0071] Load the sample solution onto a Q-Sepharose fast flow (GE Bioscience) column and wash the column with 20 mM Tris-HCl pH 7.5. Then wash the column with 20 mM Tris-HCl pH 7.5 containing 200 mM sodium chloride to further remove adsorbed protein impurities. Elute the pre-F protein with a solution with the sodium chloride solution concentration increased to 300 mM. Add ammonium sulfate to the combined solution to a concentration of 800 mM, load it onto a Butyl-Sepharose (GE Bioscience) column, wash the column with phosphate buffer (PBS, 6 mM Na2HPO4, 1.5 mM KH2PO4, 0.15 M sodium chloride pH 6.8) containing 800 mM ammonium sulfate, wash the column with PBS solution containing 400 mM ammonium sulfate, and finally elute the pre-F protein with purified water. Finally, load it onto Sephacryl S-400HR (GE Bioscience), wash the column with PBS, collect the protein peak, add a cosolvent, freeze-dry it on a vacuum freeze dryer, and store it at -70 °C for later use.

[0072] Both the pre-F protein before RSV modification and the pre-F protein after RSV modification are prepared separately by the above method.

[0073] Example 2: Detection of the temperature stability of the pre-F protein before and after modification obtained in Example 1

[0074] 1) Dilute the proteins to be tested (the pre-modification Pre-F protein prepared in Reference Example 1 and the post-modification Pre-F protein prepared in Example 1 respectively) with 1*PBS pH7.4 buffer to 20 ug / ml, place them in 1.5 mL centrifuge tubes, and the total volume is 1 mL.

[0075] 2) Incubate at different temperatures according to the following table:

[0076]

[0077] 3) After incubation, store each sample at 4°C for temporary storage.

[0078] 4) Perform ELISA test according to the protocol:

[0079] Dilute the samples stored at 4°C with 1*PBS pH7.4 to 1 ug / mL, add 100 uL / well to the ELISA plate (NUNC442404), and coat overnight at 4°C. Spin dry the ELISA plate, add 1% BSA-PBS at 150 uL / well, and incubate at 37°C for 2 hours. Wash the plate 3 times according to the program of the plate washer, add the detection antibody PA1 (Medimmune origion) at 100 uL / well according to the design, and incubate at 37°C for 2 hours. Wash the plate 3 times according to the program of the plate washer, add the AP-labeled goat anti-human secondary antibody diluted at 1:2000 at 100 uL / well, and incubate at 37°C for 1 hour. Wash the plate 3 times according to the program of the plate washer, add the pNPP substrate solution at 100 uL / well, set the wavelength of the microplate reader to 405 nm, and read the value.

[0080] The test results are as Figure 1 shown. The results show that there are significant differences in the antigen-binding activities of the pre- and post-modification Pre-F proteins after treatment at different temperatures. The detection values of the post-modification Pre-F protein at 4°C, 50°C, and 70°C are significantly higher than those of the pre-modification protein, indicating that the post-modification Pre-F protein prepared by the present invention can still maintain a high antigen-binding activity after treatment at different temperatures, that is, compared with the pre-modification Pre-F protein, the temperature stability of the post-modification Pre-F protein prepared by the present invention is significantly enhanced.

[0081] Example 3: Detection of pH stability of pre- and post-modification Pre-F proteins obtained in Example 1

[0082] 1) Solution preparation

[0083] ① 25 mM acetate buffer, pH3.5.

[0084] Preparation method (calculated based on the preparation of 100 mL volume):

[0085]

[0086] 1. Weigh accurately 98.953 mg of sodium acetate and add it to a 200 mL beaker.

[0087] 2. Weigh accurately 1.429 g of acetic acid and add it to a 5 mL centrifuge tube.

[0088] 3. Add approximately 80 mL of purified water to the sodium acetate and dissolve it thoroughly, then add the acetic acid.

[0089] 4. Adjust the pH of the solution to 3.5.

[0090] 5. Make up the volume to 100 mL, store at room temperature, and the validity period is 3 months.

[0091] ② 25 mM acetate buffer, pH 5.0.

[0092] Preparation method (calculated based on a 100 mL volume):

[0093]

[0094] 1. Weigh accurately 1.381 g of sodium acetate and add it to a 200 mL beaker.

[0095] 2. Weigh accurately 490.3 mg of acetic acid and add it to a 5 mL centrifuge tube.

[0096] 3. Add approximately 80 mL of purified water to the sodium acetate and dissolve it thoroughly, then add the acetic acid.

[0097] 4. Adjust the pH of the solution to 5.0.

[0098] 5. Make up the volume to 100 mL, store at room temperature, and the validity period is 3 months.

[0099] ③ 25 mM Tris-HCl buffer, pH 8.0.

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

[0101]

[0102] 1. Add 80 mL of purified water to a 200 mL beaker.

[0103] 2. Measure accurately 302.85 mg of Tris and add it.

[0104] 3. Take 1 mL of 1 N hydrochloric acid and add it (the theoretical value is 1.42 mL), then add it drop by drop and adjust the pH value of the solution to 8.0.

[0105] 4. Make up the volume to 100 mL, store at room temperature, and the validity period is 3 months.

[0106] ④ 25 mM Tris-HCl buffer, pH 10.0.

[0107] Preparation method (calculated based on a 100 mL volume):

[0108]

[0109] 1. Add 80 mL of purified water to a 200 mL beaker.

[0110] 2. Accurately measure and add 302.85 mg of Tris.

[0111] 3. Add 20 μL of 1 N hydrochloric acid (the theoretical value is 33 μL), and then add it drop by drop to adjust the pH of the solution to 10.0.

[0112] 4. Make up the volume to 100 mL, store at room temperature, and the validity period is 3 months.

[0113] ⑤ 25 mM PBS buffer control, pH 7.5.

[0114] Preparation method (calculated based on a 100 mL volume):

[0115]

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

[0117] 2. Accurately measure and add 142 mg of Na2HPO4, 27 mg of KH2PO4, 800 mg of NaCl, and 20 mg of KCl.

[0118] 3. Add a small amount of HCl drop by drop to adjust the pH of the solution to 7.5.

[0119] 4. Make up the volume to 100 mL, store at room temperature, and the validity period is 3 months.

[0120] 2) Dilute the proteins to be tested (the unmodified Pre-F protein prepared in Reference Example 1 and the modified Pre-F protein prepared in Example 1, respectively) to 20 μg / mL with buffers of different pH values, and place them in 1.5 mL centrifuge tubes with a total volume of 1 mL.

[0121] 3) Incubate the samples at different pH values according to the following table:

[0122]

[0123] 4) After the incubation, neutralize the pH of each sample to 7.5 with acid or base (measured with pH test paper), and place it at 4°C for temporary storage.

[0124] Perform ELISA test according to the protocol (operation is the same as temperature stability test).

[0125] Test results such as Figure 2 As shown, the results show that the antigen-binding activity of the Pre-F protein before and after modification after treatment at different pH values is significantly different. 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 protein before modification, 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, compared with the Pre-F protein before modification, the pH stability of the modified Pre-F protein prepared by the present invention is significantly enhanced.

[0126] Example 4: Osmotic Stability Testing of the Pre-F Proteins Obtained in Example 1 Before and After Modification

[0127] 1) Liquid preparation

[0128] ①10 mM Tris-HCl buffer, pH 7.5.

[0129] Preparation method (calculated based on 25 mL volume):

[0130]

[0131] 1. Add 10 mL of 25 mM Tris-HCl buffer (pH 8.0) to a 50 mL centrifuge tube.

[0132] 2. Add purified water to a volume of 25 mL.

[0133] 3. Add 6.0 mL of 1 N hydrochloric acid (theoretical value 6.45 mL), then add trace amounts to adjust the pH of the solution to 7.5.

[0134] 4. Dilute to 25 mL and store at room temperature. The shelf life is 3 months.

[0135] ②80 mM Tris-HCl buffer, pH 7.5.

[0136] Preparation method (calculated based on 100 mL volume):

[0137]

[0138] 1. Add 80 mL of purified water to a 200 mL beaker.

[0139] 2. Accurately measure 969.12 mg of Tris and add.

[0140] 3. Add 6 mL of 1 N hydrochloric acid (the theoretical value is 6.45 mL), and then add it drop by drop to adjust the pH value of the solution to 7.5.

[0141] 4. Make up the volume to 100 mL and store it at room temperature. The validity period is 3 months.

[0142] ③ 1.5 M MgCl2 solution

[0143] Preparation method (calculated based on the preparation of 100 mL volume):

[0144]

[0145] 1. Accurately measure 20 mL of 3 M MgCl2 solution and add it to a 50 mL centrifuge tube.

[0146] 2. Add purified water to 20 mL and mix well.

[0147] 3. Store it at room temperature. The validity period is 3 months.

[0148] ④ 3 M MgCl2 solution

[0149] Preparation method (calculated based on the preparation of 100 mL volume):

[0150]

[0151] 1. Accurately weigh 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 until completely dissolved.

[0153] 3. Make up the volume to 100 mL and store it at room temperature. The validity period is 3 months.

[0154] ⑤ 150 mM Tris-HCl buffer, pH 7.5.

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

[0156]

[0157] 1. Add 80 mL of purified water to a 200 mL beaker.

[0158] 2. Accurately measure 1.815 mg of Tris and add it.

[0159] 3. Take 6 mL of 1 N hydrochloric acid and add it (the theoretical value is 6.45 mL), and then add it drop by drop to adjust the pH value of the solution to 7.5.

[0160] 4. Make up to 100 mL and store at room temperature. The valid period is 3 months.

[0161] ⑥ 4 M NaCl solution

[0162] Preparation method (calculated based on a 125 mL volume):

[0163]

[0164] 1. Accurately weigh 29.22 g of sodium chloride and add it to a 200 mL beaker.

[0165] 2. Add 100 mL of purified water and stir well to dissolve.

[0166] 3. Make up to 125 mL and store at room temperature. The valid period is 3 months.

[0167] 2) Dilute the proteins to be tested (the unmodified Pre-F protein prepared in Reference Example 1 and the modified Pre-F protein prepared in Example 1 respectively) to 20 ug / mL with buffer solutions of different osmotic pressure values, and place them in 1.5 mL centrifuge tubes with a total volume of 1 mL.

[0168] 3) Incubate the samples at different osmotic pressure values according to the following table:

[0169]

[0170] 4) Dilute the groups of 10 mM and 80 mM with Tris buffer, and dilute the groups of 1500 mM and 3000 mM with MgCl2 buffer respectively.

[0171] 5) After incubation, adjust the groups of 10 mM and 80 mM to 150 mM with 4 M NaCl buffer, and dilute the groups of 1500 mM and 3000 mM 10 times and 20 times with purified water respectively, and place them for temporary storage at 4°C.

[0172] Conduct ELISA tests according to the protocol. (The operation is the same as the temperature stability test).

[0173] The results are as Figure 3 shown. The results show that there are significant differences in the antigen-binding activities of the Pre-F proteins before and after modification after incubation at different osmotic pressure values. The detection values of the modified Pre-F protein at 10 mM, 80 mM, 150 mM, 1500 mM and 3000 mM are 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 pressure values, that is, compared with the unmodified Pre-F protein, the stability of the modified Pre-F protein prepared by the present invention at different osmotic pressures is significantly enhanced.

[0174] It can be seen that, compared with the Pre-F protein before modification, the modified Pre-F protein prepared by the present invention still maintains its structural stability and antigenic cluster function under different environments, including high temperature, acidic and high osmotic pressure. Even after undergoing chemical reactions, it can still preserve its good antigenicity.

[0175] Example 5: Preparation of capsular polysaccharides of 30 serotypes of Streptococcus pneumoniae, namely 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

[0176] (1) Preparation of master seed and working seed

[0177] The capsular polysaccharides of Streptococcus pneumoniae 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 purified from the fermentation broth, and the specific method is as follows:

[0178] Streptococcus pneumoniae 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 strains in the freeze-dried seed tubes 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 bacterial solution was transferred and inoculated into 150 ml of fresh yeast-acid hydrolyzed casein culture medium and cultured at 36°C ± 2°C for 5 - 10 hours until the exponential growth phase. The culture was stopped, aliquoted and freeze-dried, and stored at 2 - 8°C as the master seed.

[0179] The bacteria in the master seed lyophilized vials of Streptococcus pneumoniae 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°C ± 2°C for 18 hours. When the bacteria grew to an OD600 reading of 1.0, the bacterial suspension was sub - inoculated into 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. Then the culture was stopped, aliquoted, lyophilized, and stored at 4°C as the working seeds of the serotypes.

[0180] (2) Bacterial fermentation

[0181] A seed tube was taken from the working seed bank and inoculated into 5 mL of yeast - acid hydrolyzed casein culture medium, and cultured at 36°C ± 2°C until the mid - exponential growth phase of the bacteria. The bacterial suspension was sub - inoculated into 150 mL of fresh yeast - acid hydrolyzed casein culture medium and cultured at 36°C ± 2°C for 5 - 10 hours until the exponential growth phase. Then 50 mL of the bacterial suspension was sub - inoculated into 2 L of yeast - acid hydrolyzed casein culture medium and cultured at 36°C ± 2°C until the mid - exponential growth phase to prepare the fermentation seed liquid. The fermentation seed liquid was inoculated into a 50 L fermenter containing 30 L of yeast - acid hydrolyzed casein culture medium. Sodium hydroxide was used to maintain the pH of the fermentation broth at 6.8 ± 0.2 until the late exponential growth phase of the bacteria.

[0182] (3) Capsular polysaccharide purification

[0183] 1. Add phosphoric acid to adjust the pH of the fermentation broth to about 4 and stir for 1 hour;

[0184] 2. Centrifuge through a disc - type centrifuge at a speed of 9600 rpm, collect the centrifugal supernatant, and discard the sediment part;

[0185] 3. Micro - filter the centrifuged liquid with a micro - filter membrane to remove the residual cell debris and insoluble small particulate matter. Micro - filter the fermentation centrifugal supernatant with a 0.22 μm membrane and collect the filtrate;

[0186] 4. Concentrate and diafilter the micro - filtrate with a 100 kD membrane package to obtain a crude bacterial capsular polysaccharide solution, and then ultra - filter and diafilter with a 30 Kd membrane using a buffer for 15 sample volumes;

[0187] 5. Further ultra - filter and diafilter the polysaccharide solution with a 50 kD membrane package for 10 sample volumes to concentrate the polysaccharide sample solution.

[0188] 6. Collect the purified polysaccharide solution into a lyophilization bottle, lyophilize it on a vacuum lyophilizer, and store it at - 70°C.

[0189] Example 6: Preparation of Pneumococcal 30 Serotype 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 Polysaccharide-RSV Recombinant Protein Conjugate (CDAP Method)

[0190] 1) Weigh out 17.5 mg of purified capsular polysaccharide of the corresponding serotype respectively and dissolve it in 4 mL of sodium phosphate buffer;

[0191] 2) Add 12 mg of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) (Sigma-Aldrich) to the polysaccharide solution, stir and react at room temperature for 1 hour;

[0192] 3) Add 3 Eqm of cystamine and react at room temperature for 1 hour;

[0193] 4) Add 0.3 mL of 1 M lysine (Sigma-Aldrich) solution for quenching reaction and react at room temperature for 1 - 2 hours;

[0194] 5) Add 8 Eqm of 3(2-chloroethyl) phosphate to the polysaccharide solution to reduce the disulfide bonds in the polysaccharide;

[0195] 6) Transfer the activated polysaccharide solution to a dialysis bag and dialyze against phosphate buffer at 4°C, changing the solution four times;

[0196] 7) Weigh out 30 mg of carrier protein and dissolve it in phosphate buffer with a protein concentration of 10 mg / mL;

[0197] 8) Add 8 mg of N-hydroxysuccinimide bromoacetate (BAANS) (Sigma-Aldrich) to the carrier protein solution, 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 solution four times;

[0198] 9) Take 4 mL of the activated polysaccharide solution and 4 mL of the activated protein solution, mix them and react at room temperature for 4 hours;

[0199] 10) Add 4 Eqm of N-acetyl-L-cysteine (Sigma-Aldrich), react at 2 - 8°C for 4 hours, add 12 Eqm of iodoacetamide (Sigma-Aldrich) and react at 2 - 8°C for 4 hours;

[0200] 11) Transfer the polysaccharide conjugate reaction solution to a dialysis bag and dialyze against phosphate buffer at 4°C;

[0201] 12) Load the sample solution onto Sepharose CL-4B, perform purification, and collect the void volume conjugate;

[0202] 13) After filtering through a 0.22 μm filter membrane, store it at 2 - 8 °C for further formulation.

[0203] The carrier protein used in step 7) is the modified Pre-F protein, and the unmodified Pre-F protein can also be used as the carrier protein.

[0204] Example 7: Preparation of Pneumococcal 30 Serotype Pn1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 1 (2) F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34 and 35B Polysaccharide-RSV Recombinant Protein Conjugate (Reductive Amination Method)

[0205] 1) Weigh 500 mg of purified capsular polysaccharide of the corresponding serotype respectively and dissolve it in 500 mL of purified water; [[ID=!4]]

[0206] 2) Degrade it through a high-pressure homogenizer at a pressure of 600 bar for three cycles, add 0.12 eqm of sodium periodate (Sigma-Aldrich), keep it in the dark, and react for 18 hours;

[0207] 3) Perform ultrafiltration washing and concentration using a 50 Kd membrane package, and lyophilize after ultrafiltration washing with purified water;

[0208] 4) Weigh 18 mg of activated polysaccharide, add 4 mL of DMSO, and stir until completely dissolved;

[0209] 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;

[0210] 6) After adding 2 Eqm of sodium borohydride (Sigma-Aldrich) to quench the reaction for 4 hours, transfer the synthesis reaction solution to a dialysis bag, dialyze against the buffer solution, and change the solution four times;

[0211] 7) Load the sample solution onto Sepharose CL4B and collect the conjugate part in the void volume.

[0212] 8) After filtering through a 0.22 μm filter membrane, store it at 4 °C for further formulation.

[0213] 9) Take samples to detect the molecular weight of the conjugate, the concentration and ratio of polysaccharide to protein. [[ID=3!]]

[0214] The carrier protein used in step 5) is the modified Pre-F protein, and the unmodified Pre-F protein can also be used as the carrier protein.

[0215] Example 8: Preparation of Immunopreparations A, B, and C

[0216] (1) Preparation of Immunopreparation A (30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine):

[0217] Detect the polysaccharide concentration in the monovalent conjugate. Respectively measure RSV recombinant protein conjugate solutions equivalent to 2.2 μg of polysaccharide (in this example, the conjugate prepared in Example 6 is used, and the carrier protein in the conjugate is 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, 23F, 24F, 33F, 34, and 35B into a sterile container. Take a sample to detect that the content of the modified Pre-F protein is about 88.6 μg. Add CpG (Genscript) with a final amount of 0.1 mg. Add phosphate buffer pH 5.8 buffer and filter through a 0.22 μm membrane for sterilization; add sterile aluminum phosphate gel (Benetag) with a final aluminum ion amount of 0.125 mg. Stir at 4°C for 1 hour, and aseptically dispense into 0.5 mL / vial and store at 4°C for immunization.

[0218] (2) Preparation B (Pfizer PCV20): In June 2021, the US Food and Drug Administration (FDA) approved Pfizer's Prevnar 20 (20-valent pneumococcal conjugate vaccine) for adults 18 years of age and older to prevent invasive diseases and pneumonia caused by 20 pneumococcal serotypes in the vaccine. In addition to the capsular polysaccharide conjugates of 13 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) already present in PREVENAR 13® (13-valent pneumococcal conjugate vaccine [diphtheria CRM197 protein]), Pfizer Prevnar 20 (PCV20) also contains capsular polysaccharide conjugates of another 7 serotypes (8, 10A, 11A, 12F, 15B, 22F, and 33F) that account for 40% of pneumococcal disease cases and deaths in the United States, and they are associated with high case fatality rates, antibiotic resistance, and / or meningitis. Among them, CRM197 used in PCV20 is the 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 consider the carrier protein itself stimulating the body to produce protective antibodies to prevent diseases. The present invention uses Pfizer Prevnar 20 (PCV20) conjugate vaccine as a control for detecting polysaccharide antibody titers after immunization with the preparation A (30-valent pneumococcal capsular polysaccharide-RSV recombinant protein conjugate vaccine) of the present invention.

[0219] (3) Preparation C (RSV Pre-F): Sampling content of the modified Pre-F protein obtained in Example 1 was 25.0 μg, CpG (Genscript) was added, and the final amount was 0.1 mg. Phosphate buffer pH 5.8 buffer was added, and it was sterilized by filtration through a 0.22 μm membrane; sterile aluminum phosphate gel (Benetag) was added, and the final aluminum ion amount was 0.125 mg. It was stirred at 4°C for 1 hour, aseptically dispensed at 0.5 mL / vial, and stored at 4°C for immunization.

[0220] Example 9: Immunization of rabbits with the preparation and blood collection

[0221] Ten New Zealand white rabbits weighing 2.5 - 3.5 kg were taken, divided into groups of 5. One group was immunized with Preparation A (prepared in Example 8), and the other group was immunized with Preparation B (Pfizer PCV20). Each rabbit was injected with 0.5 mL each time, and immunization was carried out once at 0 week and once at 2 weeks; Blood collection times were before immunization at 0 week, 1 week after immunization, and 1 week after the second immunization, for a total of three times. A part of the blood was prepared into PBMC, snap-frozen on dry ice and stored at low temperature, and another part of the blood was placed at room temperature for 4 hours, centrifuged at 10,000 RPM at room temperature, and the centrifuged supernatant serum was aspirated and stored at -70°C for testing.

[0222] Example 10: Detection of Polysaccharide Antibody Titers in Rabbit Immune Sera against 30-Valent Pneumococcal Polysaccharide-RSV Recombinant Protein Vaccine by ELISA

[0223] Prepare pneumococcal polysaccharides of different serotypes (1×PBS solution) respectively and store them in a refrigerator at 4°C. Dilute the pneumococcal polysaccharide of the serotype to be detected to 4 μg / mL, add 100 μL of the coating solution to each well to coat the ELISA plate, and incubate overnight at room temperature. Wash 4 times with the wash buffer, add 100 μL of the blocking buffer, incubate for 2 hours at room temperature, wash 4 times with the wash buffer, and it can be stored at 4°C for one week.

[0224] Dilute the corresponding test sera obtained from rabbits injected with the vaccine and control samples 1:10 to working sample sera, dilute by an appropriate multiple, add them to the wells in the first row of the ELISA plate, with a total volume of 200 μL, and perform two-fold serial dilutions downward starting from the first row. Incubate for 2 hours at room temperature. Wash 4 times with the wash buffer, add 100 μL of alkaline phosphatase-labeled goat anti-rabbit antibody (diluted 1:2000), and incubate for 4 hours at room temperature. Wash 4 times with the wash buffer, add 100 μL of p-nitrophenyl phosphate disodium salt substrate (Sigma-Aldrich) solution, and read the plate at 405 nm.

[0225] Table 1: Detection Results of IgG Antibody Titers against 30 Kinds of Pneumococcal Polysaccharides in Rabbit Immune Antisera of Formulation A and Formulation B

[0226]

[0227]

[0228] The primary difference between Formulations A and B lies in the carrier protein. IgG antibody titers in rabbit antisera immunized with the pneumococcal capsular polysaccharide conjugate vaccine showed a clear upward trend in IgG titers of antibodies against the 30 serotypes of polysaccharides stimulated by the 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine (Formulation A) after the first (D14) and second (D28) doses. The second dose was significantly more effective than the first, indicating that antibody titers increased significantly with increasing immunization times. In addition, after the second immunization (D28), the IgG titers of polysaccharide antibodies produced by the 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine (preparation A) and PCV20 Pfizer (preparation B) in animals varied according to 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 preparation A had significantly higher titers than preparation B, accounting for 90% of the components. Only for two serotypes, 12F and 22F, did preparation B have higher titers than preparation A, accounting for 10% of the components. This indicates that the immune effect of preparation A, prepared using RSV Pre-F recombinant protein as a carrier protein, was superior to that of preparation B using CRM197 as a carrier protein, demonstrating that the RSV Pre-F recombinant protein carrier in the conjugate vaccine prepared by the present invention has a good promoting and enhancing effect on the antigenicity of 30 pneumococcal polysaccharides.

[0229] At the same time, compared to formulation B (PCV20), the GM test values of anti-serum IgG antibody titers against the 10 newly added serotypes Pn2, Pn6C, Pn9N, Pn15A, Pn17F, Pn20, Pn22A, Pn24F, Pn34, and Pn35B in the conjugate vaccine formulation A of the present invention were significantly higher than the average IgG antibody titer of the 20 polysaccharide types in formulation B (168.06). This also shows that the 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine prepared by the present invention can achieve better immune effects while covering more pneumococcal serotypes and expanding the vaccine's protection range.

[0230] Example 11: Detection of pre-F protein antibody titers in sera of mice immune to RSV recombinant protein and 30-valent pneumococcal polysaccharide-RSV recombinant protein combined vaccine

[0231] (1) ELISA detection of IgG antibody titer

[0232] 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 (prepared in Example 8) and 0.1 mL of immunization preparation C (prepared in Example 8) in one group for a total of two immunizations.

[0233] Blood samples were collected one week after immunization at three time points: before the immunization at week 0, two weeks after the first immunization, and four weeks after the second immunization. A portion of the blood samples was used to prepare PBMCs, which were snap-frozen on dry ice and stored at low temperature. The other portion of the blood samples was left 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 for further detection.

[0234] Prepare a purified pre-F protein stock solution at 1 mg / mL (in 1×PBS solution) and store it in a 4°C refrigerator. Dilute the protein stock solution to 4 μg / mL in coating buffer. Add 100 μL of the coating solution to each well to coat the ELISA plate and incubate overnight at room temperature. Wash the plate 4 times with wash buffer, add 100 μL of blocking buffer, incubate for 2 hours at room temperature, wash the plate 4 times with wash buffer, and it can be stored at 4°C for one week.

[0235] Dilute the corresponding test sera obtained from mice injected with the vaccine and control samples 1:10 to prepare working sample sera. Dilute appropriately and add 200 μL in total to the first row of wells on the ELISA plate. Perform a two-fold serial dilution starting from the first row downwards and incubate for 2 hours at room temperature. Wash the plate 4 times with wash buffer, add 100 μL of alkaline phosphatase-labeled goat anti-mouse antibody (diluted 1:2000), and incubate for 4 hours at room temperature. Wash the plate 4 times with wash buffer, add 100 μL of p-nitrophenyl phosphate disodium salt substrate solution, and read the plate at 405 nm.

[0236] (2)Detection of RSV A2 virus neutralization titer

[0237] Culture RSV virus type A using Hep-2 cells in DMEM medium containing 10% bovine serum. Inoculate 96-well plates at a density of 20,000 Hep2 cells / well with 100 μL of complete medium per well and culture at 37°C and 5% CO2 for 24 hours until the cell density reaches 60 - 80%; replace the medium, aspirate the medium in the 96-well plates, and add maintenance medium, 100 μL per well; heat-inactivate the serum in a 56°C water bath for 30 minutes. Aliquot into at least 4 tubes at 15 μL per tube and store the serum at -80°C; dilute the antiserum in a 96-well V-shaped plate at an appropriate dilution factor, with 70 μL per well after dilution and 2 replicate wells for each dilution; mix the virus with different dilutions of the antiserum, dilute the virus to 100 TCID50 with DMEM, and add 70 μL per well to the above 96-well V-shaped plate. Incubate at 37°C for 1 hour; aspirate and transfer 100 μL per well to the above Hep2 cell plates; culture at 37°C and 5% CO2 for about 3 - 5 days; observe the CPE situation daily, and stain the cells with 5% glutaraldehyde containing 0.25% crystal violet.

[0238] Table 2: Geometric mean of IgG antibody titers (Eu) in the immune sera of mice vaccinated with RSV Pre-F protein and 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine

[0239]

[0240] The test results in Table 2 show that the geometric mean of IgG antibody titers (Eu) in the immune sera of mice vaccinated with 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine is significantly higher than that of RSV Pre-F recombinant protein. Among them, the IgG antibody titers in the immune sera of the 30-valent conjugate (Pn-RSV Pre-F) after the first immunization and the second immunization are approximately 1.6 and 2.4 times higher than those of RSV Pre-F (Formulation C), respectively.

[0241] The test results of the RSV A2 virus neutralization titer show that the mean neutralization titer of the mouse sera of the 30-valent conjugate (Pn-RSV Pre-F) is 12,600, which is approximately 1.9 times higher than the mouse sera neutralization titer of RSV Pre-F. Thus, it can be seen that after immunizing mice with the 30-valent pneumococcal polysaccharide-RSV recombinant protein conjugate vaccine prepared by the present invention, high-protection-titer sera can be obtained, and the mouse sera can produce relatively high neutralizing antibody titers against the main epidemic strain A of RSV.

[0242] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that the specific implementation manners of the present invention can still be modified or equivalently replaced. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention shall be covered by 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 the Pre-F protein, and its amino acid sequence is as 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, wherein 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 as 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 in the form of an aqueous solution or a freeze-dried preparation.

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

5. The multivalent pneumococcal polysaccharide-protein conjugate vaccine according to claim 4, 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.

6. The preparation method of 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 the pneumococcal polysaccharide and the RSV recombinant protein in a buffer solution or an organic solvent, and covalently binding the pneumococcal polysaccharide to the RSV recombinant protein through the reaction to obtain a conjugate.

7. The preparation method according to claim 6, characterized in that, The preparation method also includes the pretreatment of the pneumococcal polysaccharide, the expression and purification of the RSV recombinant protein, and the 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 method used for degradation is selected from high-pressure homogenizer degradation, acid hydrolysis method, or enzymatic digestion method.

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, wherein The chemical synthesis reaction is any one of the reductive amination method, 1-cyano-4-dimethylamino pyridinium tetrafluoroborate method, adipic dihydrazide method, or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride method.

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

3.

12. A recombinant expression vector containing the nucleic acid molecule according to claim 11.

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

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