Composition containing multivalent pneumococcal polysaccharide-protein conjugate as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a polyvalent pneumococcal polysaccharide-protein conjugate composition for preventing pneumococcal infection and VZV infection as well as a preparation method and application of the polyvalent pneumococcal polysaccharide-protein conjugate composition. According to the composition containing the multivalent pneumococcal polysaccharide-protein conjugate, an adopted carrier protein is a VZV recombinant protein with immunogenicity; the pneumococcal polysaccharide is connected with the VZV recombinant protein through a covalent bond; the VZV recombinant protein is selected from gE protein, and the amino acid sequence of the VZV recombinant protein is shown as SEQ ID NO: 3 or SEQ ID NO: 4. Aiming at the main epidemic condition of specific serotype pneumococcus in China, serotype combinations of 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 are creatively selected, so that the composition containing the multivalent pneumococcus polysaccharide-protein conjugate, which is designed aiming at the epidemic condition of the specific serotype pneumococcus in China, is developed.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a composition of a multivalent pneumococcal polysaccharide-protein conjugate for preventing pneumococcal infection and VZV infection, as well as a preparation method and application thereof. Background Art

[0002] Streptococcus pneumoniae (also known as the pneumococcus) is a Gram-positive diplococcus with a capsule. The bacterial outer membrane is encapsulated by a layer of capsular polysaccharides. 91 different pneumococcal serotypes have been identified, based on differences in the composition of these polysaccharides, with capsular polysaccharides being a key pathogenic factor. Normally, pneumococci parasitize the nasopharynx of healthy individuals and generally do not cause clinical symptoms. However, when the host environment changes, such as decreased immunity, respiratory viral infections such as measles and influenza, or malnutrition or the elderly, pneumococcal disease can penetrate the mucosal defense system and cause invasive infection. Based on the site of infection, pneumococcal disease can be categorized into invasive pneumococcal disease (IPD), including meningitis, bacteremia, and bacteremic pneumonia, and non-invasive pneumococcal disease (NIPD), including acute otitis media, sinusitis, and non-bacteremic pneumonia.

[0003] Pneumococcal disease, caused by the pneumococcus bacteria, is a global public health challenge, posing a serious threat to human health and a growing burden of disease. According to statistics, in 2019, 33 bacterial infections caused 7.7 million deaths worldwide, with pneumococcal infections ranking third and causing 829,000 deaths. When converted to years of life lost, pneumococcal infection ranks first. Deaths from pneumococcal infection are primarily among children under five years of age and those over 60 years of age. Infants and young children, particularly those under two years of age, are highly susceptible to pneumococcal infection and have a high mortality rate. Approximately 75% of invasive pneumococcal disease and 83% of pneumococcal meningitis occur in this age group (see the 2019 WHO Position Paper). Similarly, the morbidity and mortality rates of invasive pneumococcal infection in the elderly are also significant. According to reports, pneumonia is the fourth leading cause of death among the elderly in China, killing approximately 125,000 people annually. Pneumonia infection rates vary by region, ranging from 28.0% to 71.5%, and the incidence of pneumonia in the elderly increases sharply with age. Meanwhile, in today's increasingly aging global population, the United Nations' 2023 World Social Report indicates that the global population aged 65 and over will reach 761 million in 2021, and this number will increase to 1.6 billion by 2050, with the population aged 80 and over growing even faster. Therefore, as the elderly are a high-risk group for pneumococcal infection, the increasing aging of the population will inevitably lead to an increasing burden of pneumococcal disease.

[0004] The most common treatment for pneumococcal disease is antibiotic therapy, but worldwide, the development of pneumococcal resistance to commonly used antimicrobial drugs has become an increasingly serious problem. Years of clinical practice have proven that pneumococcal vaccination is the most cost-effective way to prevent pneumococcal disease. Currently available vaccines are the pneumococcal polysaccharide vaccine (PPV) and the 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 approximately 90% against the dominant serotype worldwide, but it still has limitations. Studies have found that bacterial polysaccharides are T-cell-independent antigens that can induce short-term immune effects in children and adults, but are ineffective in infants and young children, especially in children under two years of age. Through development, covalently linking polysaccharides to carrier proteins can convert T-cell-independent antigens into T-cell-dependent antigens. Pneumococcal polysaccharide-protein conjugate vaccines (PCVs) prepared using this technology can be administered to infants as early as two months of age, promoting strong immune and memory responses, and offering a promising approach for faster and earlier protection against pneumococcal disease. The first PCV to receive FDA approval in 2000 was PCV7 (Prevnar 7) produced by Wyeth. In 2010, Pfizer launched PCV13 (Prevnar 13), which covers a wider range of pathogens, replacing PCV7 (Prevnar 7) for routine childhood vaccination. Currently, three PCVs have received WHO prequalification: PCV10 (Synflorix) from GlaxoSmithKline, PCV13 (Prevnar 13) from Pfizer, and PCV10 (PNEUMOSIL) from the Serum Institute of India. In 2021, the FDA approved several more expensive PCVs, including Pfizer's PCV20 (Prevnar 20) and Merck's PCV15 (VAXNEUVANCE). In China, two pneumococcal polysaccharide conjugate vaccines, Watson Biopharma's PCV13 and Minhai Biopharma's PCV13, are now available. Studies have shown that conjugate vaccines can be used in all age groups and offer longer-lasting effects than polysaccharide vaccines. Therefore, polysaccharide-protein conjugate vaccines will become the dominant development trend in pneumococcal vaccines.

[0005] With the widespread use of pneumococcal conjugate vaccines both domestically and internationally, the prevalence of serotypes in different regions has shifted significantly, with a significant increase in the incidence of some non-vaccine-covered serotypes. Therefore, the development of higher-dose pneumococcal conjugate vaccines is urgent. However, a common drawback of currently available conjugate vaccines is that the carrier protein lacks immunogenic protective properties. In other words, while the conjugate vaccine carrier can stimulate antibody production, vaccine designers have not yet been able to utilize the antibodies generated by the carrier protein to prevent disease. Tetanus toxoid, diphtheria toxoid, and avirulent variant diphtheria toxins are widely used as carrier proteins not because the antibodies they produce are protective, but rather because of their safety and ability to enhance the immunogenicity of the polysaccharide in the conjugate. Furthermore, tetanus and diphtheria toxoids are already components of the DPT vaccine and are routinely used, making the ability of the carrier protein in pneumococcal conjugate vaccines to stimulate protective antibody production less crucial. Therefore, the choice of carrier protein is crucial in improving the efficacy and coverage of pneumococcal vaccines.

[0006] Varicella zoster virus (VZV) belongs to the alphaherpesvirinae subfamily of the Herpesviridae family and is one of eight human herpesviruses, also known as human herpesvirus type 3. VZV consists of a linear, double-stranded DNA encapsidated by a nucleocapsid, separated from the lipid envelope by a protein layer that comprises the primary viral glycoproteins. VZV produces approximately six glycoproteins, currently designated gB, gC, gE, gH, gI, and gL. These proteins are also expressed on the surface of infected cell membranes during viral replication. However, gE is the most abundant protein produced in infected cells. It non-covalently binds to gI and can bind to the Fc fragment of antibody G (IgG), making it a crucial antigenic protein.

[0007] VZV is a highly contagious virus found worldwide. To date, only one serotype has been identified, and it infects only humans in nature. Primary infection caused by VZV manifests as varicella, commonly seen in childhood. Following primary infection, the virus can remain latent within host sensory neurons. Furthermore, studies have shown that the FDA-approved live-attenuated Oka strain vaccine for varicella prevention can establish latent infection, just as the wild-type virus does. Aging and impaired immune function can weaken the cellular immune response, leading to VZV reactivation and the development of herpes zoster (HZ), a condition most common in adults and the elderly. Symptoms include a unilateral vesicular rash, often accompanied by fever and fatigue, followed by localized redness, burning sensation, and neuralgia. Approximately 9% to 34% of patients with HZ develop post-herpetic neuralgia (PHN), which can reach a pain rating of 7 or higher, severely impacting patients' quality of life.

[0008] With the increasing global aging of the population, the incidence and burden of herpes zoster are also showing a significant upward trend. In the Asia-Pacific region, the incidence of herpes zoster is approximately 1%, increasing with age, reaching over 5% after the age of 50. With the continued annual increase in VZV infection, the incidence rate is expected to increase by 35%-100%. According to statistics, by the end of 2023, the number of people aged 60 and over in my country will reach nearly 300 million, accounting for 21.1% of the total population, of which those aged 65 and over account for 15.4%. The number of herpes zoster patients in my country each year is 2.77 million, of which over 1.5 million new cases occur in people aged 50 and over, resulting in a total cost of approximately 1.3 billion yuan. In 2010, approximately 9 million people aged 50 and over in China suffered from herpes zoster, resulting in an economic burden of over 7.7 billion yuan. However, there is currently no specific medicine for the treatment of shingles and postherpetic neuralgia. Most clinical treatments use broad-spectrum antiviral drugs such as acyclovir. Some anesthetic or non-anesthetic analgesics and anticonvulsant and antidepressant drugs are also used to relieve the severe neuralgia caused by shingles. Even if shingles is cured, it may still recur. Therefore, vaccination with VZV vaccine is an effective means of preventing shingles.

[0009] Currently, there are two FDA-approved VZV vaccines: Merck's Zostavax and GlaxoSmithKline's Shingrix. Zostavax, a live attenuated vaccine based on the Oka strain, has a 64% preventive efficacy in people aged 60 to 69 years with a single subcutaneous injection. However, Zostavax's efficacy decreases with age, and in adults over 60 years old, its efficacy against PHN is only 39%. Shingrix, a recombinant protein subunit vaccine from GSK, contains the gE glycoprotein and the AS01B adjuvant. gE is the most abundant VZV glycoprotein and contains potential neutralizing and T cell epitopes, which are critical for viral replication and transmission between ganglion cells. This vaccine, administered intramuscularly, has demonstrated 97.4% efficacy against HZ in individuals aged 60 to 69 years and >90% efficacy across all age groups tested, including those aged 80 years and older. This efficacy is superior to Merck's live-attenuated Zostavax vaccine. While Shingrix's recombinant protein vaccine is significantly more effective than Zostavax, it is complex in formulation, has high production costs, and is expensive. Therefore, the development of a domestically developed vaccine to prevent VZV infection is crucial.

[0010] Vaccines are characterized by a single vaccine that can prevent infection by specific pathogens. For example, the pneumococcal polysaccharide conjugate vaccine can prevent pneumonia, meningitis, and otitis media caused by pneumococcal infection, and the herpes zoster virus recombinant protein vaccine can prevent shingles caused by reactivated herpes zoster virus. To achieve the goal of a single vaccine being able to prevent infection by multiple pathogens, combination vaccines are currently being used. For example, a triple vaccine containing diphtheria, pertussis, and tetanus for children; a pentavalent vaccine containing diphtheria, pertussis, tetanus, polio, and Haemophilus influenzae for children; and a hexavalent vaccine containing diphtheria, pertussis, tetanus, polio, Haemophilus influenzae, and hepatitis B for children. These vaccines are prepared by mixing single vaccines. Clinical evaluations to date have shown that the preventive efficacy of some individual vaccines in these combination vaccines is poor.

[0011] Currently, children of appropriate age in my country need to receive at least 15 different vaccines, a frequency that far outstrips the number of vaccines available. Furthermore, with the aging population, by the end of 2024, the number of people aged 60 and above in my country will have reached 310 million, representing 22% of the total population. Therefore, protecting the health of the elderly and developing vaccines specifically for them are crucial. Therefore, developing multi-component and multivalent conjugate vaccines to replace single-species vaccines and achieving universal vaccine coverage will undoubtedly be a top priority for future vaccine research and development.

[0012] However, in the development of new vaccines, the choice of carrier protein has a significant impact on the immune effect of the vaccine, especially as the price of vaccines increases. How to choose the carrier protein in the polysaccharide-protein conjugate vaccine to enhance the immune effect of the polysaccharide while avoiding the inhibitory effect of the protein carrier; and how to design a stable and highly expressed carrier protein, and at the same time combine the immunogenic carrier protein with other immunogenic substances (such as capsular polysaccharides) to prepare a vaccine with dual immunogenicity and achieve the goal of preventing two diseases with one vaccine. These are the key challenges faced in the design and development of polysaccharide-protein conjugate vaccines. Further development of multivalent pneumococcal conjugate vaccines to cover a wider range of pathogenic serotypes of pneumococci, increase non-vaccine serotypes, improve the coverage protection rate and immunogenicity of new multivalent vaccines, and reduce the risk of immunosuppressive effects of carrier proteins has important clinical value. Summary of the Invention

[0013] To overcome the shortcomings of the existing technology, the present invention utilizes polysaccharide-protein conjugate technology and innovatively uses gE protein, whose stability has been enhanced through amino acid mutation, as a carrier protein to develop a composition containing a multivalent pneumococcal polysaccharide-protein conjugate. This composition possesses dual immunogenicity and can effectively prevent infections caused by 24 pneumococcal serotypes as well as diseases caused by VZV infection, significantly reducing the number of vaccinations required, lowering vaccination costs, and improving immune coverage and efficacy.

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

[0015] In a first aspect of the present invention, a composition comprising a multivalent pneumococcal polysaccharide-protein conjugate is provided, wherein the carrier protein used in the pneumococcal polysaccharide-protein conjugate in the composition is an immunogenic VZV recombinant protein; the pneumococcal polysaccharide is covalently linked to the VZV recombinant protein; the VZV recombinant protein is selected from gE protein, whose amino acid sequence is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

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

[0017] In one embodiment of the present invention, the composition comprises a 24-valent pneumococcal polysaccharide-VZV recombinant protein conjugate, wherein the amino acid sequence of the VZV recombinant protein is shown in SEQ ID NO: 3, and the pneumococcal polysaccharide comprises 24 serotypes, namely 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.

[0018] In another embodiment of the present invention, the composition comprises a 24-valent pneumococcal polysaccharide-VZV recombinant protein conjugate, wherein the amino acid sequence of the VZV recombinant protein is shown in SEQ ID NO: 4, and the pneumococcal polysaccharide comprises 24 serotypes, namely 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.

[0019] Preferably, the composition comprising the multivalent pneumococcal polysaccharide-protein conjugate of the present invention contains an adjuvant. More preferably, 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.

[0020] Preferably, the composition comprising the multivalent pneumococcal polysaccharide-protein conjugate of the present invention can be prepared as any one of a spray, an injection, a lyophilized preparation, a capsule, a tablet or a pill.

[0021] In a second aspect of the present invention, a method for preparing a composition comprising a multivalent pneumococcal polysaccharide-protein conjugate is provided. The method comprises the following steps: subjecting pneumococcal polysaccharide and VZV recombinant protein to a chemical synthesis reaction in a buffer or an organic solvent, and covalently binding the pneumococcal polysaccharide to the VZV recombinant protein through the reaction to obtain a conjugate.

[0022] Preferably, the preparation method further comprises pre-treatment of pneumococcal polysaccharide, expression and purification of VZV recombinant protein, and purification of the conjugate.

[0023] Preferably, the polysaccharide pretreatment includes degradation and activation, and the degradation method is selected from high-pressure homogenizer degradation, acid hydrolysis or enzyme digestion.

[0024] Preferably, the chemical synthesis reaction is selected from one of the reductive amine method, the CDAP method, the adipic acid dihydrazide method or the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride method.

[0025] Preferably, the organic solvent is selected from dimethyl sulfoxide or dimethylformamide.

[0026] In the third aspect of the present invention, a nucleic acid molecule encoding the VZV recombinant protein of the amino acid sequence as described in the first aspect, such as SEQ ID NO: 3 or SEQ ID NO: 4, is provided.

[0027] In a fourth aspect of the present invention, a recombinant expression vector containing the nucleic acid molecule according to the second aspect is provided.

[0028] In a fifth aspect, the present invention provides use of a composition comprising a multivalent pneumococcal polysaccharide-protein conjugate as described in the first aspect in the preparation of a vaccine or a medicament for preventing or treating a disease, wherein the disease is a disease caused by Streptococcus pneumoniae infection or a disease caused by VZV infection.

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

[0030] (1) The present invention is based on the main prevalence of pneumococcal serotypes unique to my country, and creatively selects a combination of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, thereby developing a composition comprising a multivalent pneumococcal polysaccharide-protein conjugate designed to target the prevalence of pneumococcal serotypes unique to my country. The composition of the present invention has the ability to effectively prevent invasive infections of 24 different serotypes of pneumococci and can induce relatively balanced and relatively high immunogenicity against all 24 serotypes of pneumococci.

[0031] (2) The present invention conducts immunogenicity research on the combination of pneumococcal capsular polysaccharide and carrier protein, and creatively designs a carrier protein suitable for binding to multiple serotype polysaccharides, namely VZV gE protein. By performing amino acid mutation modification on the gE carrier protein, on the one hand, it ensures that the carrier protein itself still maintains its structural stability and antigen cluster function under different temperature environments, and even after undergoing chemical reactions, it can still maintain the antigenicity of the gE carrier protein itself; on the other hand, the gE recombinant protein carrier has a good enhancing effect on the antigenicity of 24 serotypes of pneumococcal polysaccharides.

[0032] (3) A composition comprising a multivalent pneumococcal polysaccharide-protein conjugate prepared by the present invention has a synergistic and effective technical effect: On the one hand, it can be seen from Example 10 that the polysaccharide-protein conjugate composition prepared by the present invention uses a VZV recombinant protein modified by amino acid mutation as a carrier protein, and has a better effect on enhancing the antigenicity of 24 serotypes of pneumonia polysaccharides. Immunization of animals with the preparation c prepared by the present invention can effectively increase the IgG antibody titer in the animal body, and the serum protection titer is higher; and compared with the animals before immunization, while the immune effect is significantly enhanced, the preparation c is also better than the immune effect of Pfizer PCV20 conjugate vaccine using CRM197 as a carrier protein. Among them, before immunization with the preparation (D0), the titers of IgG antibodies to polysaccharides of various serotypes of pneumococcus originally contained in the animal serum were significantly low, making it difficult to resist the infection and invasion of pneumococcus; after the first injection (D14) and the second injection (D28) of the preparation, the 24-valent pneumococcal polysaccharide gE protein conjugate vaccine (preparation c) of the present invention stimulated the animals to produce the corresponding 24 serotypes of polysaccharide antibodies, and the titers of IgG showed a significant upward trend. The immune effect of the second injection was significantly better than that of the first injection, that is, as the number of immunizations increased, the antibody titer was significantly enhanced. At the same time, after the second immunization (D28), the 24-valent pneumococcal gE protein conjugate vaccine (preparation c) and PCV20 Pfizer (preparation d) stimulated animals to produce corresponding serotype polysaccharide antibody IgG titers, which varied according to the serotype. The results of serotypes Pn1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 14, 15B, 18C, 19A, 23F and 33F showed that preparation c was significantly higher than preparation d, accounting for 80% of the components.

[0033] On the other hand, it can be seen from Example 12 that the gE recombinant protein prepared by the present invention, in addition to enhancing the immune effect of the protein itself, can also be used as a carrier protein to prepare a 24-valent pneumococcal polysaccharide-VZV gE recombinant protein conjugate preparation to obtain serum with higher protective titers and better immunogenicity. Compared with the control PBS (preparation e), the IgG antibody titer against gE protein in the rabbit immune antiserum of the adjuvanted 24-valent polysaccharide-VZV gE protein conjugate vaccine (preparation c), the adjuvanted gE protein (preparation g), and the unadjuvanted gE protein (preparation f) were significantly enhanced, and the total IgG, IgG1, and IgG2a antibody titers were significantly increased; and whether it was total IgG, IgG1, or IgG2a antibody titer, the protein antibody titer detection value in the rabbit immune serum of the adjuvanted 24-valent polysaccharide-VZV gE protein conjugate vaccine (preparation c) was the highest, significantly higher than the gE recombinant protein preparation of the non-conjugate.

[0034] At the same time, Example 13 also shows that the 24-valent polysaccharide-VZV gE protein conjugate composition prepared by the present invention can stimulate immune cells in the spleen of animals, such as NK cells and phagocytes, to produce IFN-γ. Moreover, after immunization with the 24-valent polysaccharide-VZV gE protein conjugate composition, the IFN-γ production of the sensitized immune cells under gE antigen or VZV virus stimulation is significantly higher than that of the gE protein preparation. This shows that not only does the gE recombinant protein of the present invention have a good cellular immune effect itself, but the 24-valent pneumococcal polysaccharide-VZV gE protein conjugate composition prepared using it as a carrier protein has a stronger ability to induce cellular immune responses in mice and a better effect.

[0035] (4) The present invention prepares a composition comprising a multivalent pneumococcal polysaccharide-protein conjugate, which covers a wider range of pneumococcal serotypes, expands the scope of vaccine protection, and can also achieve a better immune effect. As can be seen from Example 10, compared with PCV20 (pneumococcal 20-valent conjugate vaccine), the total average antiserum IgG antibody titer of the four newly added serotypes Pn2, Pn9N, Pn17F, and Pn20 in the present invention is approximately 271.36, which is significantly higher than the total average IgG antibody titer of the 20 serotypes of PCV20, which is 168.06.

[0036] (5) The present invention also provides a method for preparing a composition of a multivalent pneumococcal polysaccharide-protein conjugate, wherein the VZV gE recombinant protein is an amino acid sequence designed by genetic recombination technology, and the gE protein carrier with enhanced stability after amino acid mutation modification is prepared with a new adjuvant and immunized into animals. The polysaccharide antigen portion of the conjugate can stimulate the body to produce specific serotype polysaccharide antibodies, and the carrier protein portion can stimulate the body to produce humoral immunity (protective antibodies) and cellular immunity against the VZV virus. After immunization with one preparation, it can simultaneously prevent infections of 24 types of pneumococci and herpes zoster, achieving the goal of preventing two diseases with one vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a temperature stability test of gE protein before and after modification (0.06ug / mL).

[0038] Figure 2 This is a temperature stability test of gE protein before and after modification (2.0ug / mL).

[0039] Figure 3 To detect the protein antibody titer in the immune sera of mice with preparation a (gE before modification) and preparation b (gE after modification). DETAILED DESCRIPTION

[0040] 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 are described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the instruments, equipment, reagents, materials, etc. used are all available through conventional commercial means unless otherwise specified.

[0041] Example 1: Preparation of carrier protein

[0042] Varicella zoster virus (VZV) is a member of the alphaherpesvirus subfamily of the Herpesviridae genus, namely Human herpesvirus 3.

[0043] gE (glycoprotein E, gE) is encoded by the ORF68 gene, located in the short fragment region of the VZV genome. The encoded gE contains 623 amino acids. The gE protein molecule is mainly composed of the hydrophilic extracellular region composed of amino acids 1-546 (including the signal peptide), the hydrophobic transmembrane region of amino acids 547-623, and the intracellular tail.

[0044] (1) Protein construction

[0045] The full-length sequence of the VZV gE protein mentioned in the present invention is referenced by NCBI Reference Sequence: NP_040190.1, and the specific sequence is shown in SEQ ID NO. 1 (envelope glycoprotein E [Human alphaherpesvirus 3] / strain="Dumas" / 623aa).

[0046] The VZV gE protein used in the present invention is different from the original full-length protein, but is a truncated protein. The present invention selects a conserved truncated gE protein amino acid sequence as a template for design optimization. In order to improve expression efficiency and protein structural stability and preserve the antigen cluster function, only the sequences of the signal peptide region and the mature antigen region are selected. The specific sequence is shown in SEQ ID NO.2.

[0047] The VZV gE protein mutant provided herein primarily involves mutational modification of the amino acid sequence of the mature antigenic region of a selected truncated protein to optimize protein stability and immunogenicity. This mutant differs from the amino acid sequence of the VZV gE protein listed in the NCBI database, and its specific sequence is shown in SEQ ID NO. 4. Furthermore, the specific sequence of the VZV gE protein before modification provided herein is shown in SEQ ID NO. 3.

[0048] (1) Amino acid sequence design

[0049] The present invention relates to a VZV gE recombinant protein modified by amino acid mutation to obtain enhanced stability and immunogenicity, wherein the amino acid sequence is designed as follows:

[0050] The transmembrane region and intracellular region of the full-length VZV gE protein were deleted to obtain a truncated gE protein sequence as shown in SEQ ID NO.2. On this basis, amino acid point mutations were performed. Specifically, the D at position 277 of the VZV gE protein was mutated to C, and the L at position 289 was mutated to C. At the same time, Throm and 6his sequences were connected to its C-terminus to obtain the full-length mutant sequence of the VZV gE protein, whose amino acid sequence is shown in SEQ ID NO.4.

[0051] The present invention also relates to a VZV pre-modified gE recombinant protein, the amino acid sequence of which is designed as follows:

[0052] The transmembrane region and intracellular region in the full-length sequence of the VZV gE protein were deleted to obtain a truncated gE protein sequence as shown in SEQ ID NO.2. At the same time, the Throm and 6his sequences were connected to its C-terminus to obtain the full-length sequence of the VZV gE recombinant protein before modification, whose amino acid sequence is shown in SEQ ID NO.3.

[0053] SEQ ID NO.1-SEQ ID NO.4 sequences are shown below:

[0054] SEQ ID NO.1:

[0055] >VZV gE 1-623(NP_040190.1 / 623aa / envelope glycoprotein E[Humanalphaherpesvirus 3] / strain="Dumas")

[0056]

[0057] SEQ ID NO.2:

[0058] >VZV gE 1-546

[0059]

[0060]

[0061] SEQ ID NO.3:

[0062] >VZV modified gE

[0063]

[0064] SEQ ID NO.4:

[0065] >VZV modified gE D277C L289C

[0066]

[0067] (2) Synthesis of VZV gE protein target gene

[0068] Based on the designed VZV gE protein amino acid sequences SEQ ID NO.3 and SEQ ID NO.4 and the codon preference of the host cell, the corresponding gene coding sequence was determined. 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. The designed nucleotide sequence was chemically synthesized.

[0069] (3) Plasmid amplification and target gene extraction

[0070] The plasmid vector pUC19 was double-digested with EcoRI and XbaI restriction enzymes, ligated with the synthesized gene, and introduced into the amplification host DH5α. Single clones were screened using LB (Amp+) agar solid medium. Single clones containing the target gene were inoculated into LB (Amp+) liquid medium, cultured and amplified at 37°C, 200 rpm, and the plasmid pUC19-gE 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.

[0071] (4) Construction of eukaryotic expression vector

[0072] The mammalian cell expression plasmid pGN-M, containing the CMV promoter and the 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, and a single clone containing the eukaryotic expression plasmid pGN-M_gE was screened. The plasmid was inoculated in LB (Amp+) for amplification culture, and the amplified plasmid was extracted using the TaKaRa MidiBEST Endo-free Plasmid Purification Kit, and was named VZVgE.

[0073] (II) Expression and cloning of gE protein in CHO cells

[0074] CHO K1 (ATCC) cells were used as host cells. After recovery, the cells 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 2 Wells 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 a 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 gE. The clones secreting the highest amount of protein were selected for the next round of screening, and the cells were finally expanded and 30 new clones were selected and preserved.

[0075] 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.

[0076] (III) Production of gE protein in bioreactors

[0077] A 1.5-liter perfusion culture was set up in a 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.

[0078] A total of 12.5 liters of cell-free culture fluid was collected and centrifuged at 8,000 × g for 30 minutes at 4°C. The fluid was filtered through a 0.45 μm membrane and then concentrated by ultrafiltration using a 10 kDa membrane pack. Ultrafiltration was performed with buffer and the sample solution was concentrated to 0.5 liters. After adding 0.5 liters of PBS, the sample solution was concentrated to 0.5 liters. This procedure was repeated five times.

[0079] (IV) Purification of gE protein

[0080] 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 gE 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 washed with phosphate buffered saline (PBS, 6mM Na2HPO4, 1.5mM KH2PO4, 0.15M sodium chloride, pH 6.8) supplemented with 800mM ammonium sulfate. The column was then washed with a PBS solution containing 400mM ammonium sulfate, and the gE protein was finally 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.

[0081] VZV-modified gE protein and VZV-modified gE protein were prepared respectively using the above method.

[0082] Example 2: Temperature stability test of gE protein obtained in Example 1 before and after modification

[0083] 1) Dilute the test protein (the pre-modified gE protein prepared in Reference Example 1 and the modified gE 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 with a total volume of 1 mL.

[0084] 2) Incubate at different temperatures according to the table below.

[0085] Table 1: Protein incubation conditions at different temperatures

[0086] Serial number Temperature (℃) Time (min) Quantity (pieces) Concentration (ug / mL, diluted in PBS) 1 4 60 1 20 2 37 60 1 20 3 60 60 1 20 4 80 60 1 20

[0087] 3) After incubation, each sample was temporarily stored at 4°C.

[0088] 4) Preparation of detection antibodies (E5-G6)

[0089] BALB / c mice were immunized with gE protein vaccine (GSK) at a dose of 100 μg / mouse. After BALB / c mice were immunized twice, B lymphocytes were extracted from their spleens. Monoclonal antibodies (mAbs) against VZV-gE were then prepared and cloned using hybridoma cell technology (number: E5-G6).

[0090] 5) Perform ELISA test according to the following steps:

[0091] Samples stored at 4°C were diluted to 1 μg / mL with 1x PBS, pH 7.4, and 100 μL / well of the ELISA plate (NUNC442404) was added. Coat the plate overnight at 4°C. The plate was dried and 150 μL / well of 1% BSA-PBS was added. The plate was incubated at 37°C for 1 hour. The plate was washed three times using a plate washer according to the protocol. The detection antibody (i.e., primary antibody) was added at different concentrations: 0.06 μg / mL E5-G6 and 2.0 μg / mL E5-G6, at 100 μL / well. The plate was incubated at 37°C for 2 hours. The plate was washed three times using a plate washer according to the protocol. Anti-mouse secondary antibody was added at a 1:2000 dilution, at 100 μL / well. The plate was incubated at 37°C for 1 hour. The plate was washed three times using the protocol. The pNPP substrate solution was added at 100 μL / well. The plate was read on a microplate reader set to a wavelength of 405 nm.

[0092] The results of protein temperature stability test before and after modification are as follows Figure 1-2 shown.

[0093] from Figure 1 、 Figure 2 It can be seen that there are obvious differences in the binding activity of gE protein to antibodies before and after modification at different temperatures. The OD values ​​of the modified gE protein detected at 4°C, 37°C, 60°C, and 80°C using different concentrations of detection antibodies (0.06ug / mL E5-G6, 2.0ug / mL E5-G6) are significantly higher than those of the gE protein before modification. This shows that the modified gE protein prepared by the present invention can still maintain a high antigen binding activity after treatment at different temperatures, that is, compared with the gE protein before modification, the stability of the modified gE protein prepared by the present invention is further enhanced.

[0094] Example 3: Preparation of immune preparations a and b

[0095] (1) Preparation a:

[0096] 0.1 mg / mL of VZV pre-modified gE protein obtained by the method of Example 1 was added to phosphate buffer pH 5.8 buffer, sterilized and filtered with a 0.22 μm membrane, and sterile aluminum phosphate gel (Benetag) was added. The mixture was stirred at 4°C for 1 hour, and aseptically divided into 0.8 mL / bottles and stored at 4°C for immunization.

[0097] (2) Preparation b:

[0098] 0.1 mg / mL of the modified VZV gE protein obtained by the method of Example 1 was added to a phosphate buffer solution at pH 5.8, sterilized and filtered using a 0.22 μm membrane, and sterile aluminum phosphate gel (Benetag) was added. The solution was stirred at 4°C for 1 hour, and aseptically dispensed into 0.8 mL / bottles and stored at 4°C for use in immunization.

[0099] Example 4: Detection of protein antibody titers in immune sera of mice with preparations a and b

[0100] Female BALB / c mice aged 4-6 weeks were randomly divided into two groups. Each group was immunized subcutaneously with 0.1 mL of the immune preparation prepared in Example 3: Preparation a and Preparation b, once every two weeks, for a total of two immunizations. Blood was collected 28 days after immunization, and the blood was then allowed to stand at room temperature for 4 hours and centrifuged at 10,000 RPM at room temperature. The supernatant serum was aspirated and stored at -70°C for testing.

[0101] Prepare a 1 μg / mL stock solution of purified gE 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 three times with plate wash buffer, add 150 μL of blocking buffer, incubate at 37°C for 1 hour, and wash three times with 300 μL per well of plate wash buffer. Store at 4°C for one week.

[0102] Dilute the corresponding test serum obtained from immunized mice to form the working sample serum. Dilute the serum to the appropriate multiple and add 100 μL per well to the first row of wells of the ELISA plate. Perform a 2-fold serial dilution from the first row downwards and incubate at 37°C for 2 hours. Wash each well three times with 300 μL of plate wash buffer. Add 100 μL / well of AP-conjugated goat anti-mouse secondary antibody at a 1:1000 dilution and incubate at 37°C for 1 hour. Wash the plate three times using a plate washer according to the program. Add 100 μL / well of pNPP substrate solution and read the plate on a microplate reader set to a wavelength of 405 nm.

[0103] Figure 3 The test results showed that there were differences in the antibody titers of mice immunized with the gE protein preparations before and after modification. The antibody titer of mice immunized with the gE protein after amino acid modification (preparation b) was significantly higher than that of the gE protein before modification (preparation a). Moreover, after the serum of the immunized mice was diluted at different multiples, the antibody titer of the gE protein after amino acid modification (preparation b) was still significantly higher than that of the gE protein before modification (preparation a) at each concentration. Moreover, as the serum dilution multiple gradually increased, the antibody titers of the gE proteins before and after modification showed a downward trend.

[0104] It can be seen that compared with the gE protein without amino acid modification, the antibody titer detection value of mice immunized with the amino acid modified gE recombinant protein preparation prepared by the present invention is higher, serum with higher protective titer can be obtained, and the immune effect is better.

[0105] Example 5: Preparation of Pneumococcal Capsular Polysaccharide

[0106] (1) Preparation of master seeds and working seeds

[0107] 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:

[0108] 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.

[0109] 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.

[0110] (2) Bacterial fermentation

[0111] Remove a seed tube from the working seed bank and inoculate it into 5 mL of yeast-acid hydrolyzed casein broth. Cultivate at 36°C ± 2°C until the bacterial growth phase reaches the mid-exponential phase. Transfer the bacterial broth into 150 mL of fresh yeast-acid hydrolyzed casein broth and incubate at 36°C ± 2°C for 5-10 hours until the exponential growth phase begins. Transfer 50 mL of the bacterial broth into 2 L of yeast-acid hydrolyzed casein broth and incubate at 36°C ± 2°C until the mid-exponential growth phase to prepare the fermentation seed broth. This fermentation seed broth is then inoculated into a 50 L fermentor containing 30 L of yeast-acid hydrolyzed casein broth. Maintain the fermentation broth pH at 6.8 ± 0.2 with sodium hydroxide until the bacterial growth phase reaches the late exponential phase.

[0112] (3) Capsular polysaccharide purification

[0113] 1. Add phosphoric acid to adjust the pH of the fermentation liquid to between 4 and stir for 1 hour;

[0114] 2. Centrifuge in a disc centrifuge at 9600 rpm, collect the supernatant and discard the residue;

[0115] 3. 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;

[0116] 4. Use a 100kD membrane to concentrate and diafiltrate the microfiltrate to obtain a crude bacterial capsular polysaccharide solution, and then use a buffer to ultrafilter 15 sample volumes using a 30Kd membrane;

[0117] 5. Use a 50kD membrane package to further filter the polysaccharide solution for 10 sample volumes to concentrate the polysaccharide sample solution.

[0118] 6. Collect the purified polysaccharide solution into a freeze-drying bottle, freeze-dry it in a vacuum freeze dryer, and store it at -70°C.

[0119] Example 6: Preparation of Pneumococcal 24 serotypes Pn1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F polysaccharide-VZV recombinant protein conjugates (CDAP method)

[0120] 1) Weigh 17.5 mg of purified capsular polysaccharide of the corresponding serotype and dissolve it in 4 mL of sodium phosphate buffer;

[0121] 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;

[0122] 3) Add 3Eqm cystamine and react at room temperature for 1 hour;

[0123] 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;

[0124] 5) adding 8 Eqm of 3(2-chloroethyl) phosphate to the polysaccharide solution to reduce the disulfide bonds in the polysaccharide;

[0125] 6) Transfer the activated polysaccharide solution to a dialysis bag and dialyze against phosphate buffer at 4°C, changing the buffer four times;

[0126] 7) Weigh 30 mg of carrier protein and dissolve it in phosphate buffer to a protein concentration of 10 mg / mL;

[0127] 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.

[0128] 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;

[0129] 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.

[0130] 11) Transfer the polysaccharide conjugate reaction solution to a dialysis bag and dialyze against phosphate buffer at 4°C;

[0131] 12) The sample solution was loaded onto Sepharose CL-4B, purified, and the bound product in the void volume was collected.

[0132] 13) After filtering with a 0.22 μm filter membrane, store the product at 2-8°C.

[0133] The carrier protein used in step 7) is the modified gE protein, and the gE protein before modification can also be used as the carrier protein.

[0134] Example 7: Preparation of Pneumococcal 24 Serotypes Pn1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F Polysaccharide-VZV Recombinant Protein Conjugates (Reductive Amine Method (Aqueous Phase))

[0135] 1) Weigh 500 mg of purified capsular polysaccharide of the corresponding serotype and dissolve it in 500 mL of purified water;

[0136] 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;

[0137] 3) Using an ultrafiltration filter with a molecular weight of 50Kd membrane, the purified water was ultrafiltered, concentrated, and freeze-dried;

[0138] 4) Weigh 18 mg of activated polysaccharide, add 4 mL of DMSO, and stir to dissolve completely;

[0139] 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;

[0140] 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;

[0141] 7) The sample solution was loaded onto Sepharose CL4B, and the bound fraction (the void volume) was collected.

[0142] 8) After filtering with a 0.22 μm filter membrane, store at 4°C for preparation.

[0143] 9) Take samples to detect the molecular weight of the conjugate, the concentration and ratio of polysaccharide and protein.

[0144] The carrier protein used in step 5) is the modified gE protein, and the gE protein before modification can also be used as the carrier protein.

[0145] Example 8: Preparation of immune preparations c, d, e, f, g

[0146] (1) Preparation c (24-valent pneumococcal polysaccharide-VZV recombinant protein conjugate vaccine)

[0147] The polysaccharide concentration in the monovalent conjugate was detected, and the VZV recombinant protein conjugate solution (the polysaccharide-VZV recombinant protein conjugate was prepared using Example 6, or the polysaccharide-VZV recombinant protein conjugate of Example 7) equivalent to 2.2 μg of polysaccharide (excluding the polysaccharide amount of Pn6B, which was 4.4 μg) was measured, including Pn1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19 A, 19F, 20, 22F, 23F and 33F were placed in a sterile container, and the sampled protein content was about 68 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 mixture was stirred at 4°C for 1 hour, and the mixture was aseptically divided into 0.5 mL / bottle portions and stored at 4°C until use for immunization.

[0148] (2) Formulation d (Pfizer PCV20)

[0149] In June 2021, the U.S. Food and Drug Administration (FDA) approved Pfizer's Prevnar 20 (Pneumococcal 20-valent conjugate vaccine) for adults aged 18 years and above to prevent invasive diseases and pneumonia caused by the 20 pneumococcal serotypes in the vaccine. The 13-valent pneumococcal conjugate vaccine (diphtheria CRM 197 protein) contains capsular polysaccharide conjugates of 13 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F). It also contains capsular polysaccharide conjugates of seven additional serotypes (8, 10A, 11A, 12F, 15B, 22F, and 33F) that cause 40% of pneumococcal disease cases and deaths in the United States and are associated with high mortality, antibiotic resistance, and / or meningitis. The CRM197 used in PCV20 is a carrier protein used in traditional polysaccharide-protein conjugate preparations. This carrier protein enhances the immunogenicity of the polysaccharide in the conjugate, but does not consider the ability of the carrier protein itself to stimulate the body to produce protective antibodies to prevent disease.

[0150] (3) Preparation e (PBS control)

[0151] The protein sampling content is 0 μg, CpG (Genscript) is added to a final amount of 0.1 mg, phosphate buffer pH 5.8 buffer is added, and sterile filtration is performed with a 0.22 μm membrane; sterile aluminum phosphate gel (Benetag) is added to a final aluminum ion amount of 0.125 mg, and the mixture is stirred at 4°C for 1 hour, and aseptically divided into 0.5 mL / bottles and stored at 4°C for immunization.

[0152] (4) Formulation f (gE protein without adjuvant)

[0153] The modified gE protein obtained in Example 1 was sampled at a content of 55 μg, phosphate buffer solution at pH 5.8 was added, sterilized and filtered using a 0.22 μm membrane, stirred at 4°C for 1 hour, aseptically divided into 0.5 mL / bottles, and stored at 4°C for use in immunization.

[0154] (5) Preparation g (containing adjuvant gE protein)

[0155] The modified gE protein obtained in Example 1 was sampled at a content of 55 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 / bottles, and stored at 4°C for immunization.

[0156] Example 9: Immunization of rabbits with preparations and blood collection

[0157] Ten 2.5-3.5 kg New Zealand white rabbits were collected, divided into groups of five. One group received immune preparation c (prepared in Example 8) and the other received immune preparation d (Pfizer PCV20). 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.

[0158] Example 10: ELISA for detecting polysaccharide antibody titers in rabbit immune sera of 24-valent pneumococcal polysaccharide-VZV recombinant protein conjugate vaccine

[0159] Prepare different serotypes of pneumococcal polysaccharide (1× PBS solution) and store in a 4°C refrigerator. Dilute the pneumococcal polysaccharide of the serotype to be tested to 4 μg / mL, add 100 μL of coating solution to each well of the ELISA plate, and 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.

[0160] 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.

[0161] Table 2: Results of IgG antibody titers against 24 serotypes of pneumonia polysaccharide in antisera from rabbits immunized with preparations c and d

[0162]

[0163]

[0164] The results of the IgG antibody titer test of rabbit immunization antisera against pneumococcal polysaccharide-protein conjugate vaccine showed that before immunization with the preparation (D0), the IgG antibody titers of pneumococcal polysaccharide of each serotype originally contained in the rabbit serum were low; after the first (D14) and second (D28) immunizations with the preparation, the 24-valent pneumococcal polysaccharide gE protein conjugate vaccine (preparation c) stimulated the animals to produce the corresponding 24 serotype polysaccharide antibody IgG titers, which showed a significant upward trend. The immune effect of the second injection was significantly better than the first injection, that is, as the number of immunizations increased, the antibody titer increased significantly. In addition, after the second injection (D28), the 24-valent pneumococcal polysaccharide gE protein conjugate vaccine (preparation c) and PCV20 Pfizer (preparation d) stimulated animals to produce corresponding serotype polysaccharide antibody IgG titers. The titers varied according to the serotype. The results for serotypes Pn1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 14, 15B, 18C, 19A, 23F, and 33F showed that preparation c was significantly higher than preparation d, accounting for 80% of the components; serotypes Pn3, Pn12F, Pn19F, and Pn22F preparation d were higher than preparation c, accounting for only 20% of the components. It can be seen that after immunizing animals with preparation c prepared by the present invention, the IgG antibody titer in the animal body can be effectively increased, and the serum protection titer is higher; and compared with the animals before immunization, while the immune effect is significantly enhanced, preparation c is also better than the immune effect of PCV20 preparation d with CRM197 as the carrier protein. It is further illustrated that the gE recombinant protein vector in the polysaccharide-protein conjugate vaccine prepared by the present invention has a good enhancement effect on the antigenicity of 24 types of pneumococcal polysaccharides.

[0165] At the same time, the total average IgG antibody titer against the four newly added serotypes Pn2, Pn9N, Pn17F, and Pn20 in this conjugate vaccine was approximately 271.36, significantly higher than the total average IgG antibody titer of 168.06 against the 20 serotypes of PCV20. This also demonstrates that the 24-valent pneumococcal polysaccharide gE protein conjugate vaccine prepared by the present invention can achieve a better immune effect while covering more pneumococcal serotypes and expanding the vaccine's protection range.

[0166] Example 11: Immunization of rabbits with VZV gE recombinant protein and 24-valent pneumococcal polysaccharide gE protein conjugate and blood collection

[0167] Ten New Zealand white rabbits weighing 2.5-3.5 kg were divided into four groups of five. Each group was immunized with formulations c, e, f, and g prepared in Example 8, respectively. Each rabbit was subcutaneously immunized once every two weeks with 0.5 mL injected per time for a total of two immunizations. One week after immunization, blood was collected and placed 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 testing.

[0168] Example 12: ELISA for detecting gE protein and protein antibody titers in rabbit immune serum of 24-valent pneumococcal gE protein conjugate vaccine

[0169] Prepare a 1 mg / mL stock solution of purified gE 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.

[0170] Serum obtained from rabbits injected with the vaccine and control samples was diluted 1:10 to prepare working serum 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-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 solution was added. The plate was read at 405 nm.

[0171] Table 3: Geometric mean (Eu) of anti-gE IgG antibody titers in immune antisera from different preparations

[0172]

[0173] The test results in Table 3 show that compared with the PBS control (preparation e), the adjuvanted 24-valent polysaccharide-VZV gE protein conjugate vaccine (preparation c), the adjuvanted gE protein (preparation g), and the unadjuvanted gE protein (preparation f) all significantly increased the IgG antibody titers against gE protein in the rabbit immune antisera, with significant increases in total IgG, IgG1, and IgG2a antibody titers. Among them, the protein antibody titers in the rabbit immune antisera of the adjuvanted 24-valent polysaccharide-VZV gE protein conjugate vaccine (preparation c) were the highest, for both total IgG, IgG1, and IgG2a antibody titers, significantly higher than those of the adjuvanted gE protein (preparation g) and the unadjuvanted gE protein (preparation f). The protein antibody titers in the rabbit immune antisera of the adjuvanted gE protein (preparation g) were second highest. This indicates that the gE recombinant protein prepared by the present invention, in addition to enhancing the immune effect of the protein itself, can also be used as a carrier protein to prepare a 24-valent pneumococcal polysaccharide-VZV gE recombinant protein conjugate preparation, which can obtain serum with higher protective titer and has better immunogenicity.

[0174] Example 13: IFN-γ ELISpot and cytokine detection

[0175] Six-week-old female BALB / c mice were randomly divided into four groups of eight mice each. Each group was immunized with Formulation c, Formulation e, Formulation f, and Formulation g prepared in Example 8, respectively. The mice were subcutaneously immunized once every two weeks with 0.1 mL each time for a total of two immunizations. The spleens were collected one week after immunization.

[0176] The harvested spleens were ground, suspended in PBS (pH 7.4), and serially diluted. The resulting single-cell suspension was then applied to an ELISpot plate pre-coated with capture antibody. 5 μg / mL of gE protein, VZV (100 pfu / mL), and concanavalin A (1 μg / mL, positive control) (Sigma-Aldrich) were added and incubated at 37°C, 5% CO₂ for 24 hours. IFN-γ-secreting cells were detected using an ELISpot kit (MabTech), and positive spots were determined using a CTL ImmunoSpot S5UV Micro analyzer.

[0177] Table 4: IFN-γ SFC / 10 in spleen cells of mice immunized with different preparations 6 Cell number

[0178]

[0179] The test results in Table 4 show that, compared to the control PBS (preparation e), the adjuvanted 24-valent polysaccharide-VZV gE protein conjugate vaccine (preparation c), the adjuvanted gE protein (preparation g), and the unadjuvanted gE protein (preparation f) all significantly stimulated immune cells in the spleen of animals, such as NK cells and phagocytes, to produce IFN-γ. The adjuvanted 24-valent polysaccharide-VZV gE protein conjugate vaccine (preparation c) induced higher levels of IFN-γ production in immune cells sensitized with either gE antigen or VZV virus. Furthermore, the IFN-γ production by immune cells sensitized with the adjuvanted 24-valent polysaccharide-VZV gE protein conjugate vaccine (preparation c) was significantly higher than that produced by the adjuvanted gE protein (preparation g) and the unadjuvanted gE protein (preparation f). The amount of IFN-γ produced by splenic immune cells sensitized with the adjuvanted gE protein (preparation g) was higher than that produced by the unadjuvanted gE protein (preparation f). This shows that the gE recombinant protein prepared by the present invention not only has a good immune effect itself, but also the 24-valent pneumococcal polysaccharide-VZV gE recombinant protein conjugate preparation prepared by using it as a carrier protein has a stronger ability to induce cellular immune response in mice and a better effect.

[0180] 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. A composition comprising a multivalent pneumococcal polysaccharide-protein conjugate, characterized in that The carrier protein used in the pneumococcal polysaccharide-protein conjugate in the composition is an immunogenic VZV recombinant protein; the pneumococcal polysaccharide is covalently linked to the VZV recombinant protein; the VZV recombinant protein is selected from gE protein, and its amino acid sequence is shown in SEQ ID NO: 3 or SEQ ID NO:

4.

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

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

4. A composition comprising a multivalent pneumococcal polysaccharide-protein conjugate according to claim 2, characterized in that: The composition comprises a 24-valent pneumococcal polysaccharide-VZV recombinant protein conjugate, wherein the amino acid sequence of the VZV recombinant protein is shown in SEQ ID NO: 4, and the pneumococcal polysaccharide comprises 24 serotypes, namely 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.

5. A composition comprising a multivalent pneumococcal polysaccharide-protein conjugate according to claim 1, characterized in that: The composition contains an adjuvant.

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

7. A composition comprising a multivalent pneumococcal polysaccharide-protein conjugate according to claim 1, characterized in that: The composition is any one of a spray, an injection, a lyophilized agent, a capsule, a tablet or a pill.

8. A method for preparing a composition comprising a multivalent pneumococcal polysaccharide-protein conjugate according to any one of claims 1 to 7, characterized in that: The preparation method comprises the following steps: allowing pneumococcal polysaccharide and VZV recombinant protein to undergo a chemical synthesis reaction in a buffer or an organic solvent, and covalently binding the pneumococcal polysaccharide to the VZV recombinant protein through the reaction to obtain a conjugate.

9. The preparation method according to claim 8, characterized in that The preparation method also includes pre-treatment of pneumococcal polysaccharide, expression and purification of VZV recombinant protein and purification of the conjugate.

10. The preparation method according to claim 9, characterized in that The polysaccharide pretreatment includes degradation and activation, and the degradation method is selected from high-pressure homogenizer degradation, acid hydrolysis or enzyme digestion.

11. The preparation method according to claim 8, characterized in that The chemical synthesis reaction is selected from one of the reductive amine method, the CDAP method, the adipic acid dihydrazide method or the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride method.

12. The preparation method according to claim 8, characterized in that The organic solvent is selected from dimethyl sulfoxide or dimethylformamide.

13. A nucleic acid molecule encoding the VZV recombinant protein according to claim 1, wherein the amino acid sequence is as shown in SEQ ID NO: 3 or SEQ ID NO:

4.

14. A recombinant expression vector containing the nucleic acid molecule according to claim 13.

15. Use of a composition comprising a multivalent pneumococcal polysaccharide-protein conjugate according to any one of claims 1 to 7 in the preparation of a vaccine or a drug for preventing or treating a disease, characterized in that: The disease is a disease caused by Streptococcus pneumoniae infection or a disease caused by VZV infection.

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