A composition comprising a multivalent pneumococcal polysaccharide-protein conjugate, and methods of making and using the same

Abstract

This invention relates to the field of biotechnology, specifically to a composition containing a multivalent pneumococcal polysaccharide-protein conjugate for the prevention of pneumococcal infection and VZV infection, its preparation method, and its application. The composition of this invention, comprising a multivalent pneumococcal polysaccharide-protein conjugate, uses an immunogenic VZV recombinant protein as the carrier 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. This invention addresses the prevalence of serotypes of pneumococcus unique to my country by creatively selecting 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. This results in a composition containing polyvalent pneumococcal polysaccharide-protein conjugates designed specifically for the prevalence of these serotypes in my country.

Description

Technical Field

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

[0002] Streptococcus pneumoniae is a capsule-forming Gram-positive diplococcus. Its outer membrane is composed of a capsular polysaccharide. Based on the differences in the composition of this capsular polysaccharide, 91 different serotypes of Streptococcus pneumoniae have been identified, with the capsular polysaccharide being an important pathogenic factor. Under normal circumstances, Streptococcus pneumoniae resides in the nasopharynx of healthy individuals and generally does not cause clinical symptoms. However, when the parasitic environment changes, such as a weakened immune system, respiratory viral infections like measles or influenza, or conditions such as malnutrition or old age, it can cause invasive infection through the mucosal defense system. Based on the site of infection, pneumococcal disease can be classified 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 Streptococcus pneumoniae, is a global public health challenge posing a serious threat to human health, with an increasingly severe disease burden. Statistics show that in 2019, 33 bacterial infections caused 7.7 million deaths globally, with pneumococcus ranking third, accounting for 829,000 deaths. In terms of lost years of life, pneumococcus ranks first, and deaths from pneumococcal infection are primarily concentrated in children under 5 years old and the elderly over 60 years old. Infants, especially those under 2 years old, are highly susceptible to pneumococcal infection, and their mortality rate is extremely high; approximately 75% of invasive pneumococcal disease and 83% of pneumococcal meningitis occur in this age group (see WHO Position Paper 2019). Similarly, the morbidity and mortality rates of invasive pneumococcal disease in the elderly are also significant. According to reports, pneumonia is the fourth leading cause of death among the elderly in China, causing approximately 125,000 deaths annually. The infection rate varies by region, ranging from 28.0% to 71.5%, and the incidence of pneumonia increases dramatically with age. Meanwhile, in an era of increasingly severe global population aging, the UN's "World Society 2023" report indicates that the global population aged 65 and over was 761 million in 2021, and this number is projected to increase to 1.6 billion by 2050, with the population aged 80 and over growing at an even faster rate. Therefore, the elderly are a high-risk group for pneumococcal infection, and the aging population will inevitably lead to an increasingly heavy burden of pneumococcal disease.

[0004] Antibiotic therapy is a common treatment for pneumococcal disease, but drug resistance to commonly used antibiotics has become an increasingly serious problem globally. Years of clinical practice have proven that pneumococcal vaccination is the most economical and effective way to prevent pneumococcal disease. Currently available vaccines include pneumococcal polysaccharide vaccine (PPV) and pneumococcal conjugate vaccine (PCV). In the 1980s, Wyeth's (acquired by Pfizer in 2009) 23-valent pneumococcal polysaccharide vaccine (PPV23) achieved approximately 90% global immunization coverage of the dominant serotypes, but limitations remain. Studies have found that bacterial polysaccharides are non-T-cell dependent antigens, inducing short-term immune responses in children and adults, but ineffective in infants, especially in children under 2 years old. Through development, polysaccharides can be covalently bound to carrier proteins to convert non-T cell-dependent antigens into T cell-dependent antigens. Pneumococcal polysaccharide-protein conjugate vaccines (PCVs) prepared using this technology can be administered to infants as early as 2 months of age, promoting a stronger immune response and memory response. This is a beneficial immunization method for faster and earlier prevention of pneumococcal disease. The world's first PCV to receive FDA approval in 2000 was PCV7 (Prevnar 7) manufactured by Wyeth. In 2010, Pfizer launched PCV13 (Prevnar 13), which covers more types, replacing PCV7 (Prevnar 7) for routine childhood vaccinations. Currently, three PCVs have received WHO prequalification: GSK's PCV10 (Synflorix), Pfizer's PCV13 (Prevenar 13), and the Serum Institute of India's PCV10 (PNEUMOSIL). In 2021, the FDA approved several higher-priced PCV vaccines, including Pfizer's PCV20 (Prevnar 20) and Merck's PCV15 (VAXNEUVANCE). Domestically, pneumococcal polysaccharide conjugate vaccines, developed by Watson Bio and Minhai Biotechnology, are available. Research has found that conjugate vaccines can be used in any age group and have a longer-lasting and more potent effect than polysaccharide vaccines. Therefore, polysaccharide-protein conjugate vaccines will become the main development trend for pneumococcal vaccines.

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

[0006] Varicella-zoster virus (VZV) belongs to the subfamily Alphaherpesvirinae of the family Herpesviridae and is one of the eight human herpesviruses, also known as human herpesvirus type 3. VZV consists of a nucleocapsid enclosing a linear, double-stranded DNA molecule. A protein layer separates the nucleocapsid from the lipid envelope; this protein layer constitutes the main 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, in infected cells, gE protein is the most abundant protein produced. It adsorbs non-covalently to gI and can adsorb to the Fc fragment of antibody G (IgG), making it a very important antigenic protein.

[0007] The varicella-zoster virus (VZV) is found globally and is highly contagious. To date, only one serotype has been identified, and it infects only humans in nature. Primary infection with VZV manifests as chickenpox, commonly in childhood. After primary infection, the virus can remain latent in the host's sensory neurons. Furthermore, studies have found that the FDA-approved Oka strain live attenuated vaccine for chickenpox prevention, like the wild-type virus, also establishes latent infection. With age and impaired immune function, cellular immune responses weaken, which can induce VZV reactivation and trigger herpes zoster (HZ), which is more common in adults and the elderly. Symptoms include a unilateral vesicular rash, usually accompanied by fever and fatigue. The affected skin initially reddens, accompanied by a burning sensation and nerve pain. Approximately 9% to 34% of shingles patients develop postherpetic neuralgia (PHN), with pain ratings reaching 7 or higher, indicating severe pain that significantly impacts patients' quality of life.

[0008] With the increasing aging of the global population, the incidence and burden of shingles are showing a significant upward trend. In the Asia-Pacific region, the incidence of shingles is approximately 1%, increasing with age, reaching over 5% after age 50. Furthermore, with the continued increase in VZV infection each year, the incidence rate is projected to increase by 35%-100%. Statistics show that by the end of 2023, my country's population aged 60 and above reached nearly 300 million, accounting for 21.1% of the total population, with those aged 65 and above accounting for 15.4%. my country sees 2.77 million shingles patients annually, with over 1.5 million new cases among those aged 50 and above, incurring approximately 1.3 billion yuan in medical expenses. In 2010, there were approximately 9 million shingles patients aged 50 and above in China, with an economic burden exceeding 7.7 billion yuan. However, there is currently no specific drug for treating shingles and postherpetic neuralgia. In clinical practice, broad-spectrum antiviral drugs such as acyclovir are mostly used for treatment. Some anesthetic or non-anesthetic analgesics, as well as anticonvulsants and antidepressants are also used to relieve the severe neuralgia caused by shingles. Moreover, even if shingles is cured, it may still recur. Therefore, VZV vaccination is an effective means of preventing shingles.

[0009] Currently, there are two FDA-approved vaccines for VZV: Merck's Zostavax and GSK's Shingrix. Zostavax is a live attenuated vaccine based on the Oka strain. In individuals aged 60-69, a single subcutaneous injection of Zostavax achieves a 64% efficacy rate. However, the effectiveness of Zostavax declines with age; in adults over 60, the vaccine's efficacy against PHN is only 39%. The other vaccine is GSK's recombinant protein subunit vaccine, Shingrix, which contains gE glycoprotein and AS01B adjuvant. gE is the most abundant glycoprotein in VZV, containing potential neutralizing epitopes and T-cell epitopes, playing a crucial role in viral replication and transmission between ganglion cells. This vaccine, administered via intramuscular injection, showed a 97.4% efficacy against HZV infection in individuals aged 60-69, exceeding 90% in all tested age groups, including those ≥80 years old. This efficacy is superior to Merck's live attenuated vaccine, Zostavax. While the Shingrix recombinant protein vaccine is significantly more effective than Zostavax, its complex formulation, higher production costs, and higher price make it more expensive. Therefore, it is essential for China to independently develop a vaccine product capable of preventing VZV infection.

[0010] For vaccines, a key characteristic is that a single vaccine can prevent infection by a specific pathogen. For example, pneumococcal polysaccharide conjugate vaccine can prevent pneumonia, meningitis, and otitis media caused by pneumococcal infection, while varicella-zoster virus recombinant protein vaccine can prevent shingles caused by varicella-zoster virus reactivation. To achieve the goal of preventing infection by multiple pathogens with a single formulation, combination vaccines are currently used. Examples include trivalent vaccines for diphtheria, pertussis, and tetanus in children; pentavalent vaccines for diphtheria, pertussis, tetanus, poliomyelitis, and Haemophilus influenzae in children; and hexavalent vaccines for diphtheria, pertussis, tetanus, poliomyelitis, Haemophilus influenzae in children, and hexavalent vaccines for hepatitis B. These vaccines are prepared by mixing single-product vaccines. Current clinical evaluations show that the preventive efficacy of some single-product components in combination vaccines is not ideal.

[0011] Currently, children of appropriate age in my country need to receive at least 15 different vaccines, far exceeding the number of vaccine types. Furthermore, with the aging population, by the end of 2024, my country's population aged 60 and above had reached 310 million, accounting for 22% of the total population. Therefore, protecting the health of the elderly and developing vaccine products specifically for them is indispensable. Thus, developing multivalent conjugate vaccines to replace single-drug vaccines and achieving full coverage of vaccine protection will inevitably be the primary task of future vaccine research and development.

[0012] However, in the development of new vaccines, the choice of carrier protein has a significant impact on the vaccine's immunogenicity. Especially with increasing vaccine valence, key challenges in the design and development of polysaccharide-protein conjugate vaccines include: how to select carrier proteins that enhance polysaccharide immunity while avoiding the inhibitory effects of protein carriers; and how to design and obtain stable and highly expressed carrier proteins, while simultaneously combining immunogenic carrier proteins with other immunogenic substances (such as capsular polysaccharides) to prepare vaccines with dual immunogenicity, achieving the goal of protecting against two diseases with a single vaccine. Further development of multivalent pneumococcal conjugate vaccines, covering a wider range of pathogenic serotypes of pneumococcus, including non-vaccine serotypes, to improve the coverage and immunogenicity of novel multivalent vaccines and reduce the risk of immunosuppressive effects from carrier proteins, has significant clinical value. Summary of the Invention

[0013] To overcome the shortcomings of existing technologies, this invention innovatively utilizes polysaccharide-protein binding technology, employing gE protein with enhanced stability through amino acid mutation modification as a carrier protein to develop a composition containing a polyvalent pneumococcal polysaccharide-protein conjugate. The composition prepared by this invention possesses dual immunogenicity, effectively preventing infection by 24 serotypes of pneumococcus and diseases caused by VZV infection simultaneously, significantly reducing the number of vaccinations, lowering immunization costs, and improving the scope and efficacy of immunization coverage.

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

[0015] In a first aspect of the invention, a composition comprising a multivalent pneumococcal polysaccharide-protein conjugate is provided, wherein the pneumococcal polysaccharide-protein conjugate in the composition uses an immunogenic VZV recombinant protein as a carrier 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.

[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 is a composition comprising 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 is a composition comprising a 24-valent pneumococcal polysaccharide-VZV recombinant protein conjugate, wherein the amino acid sequence of the VZV recombinant protein is as 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 of the present invention comprising a polyvalent pneumococcal polysaccharide-protein conjugate 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 of the present invention comprising polyvalent pneumococcal polysaccharide-protein conjugate can be prepared as any one of a spray, injection, lyophilized form, capsule, tablet or pill.

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

[0022] Preferably, the preparation method further includes pretreatment 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 homogenization degradation, acid hydrolysis or enzymatic digestion.

[0024] Preferably, the chemical synthesis reaction is selected from one of the following: the reducing amine method, the CDAP method, the adipic 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 a third aspect of the invention, a nucleic acid molecule is provided that encodes a VZV recombinant protein with an amino acid sequence as described in the first aspect, such as SEQ ID NO: 3 or SEQ ID NO: 4.

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

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

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

[0030] (1) This invention, in response to the prevalence of pneumococcal serotypes unique to my country, creatively selects 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, thereby developing a composition containing polyvalent pneumococcal polysaccharide-protein conjugates designed specifically for the prevalence of pneumococcal serotypes unique to my country. The composition of this invention can effectively prevent invasive infections of 24 different serotypes of pneumococcus and induces relatively balanced and high immunogenicity against all 24 serotypes.

[0031] (2) This invention, through immunogenicity studies of the combination of pneumococcal capsular polysaccharide and carrier protein, creatively designed a carrier protein suitable for binding with polysaccharides of multiple serotypes, namely VZV gE protein. By modifying the gE carrier protein with amino acid mutations, on the one hand, the carrier protein itself maintains its structural stability and antigenic cluster function under different temperature environments, and can still maintain its antigenicity even after undergoing chemical reactions; on the other hand, the gE recombinant protein carrier has a good enhancing and promoting effect on the antigenicity of polysaccharides of 24 serotypes of pneumococcus.

[0032] (3) The composition containing polyvalent pneumococcal polysaccharide-protein conjugate prepared by the present invention has a synergistic effect: On the one hand, as can be seen from Example 10, the polysaccharide-protein conjugate composition prepared by the present invention uses VZV recombinant protein modified by amino acid mutation as the carrier protein, which has a better enhancement effect on the antigenicity of 24 serotypes of pneumococcal polysaccharides. Immunizing animals with formulation c prepared by the present invention can effectively increase the IgG antibody titer in the animals and the serum protective titer is higher; and compared with the animals before immunization, while the immunization effect is significantly enhanced, formulation c is also superior to the immunization effect of Pfizer PCV20 conjugate vaccine with CRM197 as the carrier protein. Before immunization with the formulation (D0), the titers of pneumococcal polysaccharide IgG antibodies in the animal serum were significantly low, making it difficult to resist pneumococcal infection. After the first dose (D14) and the second dose (D28), the 24-valent pneumococcal polysaccharide gE protein conjugate vaccine (formulation c) stimulated the animals to produce corresponding 24-serotype polysaccharide antibody IgG titers, which showed a significant upward trend. The immunization effect of the second dose was significantly better than that of the first dose, that is, the antibody titer was significantly enhanced with the increase of the number of immunizations. Meanwhile, after the second immunization (D28), the 24-valent pneumococcal polysaccharide gE protein conjugate vaccine (formulation c) and PCV20 Pfizer (formulation d) stimulated the animals to produce corresponding serum polysaccharide antibody IgG titers. The titers varied depending on 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 formulation c was significantly higher than formulation d, accounting for 80% of the components.

[0033] On the other hand, as can be seen from Example 12, the gE recombinant protein prepared in this invention, in addition to enhancing the immune effect of the protein itself, can produce serum with higher protective titers and better immunogenicity when used as a carrier protein in the preparation of 24-valent pneumococcal polysaccharide-VZV gE recombinant protein conjugate formulations. Specifically, compared to the control PBS (formulation e), the IgG antibody titers against gE protein in rabbit immune antiserum were significantly enhanced in the adjuvanted 24-valent polysaccharide-VZV gE protein conjugate vaccine (formulation c), the adjuvanted gE protein (formulation g), and the unadjuvanted gE protein (formulation f). The titers of total IgG, IgG1, and IgG2a antibodies were also significantly increased. Furthermore, the protein antibody titers detected in the rabbit immune serum of the adjuvanted 24-valent polysaccharide-VZV gE protein conjugate vaccine (formulation c) were the highest, significantly higher than those of the unconjugate gE recombinant protein formulation.

[0034] Meanwhile, as can be seen from Example 13, the 24-valent polysaccharide-VZV gE protein conjugate composition prepared by this invention can stimulate immune cells in the spleen of animals, such as NK cells and phagocytes, to produce IFN-γ. Furthermore, after immunization with the 24-valent polysaccharide-VZV gE protein conjugate composition, the IFN-γ produced by sensitized immune cells under stimulation by gE antigen or VZV virus is significantly higher than that produced by the gE protein preparation. This indicates that not only does the recombinant gE protein itself have good cellular immune effects, 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, resulting in superior efficacy.

[0035] (4) The composition containing multivalent pneumococcal polysaccharide-protein conjugate prepared by the present invention can achieve better immunization effects while covering more valences of pneumococcal serotypes and expanding the scope of vaccine protection. 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 about 271.36, which is significantly higher than the total average antiserum IgG antibody titer of 20 serotypes of PCV20 (168.06).

[0036] (5) The present invention also provides a method for preparing a composition of polysaccharide-protein conjugates of multivalent pneumococcus, wherein the VZV gE recombinant protein is an amino acid sequence designed by gene recombination technology. The stability of the gE protein carrier is enhanced after the amino acid is mutated and modified. After the conjugate is combined with a novel adjuvant preparation and immunized with animals, the polysaccharide antigen part of the conjugate can stimulate the body to produce specific serum polysaccharide antibodies, and the carrier protein part can stimulate the body to produce humoral immunity (protective antibodies) and cellular immunity against VZV virus. After immunization with one preparation, it can simultaneously prevent infection with 24 kinds of pneumococcus and herpes zoster, achieving the goal of one vaccine preventing two diseases. Attached Figure Description

[0037] Figure 1 Temperature stability of gE protein before and after modification (0.06 ug / mL).

[0038] Figure 2 Temperature stability of gE protein before and after modification (2.0 ug / mL).

[0039] Figure 3 To detect the protein antibody titers in mouse immune serum of formulation a (unmodified gE) and formulation b (modified gE). Detailed Implementation

[0040] To more concisely and clearly demonstrate the technical solution, purpose, and advantages of the present invention, the technical solution of the present invention is described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the instruments, equipment, reagents, materials, etc., used can all be obtained through conventional commercial means unless otherwise specified.

[0041] Example 1: Preparation of carrier protein

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

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

[0044] (I) Protein Construction

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

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

[0047] The VZV gE protein mutant provided by this invention mainly involves modifying the amino acid sequence of the mature antigen region of a selected truncated protein to optimize protein stability and immunogenicity. Furthermore, this mutant differs from the amino acid sequence of the VZV gE protein included in the NCBI database; its specific sequence is shown in SEQ ID NO.4. Additionally, the specific sequence of the unmodified VZV gE protein provided by this invention is shown in SEQ ID NO.3.

[0048] (1) Amino acid sequence design

[0049] This invention relates to a recombinant VZV gE protein with enhanced stability and immunogenicity obtained through amino acid mutation modification, the amino acid sequence of which is designed as follows:

[0050] The transmembrane and intracellular regions of the full-length VZV gE protein sequence were deleted to obtain the truncated gE protein sequence shown in SEQ ID NO.2. Then, amino acid point mutations were performed on this sequence. 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, the Throm and 6his sequences were attached to the C end to obtain the full-length mutant VZV gE protein sequence, the amino acid sequence of which is shown in SEQ ID NO.4.

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

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

[0053] The sequences SEQ ID NO.1 through SEQ ID NO.4 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] gE before VZV modification

[0063]

[0064] SEQ ID NO.4:

[0065] >VZV modified gE D277C L289C

[0066]

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

[0068] Based on the designed VZV gE protein amino acid sequences SEQ ID NO.3 and SEQ ID NO.4 and the codon bias of the host cell, the corresponding gene coding sequence was determined, and the restriction endonuclease EcoRI sequence was added to the C-terminus of the gene segment, and the restriction endonuclease XbaI sequence was added to the N-terminus, and the designed nucleotide sequence was chemically synthesized.

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

[0070] The plasmid vector pUC19 was digested with EcoRI and XbaI restriction enzymes and ligated to the synthesized gene. This ligation was then introduced into the host DH5α for amplification. Single clones were screened using LB (Amp+) agar solid medium. Single clones containing the target gene were inoculated into LB (Amp+) liquid medium and amplified at 37°C and 200 rpm. The plasmid pUC19-gE was extracted using the Sigma-Aldrich GenElute™ HP plasmid medium-scale preparation kit. The target gene fragment was recovered from the plasmid extracted by double digestion with EcoRI and XbaI restriction enzymes using the TaKaRaMiniBestAgaroseGel Extraction Kit.

[0071] (4) Construction of eukaryotic expression vectors

[0072] The mammalian cell expression plasmid pGN-M, containing the CMV promoter and dihydrofolate reductase (DHFR) gene, was digested with EcoRI and XbaI restriction enzymes. The vector DNA fragment was recovered using the TaKaRa MiniBEST DNA Fragment Purification Kit Ver. 4.0. The vector DNA fragment and the target gene fragment were ligated at sticky ends and introduced into the DH5α amplification host. Single clones containing the eukaryotic expression plasmid pGN-M_gE were obtained by screening. The clones were inoculated into LB (Amp+) cells for amplification culture. The amplified plasmid was extracted using the TaKaRa MidiBEST Endo-free Plasmid Purification Kit and named VZVgE.

[0073] (II) Expression and Cloning Screening of gE Protein in CHO Cells

[0074] CHO K1 (ATCC) cells were used as host cells. After cell resuscitation, the cells were cultured in DMEM medium (Sigma-Aldrich) containing 10% newborn calf serum and passaged every 3 days. After two passages, the cells showed good growth. Then, CHO K1 cells were cultured at a rate of 0.75 × 10⁻⁶ cells / year. 6 Three 9.6cm cells / wells 2 The cells were placed in wells containing Iscove's optimized DMEM medium (Sigma-Aldrich) and 10% fetal bovine serum (IMEM+FBS) (Gibco). Cells were incubated in a humidity-saturated incubator at 37°C and 5% CO2. Each well contained 4 μg of the pcDNAVZVE vector. DNA was mixed with Lipofectamine 2000 (Sigma-Aldrich) and added to two wells. Lipofectamine 2000 was added separately to a third well as a negative control. After 48 hours, the medium was removed, and the cells were centrifuged at 200×g for 5 minutes. The supernatant was stored at -20°C. Add IMDM+FBS culture medium and 10 μg / mL Blastidin-HCl (Invitrogen) to one well of transfected cells. Wash the other well with PBS, then lyse the cells with a mixture of 50 mM Tris-HCl, pH 8, 150 mM NaCl, and 1% (v / v) Triton X-100 containing complete, EDA-free protease inhibitors (Roche Diagnostics). Centrifuge at 16000×g for 10 minutes at 4°C, and store the lysate at -20°C. Use Western blot to detect the presence of recombinant protein in the supernatant and lysate. After culturing in selective medium for 5 days, wash the cells with trypsin (Invitrogen) and then seed them onto 9 cm Petri dishes for serial dilutions to isolate single clones. Over subsequent days 7-11, select 42 single clones and transfer them to wells of a 96-well plate. Use Western blot to analyze the culture supernatant and screen for high expression of the protein gE. The clone that secretes the highest amount of protein is selected for the next round of screening, and the cells are eventually expanded. Thirty new clones are then selected and preserved.

[0075] The selected clones were amplified into three T175 flasks (NETS). After trypsin digestion and washing with PBS, the clones were resuspended in 250 mL rotating flasks containing 100 mL of ProCHO4 (Lonza), 1×ProHT, 4 mM L-glutamine, and 2% FBS (Lonza). The flasks were incubated at 37°C with 5% CO2 at 90 rpm with the cap slightly ajar to allow air diffusion. Cells were sampled daily, stained with trypan blue (Sigma-Aldrich), and counted. Cells were passaged every 3–5 days. After a plateau phase (when the viable cell concentration exceeded 0.3 × 10⁶ cells / mL and the viable cell count exceeded 90%), the cells were gradually removed once they had adapted and grown well. At this point, the cells were considered fully adapted for serum-free suspension growth.

[0076] (III) Production of gE protein in a bioreactor

[0077] A 1.5-liter perfusion culture was configured in a bioreactor, equipped with a rotary filter (10 μm). Culture parameters were set as follows: temperature controlled at 37°C using a heating blanket; pH adjusted to 6.9 using CO2 or 0.3M sodium hydroxide; stirring speed at 200-300 RPM; dissolved oxygen (dO2) adjusted to 40% of saturated air using a N2 and O2 mixture at a maximum flow rate of 200 mL / min. The perfusion rate was 0.3 to 0.8 V dilutions / day, and cell counting was performed daily by sampling the culture medium. Trypan blue staining was used, and glucose and lactate concentrations in the supernatant were detected offline.

[0078] A total of 12.5 liters of cell-free culture medium was collected, centrifuged at 8000×g for 30 minutes at 4°C, filtered through a 0.45 μm membrane, and then concentrated by ultrafiltration using a 10 kDa membrane. The sample was washed with buffer and concentrated to 0.5 liters. 0.5 liters of PBS were added, and the concentration was further reduced to 0.5 liters. This process was repeated five times.

[0079] (iv) Purification of gE protein

[0080] The sample solution was loaded onto a Q-Sepharose fast flow (GE Bioscience) column and washed with 20 mM Tris-HCl pH 7.5. Then, the column was washed with 20 mM Tris-HCl pH 7.5 containing 200 mM sodium chloride to further remove adsorbed protein impurities. gE protein was eluted with a solution containing 300 mM sodium chloride. Ammonium sulfate was added to the combined solution to a concentration of 800 mM, and the solution was loaded onto a Butyl-Sepharose (GE Bioscience) column. The column was washed with phosphate-buffered saline (PBS, 6 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 0.15 M sodium chloride pH 6.8) containing 800 mM ammonium sulfate, followed by washing with PBS containing 400 mM ammonium sulfate. Finally, gE protein was eluted with purified water. Finally, the sample was loaded onto a Sephacryl S-400HR (GEBioscience), the column was washed with PBS, the protein peak was collected, a co-solvent was added, and the column was freeze-dried in a vacuum freeze dryer and stored at -70°C for later use.

[0081] Both gE proteins before and after VZV modification were prepared using the methods described above.

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

[0083] 1) Dilute the test proteins (the unmodified gE protein prepared in Example 1 and the modified gE protein prepared in Example 1) to 20ug / mL with 1*PBS pH7.4 buffer and place them in a 1.5mL centrifuge tube with a total volume of 1mL.

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

[0085] Table 1: Incubation conditions of proteins at different temperatures

[0086] Serial Number Temperature (°C) Time (min) Quantity (pieces) Concentration (ug / mL, diluted with 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℃.

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

[0089] BALB / c mice were immunized with gE protein vaccine (GSK) at a dose of 100 μg per mouse. After two immunizations, B lymphocytes were extracted from the spleen. Then, monoclonal antibody mAbs (numbered E5-G6) against VZV-gE were prepared, cloned and screened using hybridoma cell technology.

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

[0091] Dilute the sample stored at 4℃ to 1ug / mL with 1*PBS pH 7.4, add 100µL / well to a microplate (NUNC442404), and coat overnight at 4℃. Shake the plate dry, add 150µL / well of 1% BSA-PBS, and incubate at 37℃ for 1 hour. Wash the plate three times according to the programmed procedure, adding different concentrations of detection antibody (i.e., primary antibody) as designed: 0.06µg / mL E5-G6, 2.0µg / mL E5-G6, 100µL / well, and incubate at 37℃ for 2 hours. Wash the plate three times according to the programmed procedure, add anti-mouse secondary antibody at a 1:2000 dilution, 100µL / well, and incubate at 37℃ for 1 hour. Wash the plate three times according to the programmed procedure, add pNPP substrate solution, 100µL / well, set the microplate reader to 405nm, and read the value.

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

[0093] from Figure 1 , Figure 2 As can be seen, the binding activity of gE protein to antibodies before and after modification differs significantly at different temperatures. The OD values ​​of the modified gE protein, detected using different concentrations of detection antibodies (0.06 ug / mL E5-G6 and 2.0 ug / mL E5-G6) at 4℃, 37℃, 60℃, and 80℃, were significantly higher than those of the unmodified gE protein. This indicates that the modified gE protein prepared in this invention can maintain high antigen-binding activity even after treatment at different temperatures; that is, compared to the unmodified gE protein, the stability of the modified gE protein prepared in this invention is further enhanced.

[0094] Example 3: Preparation of immunomodulatory agents a and b

[0095] (1) Formulation a:

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

[0097] (2) Formulation b:

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

[0099] Example 4: Detection of protein antibody titers in mouse immune serum of formulations a and b

[0100] Female BALB / c mice aged 4-6 weeks were randomly divided into two groups. Each group received an immunization using formulations prepared in Example 3: formulation a and formulation b. Subcutaneous immunization was performed every two weeks, with 0.1 mL administered each time, for a total of two immunizations. Blood was collected 28 days after immunization. The blood was then left at room temperature for 4 hours, centrifuged at 10,000 RPM at room temperature, and the supernatant serum was collected and stored at -70°C for testing.

[0101] Prepare a purified gE protein stock solution of 1 μg / mL (1×PBS solution) and store at 4°C. Dilute the protein stock solution to 4 μg / mL coating buffer, add 100 μL of coating solution to each well to coat the ELISA plate, and incubate overnight at room temperature. Wash three times with wash buffer, add 150 μL of blocking buffer, incubate at 37°C for 1 hour, wash three times with 300 μL of wash buffer per well, and it can be stored at 4°C for one week.

[0102] Dilute the corresponding test serum obtained after mouse immunization to prepare working sample serum. Add 100 μl to each well in the first row of ELISA plates, starting with the first row and performing serial 2-fold dilutions downwards. Incubate at 37°C for 2 hours. Wash each well three times with 300 μl of wash buffer. Add 100 μL of AP-labeled goat anti-mouse secondary antibody at a 1:1000 dilution and incubate at 37°C for 1 hour. Wash the plate three times according to the programmed sequence. Add 100 μL of pNPP substrate solution per well. Set the microplate reader to 405 nm and read the value.

[0103] Figure 3 The test results showed that there were differences in antibody titers between mice immunized with gE protein preparations before and after modification. Specifically, the antibody titer of mice immunized with amino acid-modified gE protein (preparation b) was significantly higher than that of mice immunized with unmodified gE protein (preparation a). Furthermore, even after different dilutions of the immunized mouse serum, the antibody titer of amino acid-modified gE protein (preparation b) remained significantly higher than that of unmodified gE protein (preparation a) at all concentrations. Moreover, as the serum dilution factor gradually increased, the antibody titers of both unmodified and modified gE proteins showed a decreasing trend.

[0104] Therefore, compared with unmodified gE protein, the amino acid-modified recombinant gE protein preparation prepared in this invention produces higher antibody titers in mice immunized with it, resulting in serum with higher protective titers and better immunization effects.

[0105] Example 5: Preparation of Pneumococcal Capsular Polysaccharide

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

[0107] The specific method for purifying capsular polysaccharides from the fermentation broth of pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F is as follows:

[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 cultures from the lyophilized seed tubes were inoculated into 5 ml of yeast-acid-hydrolyzed casein culture medium and cultured at 36℃±2℃ for 18 hours. When the bacterial growth reached an OD600 reading of 1.0, the culture was transferred to 150 ml of fresh yeast-acid-hydrolyzed casein culture medium and cultured at 36℃±2℃ for 5-10 hours until the exponential growth phase. The culture was then stopped, aliquoted, lyophilized, and stored as the primary seed culture at 2-8℃.

[0109] The bacterial cultures of pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F were inoculated into 5 mL of yeast-acid-hydrolyzed casein culture medium and cultured at 36℃±2℃ for 18 hours. When the bacterial growth reached an OD600 reading of 1.0, the culture was transferred to 150 mL of fresh yeast-acid-hydrolyzed casein culture medium and cultured at 36℃±2℃ for 8 hours until the exponential growth phase. The culture was then stopped, aliquoted, freeze-dried, and stored at 4℃ as working seed cultures for the serotypes.

[0110] (2) Bacterial fermentation

[0111] Seed tubes were taken from the working seed bank and inoculated into 5 mL of yeast-acid-hydrolyzed casein culture medium. The culture was incubated at 36℃±2℃ until the mid-exponential growth phase. The culture was then transferred to 150 mL of fresh yeast-acid-hydrolyzed casein culture medium and incubated at 36℃±2℃ for 5-10 hours until the exponential growth phase. 50 mL of the culture was then transferred to 2 L of yeast-acid-hydrolyzed casein culture medium and incubated at 36℃±2℃ until the mid-exponential growth phase to prepare the fermentation seed culture. This seed culture was then inoculated into a 50 L fermenter containing 30 L of yeast-acid-hydrolyzed casein culture medium. The pH of the fermentation broth was maintained at 6.8±0.2 using sodium hydroxide until the bacteria reached the late exponential growth phase.

[0112] (3) Purification of capsular polysaccharides

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

[0114] 2. Centrifuge using a disc centrifuge at a speed of 9600 rpm, collect the supernatant, and discard the residue.

[0115] 3. Use a microfiltration membrane to microfilter the centrifuged liquid to remove residual cell debris and insoluble small particulate matter. Use a 0.22μm membrane to microfilter the fermentation centrifuged supernatant and collect the filtrate.

[0116] 4. The microfiltrate was concentrated and washed using a 100kD membrane to obtain a crude bacterial capsular polysaccharide solution. Then, 15 sample volumes were washed with buffer and ultrafiltration using a 30kD membrane.

[0117] 5. Use a 50kD membrane to further wash and 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℃.

[0119] Example 6: Preparation of polysaccharide-VZV recombinant protein conjugates of 24 serotypes of Streptococcus pneumoniae (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) (CDAP method)

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

[0121] 2) Add 12 mg of 1-cyano-4-dimethylammonium pyridine tetrafluoroborate (CDAP) (Sigma-Aldrich) to the polysaccharide solution, stir, and react at room temperature for 1 hour;

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

[0123] 4) Add 0.3 mL of 1M lysine (Sigma-Aldrich) solution to quench the reaction, and react at room temperature for 1-2 hours;

[0124] 5) Add 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 achieve a protein concentration of 10 mg / mL;

[0127] 8) Add 8 mg of N-hydroxysuccinimide bromoacetate (BAANS) (Sigma-Aldrich) to the carrier protein solution and react at room temperature for 2 hours. Transfer the activated protein solution to a dialysis bag (Thermo Scientific) and dialyze against phosphate buffer at 4°C, changing the buffer four times.

[0128] 9) Mix 4 mL of activated polysaccharide solution with 4 mL of activated protein solution and react at room temperature for 4 hours;

[0129] 10) Add 4 Eqm of N-acetyl-L-cysteine ​​(Sigma-Aldrich), react at 2-8℃ for 4 hours, add 12 Eqm of iodoacetamide (Sigma-Aldrich), and react at 2-8℃ for 4 hours;

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

[0131] 12) Load the sample solution onto Sepharose CL-4B, purify it, and collect the external water volume bound.

[0132] 13) After filtration through a 0.22μm filter membrane, store at 2-8℃ until preparation is complete.

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

[0134] Example 7: Preparation of polysaccharide-VZV recombinant protein conjugates of 24 serotypes of Streptococcus pneumoniae (Pn1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F) (reduced amine method (aqueous phase))

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

[0136] 2) Degradation was carried out using a high-pressure homogenizer at a pressure of 600 bar for three cycles, with the addition of 0.12 eqm sodium periodate (Sigma-Aldrich), and the reaction was carried out in the dark for 18 hours.

[0137] 3) Use ultrafiltration to wash the purified water with a membrane with a molecular weight of 50Kd, then concentrate and freeze-dry it;

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

[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 quenching the reaction with 2 Eqm sodium borohydride (Sigma-Aldrich) for 4 hours, transfer the synthesis reaction solution to a dialysis bag, dialyze against the buffer solution, and change the solution four times.

[0141] 7) Load the sample solution onto Sepharose CL4B and collect the bound portion of the external water volume.

[0142] 8) After filtration through a 0.22μm filter membrane, store at 4℃ until preparation.

[0143] 9) Sample and test the molecular weight of the conjugate, as well as the concentration and ratio of polysaccharides and proteins.

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

[0145] Example 8: Preparation of immunomodulatory agents c, d, e, f, and g

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

[0147] The polysaccharide concentration in the monovalent conjugates was determined by measuring 2.2 μg of VZV recombinant protein conjugate solution (the polysaccharide-VZV recombinant protein conjugate prepared in Example 6, or the polysaccharide-VZV recombinant protein conjugate from Example 7) equivalent to 4.4 μg of polysaccharide, 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 transferred to sterile containers. Samples were taken to determine the protein content, which was approximately 68 μg. CpG (Genscript) was added to a final volume of 0.1 mg. Phosphate buffer (pH 5.8) was added, and the mixture was sterilely filtered through a 0.22 μm membrane. Sterile aluminum phosphate gel (Benetag) was added to a final aluminum ion content of 0.125 mg. The mixture was stirred at 4°C for 1 hour, aseptically dispensed into 0.5 mL vials, and stored at 4°C for use in immunization.

[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 use in adults 18 years of age and older to prevent invasive disease and pneumonia caused by the 20 pneumococcal serotypes contained in the vaccine. Pfizer Prevnar 20 (PCV20) contains, in addition to the ingredients already listed in PREVENAR... The pneumococcal 13-valent conjugate vaccine [Diphtheria CRM 197 protein] contains capsular polysaccharide conjugates for 13 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F), as well as capsular polysaccharide conjugates for seven other serotypes (8, 10A, 11A, 12F, 15B, 22F, and 33F) that account for 40% of pneumococcal disease cases and deaths in the United States. These conjugates are associated with high mortality, antibiotic resistance, and / or meningitis. The CRM197 used in PCV20 is a carrier protein used in the preparation of conventional polysaccharide-protein conjugates. This carrier protein enhances the immunogenicity of the polysaccharides in the conjugate, but it does not consider the carrier protein itself stimulating the body to produce protective antibodies to prevent disease.

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

[0151] The protein sample was 0 μg. CpG (Genscript) was added to a final volume of 0.1 mg. Phosphate buffer (pH 5.8) was added, and the sample was sterilely filtered through a 0.22 μm membrane. Sterile aluminum phosphate gel (Benetag) was added to a final aluminum ion content of 0.125 mg. The sample was stirred at 4 °C for 1 hour, aseptically dispensed into 0.5 mL / bottle, and stored at 4 °C for use in immunization.

[0152] (4) Formulation f (adjuvant-free gE protein)

[0153] The modified gE protein obtained in Example 1 was sampled at a concentration of 55 μg. It was added to phosphate buffer (pH 5.8), sterilized and filtered through a 0.22 μm membrane, stirred at 4°C for 1 hour, aseptically dispensed into 0.5 mL / bottle, and stored at 4°C for use in immunization.

[0154] (5) Formulation g (containing adjuvant g E protein)

[0155] The modified gE protein obtained in Example 1 was sampled at a concentration of 55 μg. CpG (Genscript) was added to a final amount of 0.1 mg. Phosphate buffer (pH 5.8) was added, and the mixture was sterilely filtered through a 0.22 μm membrane. Sterile aluminum phosphate gel (Benetag) was added to a final aluminum ion concentration of 0.125 mg. The mixture was stirred at 4°C for 1 hour, aseptically dispensed into 0.5 mL / bottle, and stored at 4°C for use in immunization.

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

[0157] Ten New Zealand white rabbits weighing 2.5-3.5 kg were selected and divided into groups of five. One group received immunization agent c (prepared in Example 8), and the other group received immunization agent d (Pfizer PCV20). Each rabbit was injected with 0.5 mL of the immunization agent at two separate times: week 0 and week 2. Blood samples were collected three times: before week 0 immunization, one week after the first immunization, and one week after the second immunization. A portion of the blood was prepared into PBMCs, flash-frozen on dry ice, and stored at low temperature. The other portion of the blood was left at room temperature for 4 hours, centrifuged at 10,000 RPM at room temperature, and the supernatant serum was collected and stored at -70°C for later analysis.

[0158] Example 10: ELISA detection of polysaccharide antibody titers in rabbit serum immunized with 24-valent pneumococcal polysaccharide-VZV recombinant protein conjugate vaccine

[0159] Prepare different serotypes of pneumococcal polysaccharides (1×PBS solution) and store at 4°C. Dilute the polysaccharide of the desired serotype of pneumococcal polysaccharide to 4 μg / mL, add 100 μL of coating solution to each well to coat the ELISA plate, and incubate overnight at room temperature. Wash four times with wash buffer, add 100 μL of blocking buffer, incubate for 2 hours at room temperature, wash four times with wash buffer, and store at 4°C for one week.

[0160] The corresponding test serum obtained from rabbit vaccination and control samples was diluted 1:10 to prepare working sample serum. The appropriate dilution factor was added to the first row of wells in an ELISA plate, with a total volume of 200 μL. Serial dilutions of 2-fold were performed from the first row downwards, and the plates were incubated at room temperature for 2 hours. The plates were washed four times with wash buffer, and 100 μL of alkaline phosphatase-labeled goat anti-rabbit antibody (1:2000 dilution) was added. The plates were incubated at room temperature for 4 hours. The plates were washed four times with wash buffer, and 100 μL of disodium 4-nitrobenzene phosphate substrate (Sigma-Aldrich) solution was added. The plates were read at 405 nm.

[0161] Table 2: Detection results of 24 serum types of pneumopolysaccharide IgG antibody titers in rabbit antiserum after immunization with formulations c and d.

[0162]

[0163]

[0164] The results of the detection of IgG antibody titers in rabbit antiserum immunized with pneumococcal polysaccharide-protein conjugate vaccine showed that before immunization (D0), the titers of IgG antibodies against various serotypes of pneumococcus in rabbit serum were low. After the first dose (D14) and the second dose (D28), the titers of IgG antibodies against the corresponding 24 serotypes stimulated by the 24-valent pneumococcal polysaccharide gE protein conjugate vaccine (formulation c) showed a significant upward trend. The immunization effect of the second dose was significantly better than that of the first dose, that is, the antibody titers increased significantly with the number of immunizations. Furthermore, after the second immunization (D28), the 24-valent pneumococcal polysaccharide gE protein conjugate vaccine (formulation c) and PCV20 Pfizer (formulation d) stimulated animals to produce corresponding serum polysaccharide antibody IgG titers, which varied depending on the serotype. For serotypes Pn1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 14, 15B, 18C, 19A, 23F, and 33F, the results showed that formulation c was significantly higher than formulation d, accounting for 80% of the component; for serotypes Pn3, Pn12F, Pn19F, and Pn22F, formulation d was higher than formulation c, accounting for only 20% of the component. Therefore, immunization with formulation c prepared according to this invention can effectively increase the IgG antibody titer in animals, resulting in higher serum protective efficacy. Moreover, compared to before immunization, formulation c significantly enhances the immunization effect and is superior to the immunization effect of formulation d, which uses PCV20 with CRM197 as the carrier protein. This further demonstrates that the gE recombinant protein carrier in the polysaccharide-protein conjugate vaccine prepared by this invention has a good enhancing effect on the antigenicity of 24 kinds of pneumococcal polysaccharides.

[0165] Meanwhile, the average total titer of antiserum IgG antibodies against the four newly added serotypes Pn2, Pn9N, Pn17F, and Pn20 in this conjugate vaccine is approximately 271.36, significantly higher than the average total titer of polysaccharide IgG antibodies against the 20 serotypes of PCV20 (168.06). This also demonstrates that the 24-valent pneumococcal polysaccharide gE protein conjugate vaccine prepared in this invention, while covering more valences of pneumococcal serotypes and expanding the vaccine's protective range, also achieves superior immunization efficacy.

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

[0167] Ten New Zealand white rabbits weighing 2.5-3.5 kg were taken and divided into four groups of five. Each group was immunized with formulations c, e, f, and g prepared in Example 8, respectively. Subcutaneous immunization was performed every two weeks, with each rabbit injected with 0.5 mL per immunization, for a total of two immunizations. Blood was collected one week after immunization. The collected blood was left at room temperature for 4 hours, centrifuged at 10,000 RPM at room temperature, and the supernatant serum was collected and stored at -70°C for testing.

[0168] Example 12: ELISA detection of protein antibody titers in rabbit immune serum from gE protein and 24-valent pneumococcal polysaccharide gE protein conjugate vaccine.

[0169] Prepare a purified gE protein stock solution of 1 mg / mL (1×PBS solution) and store at 4°C. Dilute the protein stock solution to 4 μg / mL coating buffer, add 100 μL of coating solution to each well to coat the ELISA plate, and incubate overnight at room temperature. Wash four times with wash buffer, add 100 μL of blocking buffer, incubate for 2 hours at room temperature, wash four times with wash buffer, and it can be stored at 4°C for one week.

[0170] The corresponding test serum obtained from rabbit vaccination and control samples was diluted 1:10 to prepare working sample serum. The appropriate dilution factor was added to the first row of wells in an ELISA plate, with a total volume of 200 μL. Serial dilutions of 2-fold were performed from the first row downwards, and the plates were incubated at room temperature for 2 hours. The plates were washed four times with wash buffer, and 100 μL of alkaline phosphatase-labeled goat anti-rabbit antibody (1:2000 dilution) was added. The plates were incubated at room temperature for 4 hours. The plates were washed four times with wash buffer, and 100 μL of disodium 4-nitrobenzene phosphate substrate solution was added. The plates were read at 405 nm.

[0171] Table 3: Geometric mean (Eu) of anti-gE IgG antibody titers in different formulations of immunoseptic antiserum

[0172]

[0173] Table 3 shows that, compared with the control PBS (formulation e), the titers of anti-gE protein IgG antibodies in rabbit immune antiserum were significantly enhanced in all three vaccines: adjuvanted 24-valent polysaccharide-VZV gE protein conjugate vaccine (formulation c), adjuvanted gE protein (formulation g), and unadjuvanted gE protein (formulation f). The titers of total IgG, IgG1, and IgG2a antibodies were also significantly increased. Specifically, the titers of total IgG, IgG1, and IgG2a antibodies were highest in rabbit immune serum from the adjuvanted 24-valent polysaccharide-VZV gE protein conjugate vaccine (formulation c), significantly higher than those from adjuvanted gE protein (formulation g) and unadjuvanted gE protein (formulation f); the titers of adjuvanted gE protein (formulation g) were the second highest. This invention demonstrates that the gE recombinant protein prepared in this invention, in addition to enhancing the immune effect of the protein itself, can also produce serum with higher protective titers and better immunogenicity when used as a carrier protein to prepare 24-valent pneumococcal polysaccharide-VZV gE recombinant protein conjugate formulations.

[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 each. Each group was immunized with formulations c, e, f, and g prepared in Example 8, respectively. Subcutaneous immunization was performed every two weeks, with 0.1 mL administered each time, for a total of two immunizations. Spleens were collected one week after immunization.

[0176] After collecting and grinding the spleens, PBS (pH 7.4) was added for resuscitation. The cells were then serially diluted, and the resulting single-cell suspension was added to ELISpot plates pre-coated with capture antibodies. 5 μg / mL gE protein, VZV (100 pfu / mL), and concanavalin A (1 μg / mL, positive control) (Sigma-Aldrich) were added, and the plates were incubated at 37°C and 5% CO2 for 24 hours. IFN-γ secretory cells were detected using an ELISpot kit (MabTech), and positive spots were identified using a CTL ImmunoSpot S5UV Micro analyzer.

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

[0178]

[0179] Table 4 shows that, compared to the control PBS (formulation e), the adjuvanted 24-valent polysaccharide-VZV gE protein conjugate vaccine (formulation c), the adjuvanted gE protein (formulation g), and the unadjuvanted gE protein (formulation 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 (formulation c) produced higher levels of IFN-γ by sensitized immune cells under stimulation by gE antigen or VZV virus. Furthermore, the IFN-γ produced by immune cells sensitized with the adjuvanted 24-valent polysaccharide-VZV gE protein conjugate vaccine (formulation c) was significantly higher than that produced by the adjuvanted gE protein (formulation g) and the unadjuvanted gE protein (formulation f). The amount of IFN-γ produced by spleen immune cells sensitized with the adjuvanted gE protein (formulation g) was higher than that produced by the unadjuvanted gE protein (formulation f). This indicates that the gE recombinant protein prepared in this invention, in addition to having good immune effects, has a stronger ability to induce cellular immune responses in mice when used as a carrier protein to prepare 24-valent pneumococcal polysaccharide-VZV gE recombinant protein conjugate formulations.

[0180] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention pending approval.

Claims

1. A composition comprising a multivalent pneumococcal polysaccharide-protein conjugate, characterized in that, The pneumococcal polysaccharide-protein conjugate in the composition uses an immunogenic VZV recombinant protein as the carrier 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: 4; 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.

2. The composition comprising a polyvalent pneumococcal polysaccharide-protein conjugate according to claim 1, 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.

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

4. The composition comprising a polyvalent pneumococcal polysaccharide-protein conjugate according to claim 3, 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.

5. The composition comprising a polyvalent pneumococcal polysaccharide-protein conjugate according to claim 1, characterized in that, The composition is any one of a spray, injection, capsule, tablet or pill.

6. A method for preparing a composition comprising a polyvalent pneumococcal polysaccharide-protein conjugate as described in any one of claims 1-5, characterized in that, The preparation method includes the following steps: chemically synthesizing pneumococcal polysaccharide and VZV recombinant protein in a buffer or organic solvent, thereby covalently binding pneumococcal polysaccharide to VZV recombinant protein to obtain a conjugate.

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

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

9. The preparation method according to claim 6, characterized in that, The chemical synthesis reaction is selected from one of the following: the reducing amine method, the CDAP method, the adipamide dihydrazine method, or the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride method.

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

11. A nucleic acid molecule encoding the VZV recombinant protein with the amino acid sequence as described in claim 1, as shown in SEQ ID NO:

4.

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

13. The use of a composition comprising a multivalent pneumococcal polysaccharide-protein conjugate as described in any one of claims 1-5 in the preparation of a vaccine for the prevention of disease, characterized in that, The disease is caused by Streptococcus pneumoniae infection or VZV infection.

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