Immunogenic composition containing multivalent pneumococcal polysaccharide-protein conjugate as well as preparation method and application of immunogenic composition

Abstract

The invention relates to the technical field of biology, in particular to a multivalent pneumococcal polysaccharide-protein conjugate immunogenic composition for preventing pneumococcal and varicella-zoster virus infection as well as a preparation method and application of the multivalent pneumococcal polysaccharide-protein conjugate immunogenic composition. The immunogenic composition of the present invention comprises: at least one pneumococcal polysaccharide-protein conjugate wherein the pneumococcal polysaccharide is linked to a carrier protein through a covalent bond; the carrier protein is varicella-zoster virus glycoprotein E, namely VZV gE recombinant protein, and the amino acid sequence of the VZV gE recombinant protein is shown as SEQ ID NO: 3 or SEQ ID NO: 4. The immunogenic composition containing the multivalent pneumococcal polysaccharide-protein conjugate, provided by the invention, shows a remarkable synergistic interaction effect, and realizes a'dual enhancement 'effect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a polyvalent pneumococcal polysaccharide-protein conjugate immunogenic composition for preventing pneumococcal and varicella-zoster virus infections, as well as a preparation method and application thereof. Background Art

[0002] Streptococcus pneumoniae, also known as pneumococcus, is a Gram-positive diplococcus with an outer membrane encapsulated by a capsular polysaccharide. Nearly 100 serotypes can be distinguished based on the composition and structure of these capsular polysaccharides. These capsular polysaccharides not only serve as the molecular basis for serotyping but also serve as core virulence factors mediating immune evasion. Pneumococci normally inhabit the nasopharynx of healthy individuals and generally do not cause clinical symptoms. However, when the host environment changes, such as decreased immune defenses, respiratory viral infections such as measles and influenza, 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 nonbacteremic pneumonia.

[0003] Pneumococcal disease, a major global public health challenge, continues to increase in its burden. According to the 2019 Global Burden of Disease Study on Bacterial Infections, of the 33 major bacterial pathogens responsible for 7.7 million deaths, Streptococcus pneumoniae caused 829,000 deaths (ranking third). Its impact, measured by years of life lost (YLL), leaps to first place. Deaths from this disease show significant age stratification, with deaths primarily occurring in children under five years of age and those over 60 years of age. Infants and young children, especially those under two years of age, are particularly 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 (WHO, 2019). The elderly also face a significant threat from pneumococcal disease. According to relevant statistics, pneumonia is the fourth leading cause of death among the elderly in China, with an average of approximately 125,000 deaths annually. Regional infection rates vary significantly (ranging from 28.0% to 71.5%), and the incidence rate rises sharply with age. It is noteworthy that, given the accelerating aging of the 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. This indicates that with the intensifying trend of population aging, the number of people at high risk of pneumococcal infection will continue to expand, further increasing the pressure on disease prevention and control.

[0004] Common treatment options for pneumococcal disease primarily rely on antimicrobial agents, but the widespread use and misuse of antibiotics has led to the growing problem of drug resistance in pneumococci. Years of clinical practice have proven that pneumococcal vaccination is the most cost-effective way to prevent pneumococcal disease. Currently, the vaccines available 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 approximately 90% global coverage against the dominant serotype. However, limitations remain. Studies have shown that bacterial capsular polysaccharide antigens are T-cell-independent antigens that can stimulate mature B lymphocytes but not T lymphocytes. The immune response mediated by these antigens is short-lived and does not produce immune memory. Because infants under two years of age have immature immune function and respond poorly to T-cell-independent antigens, polysaccharide vaccines (PPVs) are incapable of inducing a protective immune response. Further research and development has resulted in the conjugation of polysaccharides to carrier proteins, converting T-cell-independent antigens into T-cell-dependent antigens, thereby stimulating a T-cell-dependent immune response. Pneumococcal polysaccharide-protein conjugate vaccines (PCVs), developed using this technology, can be administered to infants as early as two months of age. They promote strong immune and memory responses, 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: GlaxoSmithKline's PCV10 (Synflorix), Pfizer's PCV13 (Prevenar 13), and the Serum Institute of India's PCV10 (PNEUMOSIL). In 2021, the FDA approved higher-priced PCVs, including Pfizer's PCV20 (Prevnar 20) and Merck's PCV15 (VAXNEUVANCE). Studies have shown that conjugate vaccines (PCVs) induce more durable and effective immune responses across all age groups and are poised to gradually replace polysaccharide vaccines (PPVs) as the mainstream treatment for preventing pneumococcal infection.

[0005] With the global rollout of pneumococcal conjugate vaccines (PCVs), vaccine pressure selection has led to significant changes in serotype epidemiology, with a clear upward trend in the incidence of some non-vaccine-covered serotypes. This has necessitated the development of higher-dose pneumococcal conjugate vaccines. However, a common drawback of currently available conjugate vaccines is that the carrier protein lacks immunogenicity. In other words, while the conjugate vaccine carrier can stimulate antibody production, vaccine designers have not yet been able to leverage the antibodies generated by the carrier protein to prevent disease. Currently widely used carriers, such as tetanus toxoid (TT), diphtheria toxoid (DT), and the avirulent variant of diphtheria toxin (CRM197), effectively enhance the immunogenicity of polysaccharide antigens and are proven safe, the antibodies they elicit do not directly protect against disease. Furthermore, tetanus and diphtheria toxoids are already components of the DPT vaccine and are routinely administered, making the ability of the carrier protein in pneumococcal conjugate vaccines to stimulate protective antibody production irrelevant. Therefore, in the process of promoting the development of multivalent vaccines to expand serotype coverage, the optimal selection of carrier proteins is also an especially important consideration.

[0006] Varicella zoster virus (VZV) belongs to the alphaherpesvirinae subfamily of the Herpesviridae family. It is an ellipsoidal particle approximately 150-200 nm in diameter. Its genetic material is a linear double-stranded DNA molecule enclosed by an icosahedral nucleocapsid. The nucleocapsid is surrounded by a lipoprotein envelope containing six glycoproteins, currently designated gB, gC, gE, gH, gI, and gL. These glycoproteins are involved in viral infection, replication, assembly, and intercellular transmission. In infected cells, gE is the most abundant envelope glycoprotein. It non-covalently binds to gI and can bind to the Fc fragment of antibody G (IgG), making it a crucial antigenic protein and a key target for both cellular and humoral immune responses.

[0007] VZV is a highly contagious and globally distributed pathogen. Only one serotype has been identified, and humans are its sole natural reservoir. Primary infection with the virus presents with the common childhood varicella (chickenpox) symptoms. Following infection, the virus can remain latent in the host's sensory neurons for extended periods. Notably, studies have shown that the Oka strain of the FDA-approved live-attenuated vaccine for varicella prevention, similar to the wild-type virus, can establish latent infection. With aging or when immune function declines, resulting in a weakened cellular immune response, latent virus can reactivate and cause herpes zoster (HZ). This condition, which primarily affects adults and the elderly, presents with a unilateral vesicular rash, often accompanied by fever, fatigue, redness, swelling, and significant burning sensation in the affected area, as well as neuralgia. Studies have shown that approximately 9% to 34% of patients with HZ develop post-herpetic neuralgia (PHN), a pain level that can reach levels 7 or higher, significantly disrupting daily life.

[0008] With the increasing aging of the global 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 effective treatment for herpes zoster 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 herpes zoster. Even so, relapse is still possible even after recovery. Drug treatment cannot solve the problem of virus elimination and prevention of invisible infection. Therefore, vaccination is the most effective means to prevent and control herpes zoster and its complications.

[0009] Currently, there are two VZV vaccines approved by the US FDA: 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, the vaccine's 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 vaccine, Zostavax. 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] The core attribute of a vaccine lies in its ability to protect against specific pathogens. For example, the pneumococcal polysaccharide vaccine effectively prevents pneumonia, meningitis, and otitis media caused by pneumococci, while the VZV vaccine prevents reactivation of the latent virus, which causes herpes zoster. To achieve the goal of protecting against multiple diseases with a single vaccine, combination vaccine technology is currently being used. Examples include the DPT (Dutsubo-Pertussis-Tetrapene) triple vaccine for children, the DPT (Dutsubo-Pertussis-Polio-Haemophilus Influenzae) pentavalent vaccine, and the hexavalent vaccine containing a hepatitis B component. Notably, although these combination vaccines are prepared by mixing individual vaccines, clinical evaluations have shown that the protective efficacy of some individual vaccines in combination formulations is poor. Currently, children of appropriate age in my country are required to receive over 15 different vaccines, with the actual number of vaccinations far exceeding the number of vaccines. Furthermore, with the aging population (by 2024, the number of people aged 60 and over in my country will reach 310 million, accounting for 22% of the total population), vaccine development for the elderly has become a public health priority. Therefore, developing multivalent and combined vaccines to replace single vaccines and achieve universal population protection will undoubtedly become a key strategic direction in vaccine research and development.

[0011] However, in the development of new vaccines, the choice of carrier protein directly affects the immune effect. Especially as vaccine prices increase, polysaccharide-protein conjugate vaccines face the dual challenge of enhancing the immunogenicity of the polysaccharide while circumventing the inhibitory effects of the carrier protein. In addition, how to design a stable and highly expressed carrier protein and combine it with immunogenic substances such as capsular polysaccharides to develop an innovative "one vaccine to prevent two diseases" product with dual immunogenicity are all key challenges faced in the vaccine design and development process. Summary of the Invention

[0012] In order to solve the problems existing in the prior art, the present invention provides an immunogenic composition containing a multivalent pneumococcal polysaccharide-protein conjugate. The immunogenic composition adopts polysaccharide-protein binding technology, creatively designs a VZV gE protein that has been genetically engineered to enhance stability, and uses the protein as a carrier protein to bind to 29 serotypes of pneumococcal polysaccharides. As a result, the immunogenic composition of the present invention can not only induce protective immunity against 29 serotypes of pneumococci, but also simultaneously stimulate immune responses against varicella-zoster virus, showing excellent dual immunogenicity. The immunogenic composition provided by the present invention can significantly reduce the number of vaccinations, reduce the cost and complexity of immunization programs, and greatly broaden the breadth and efficacy of immune protection, and also provide a more efficient and economical solution for public health prevention strategies.

[0013] The technical solutions to the technical problems of the present invention are as follows: In a first aspect of the present invention, there is provided an immunogenic composition comprising: at least one pneumococcal polysaccharide-protein conjugate, wherein the pneumococcal polysaccharide is covalently linked to a carrier protein; The carrier protein is varicella-zoster virus glycoprotein E, namely VZV gE recombinant protein, whose amino acid sequence is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0014] Furthermore, the pneumococcal polysaccharide is selected from at least two of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34 and 35B.

[0015] In a preferred embodiment of the present invention, the composition comprises a 29-valent pneumococcal polysaccharide-protein conjugate, wherein the pneumococcal polysaccharide comprises 29 serotypes, namely serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34, and 35B. The amino acid sequence of the carrier protein is shown in SEQ ID NO: 3.

[0016] In a preferred embodiment of the present invention, the composition comprises a 29-valent pneumococcal polysaccharide-protein conjugate, wherein the pneumococcal polysaccharide comprises 29 serotypes, namely serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34, and 35B. The amino acid sequence of the carrier protein is shown in SEQ ID NO: 4.

[0017] Furthermore, the composition further comprises an adjuvant. Preferably, the adjuvant is selected from one or more of aluminum salt adjuvants, emulsion adjuvants, immunostimulatory complexes, Toll-like receptor agonists, saponin adjuvants, or cytokines.

[0018] In a second aspect of the present invention, a method for preparing the immunogenic composition of the first aspect is provided, comprising the following steps: subjecting pneumococcal polysaccharide to a coupling reaction with VZV gE recombinant protein under suitable reaction conditions to form a covalently linked pneumococcal polysaccharide-protein conjugate.

[0019] Furthermore, the method further comprises one or more of the following steps: activating the pneumococcal polysaccharide; expressing and purifying the VZV gE recombinant protein; and purifying the coupling reaction product to obtain the pneumococcal polysaccharide-protein conjugate. Preferably, the activation treatment of the pneumococcal polysaccharide comprises degradation and / or chemical activation of the polysaccharide. The degradation method is selected from high-pressure homogenization, acid hydrolysis, or enzymatic digestion; the chemical activation method is selected from activation using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) or cyanogen bromide (CNBr). The coupling reaction is selected from the reductive amine method, the carbodiimide method, or the adipic acid dihydrazide (ADH)-mediated coupling method.

[0020] In a third aspect of the present invention, there is provided a use of the immunogenic composition according to the first aspect in the preparation of a medicament for preventing or treating a disease caused by infection with Streptococcus pneumoniae; and / or preventing or treating a disease caused by infection with varicella-zoster virus (VZV) in a subject.

[0021] Furthermore, the diseases caused by Streptococcus pneumoniae infection include pneumonia, bacteremia, meningitis or otitis media; the diseases caused by VZV infection include chickenpox, herpes zoster or postherpetic neuralgia.

[0022] In a fourth aspect of the present invention, an isolated nucleic acid molecule is provided, which encodes a VZV gE recombinant protein having an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0023] In the fifth aspect of the present invention, a recombinant expression vector is provided, which comprises the nucleic acid molecule as described in the fourth aspect.

[0024] In the sixth aspect of the present invention, a host cell is provided, which comprises the recombinant expression vector as described in the fifth aspect or the nucleic acid molecule as described in the fourth aspect is integrated into its genome.

[0025] The present invention has the following technical effects: (1) The present invention provides an immunogenic composition containing a multivalent pneumococcal polysaccharide-protein conjugate. The design of the immunogenic composition is based on an in-depth analysis of the epidemiological characteristics of pneumococci in my country. To achieve the best protective effect, a combination of 29 core serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34 and 35B was screened and combined. The immunogenic composition of the present invention can effectively prevent invasive pneumococcal diseases caused by the above 29 serotypes and can induce a high and balanced immune response to each serotype.

[0026] (2) Based on the study of the immunogenicity of the combination of pneumococcal polysaccharide and carrier protein, the present invention innovatively screened and designed a new carrier protein (VZV gE recombinant protein). By subjecting the gE protein to specific amino acid mutations, on the one hand, the stability of the carrier protein is significantly improved, enabling it to maintain the integrity of its higher-order structure and the function of its key antigenic clusters under various temperature conditions and during subsequent chemical reactions, thereby ensuring its own immunogenicity; on the other hand, the modified gE protein, as a carrier, can significantly enhance the antigenicity of the 29 serotypes of pneumococcal polysaccharides it binds to, thereby further optimizing the overall immune efficacy of the vaccine.

[0027] (3) The immunogenic composition containing a multivalent pneumococcal polysaccharide-protein conjugate provided by the present invention exhibits significant synergistic effects, achieving a "double enhancement" effect: On the one hand, the immunogenic composition prepared by the present invention uses an optimized gE recombinant protein as a new carrier, which can significantly enhance the antigenicity of 29 serotypes of pneumococcal polysaccharides. As can be seen from Example 9, the use of the preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) prepared by the present invention to immunize animals can effectively increase the IgG antibody level in the animal body, and the serum protection titer is higher; and compared with the pre-immunization period, the antibody effect is significantly improved. The preparation D of the present invention is even better than the immunization effect of preparation C (Pfizer PCV13) using CRM197 as the carrier protein. Among them, the preparation D before immunization D0 (see Figure 4 ), the original level of pneumococcal polysaccharide IgG antibodies of various serotypes in rabbit serum was low, and after the first injection (D14), the second injection (D28), and the third injection (D35, D42) of the preparation, the preparation D (29-valent pneumococcal polysaccharide gE protein conjugate vaccine) stimulated the animals to produce the corresponding 29 serotypes of polysaccharide IgG antibodies, and the titers showed a significant upward trend. The immune effect after the third injection (D35) was significantly better than that after the first injection (D14) and the second injection (D28), that is, as the number of immunizations increased, the IgG antibody titer increased significantly; and blood tests after the completion of immunization showed that on D42 (see Figure 8 ) Although the titer of IgG antibody to pneumonia polysaccharide was higher than that of D35 (see Figure 7 ) test results decreased slightly, but the titers of IgG antibodies to 29 serotypes of pneumococcal polysaccharide remained at a stable and high level.

[0028] At the same time, on D14, D28, D35, and D42 after immunization, the titers of polysaccharide IgG antibodies produced by Preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) and Preparation C (Pfizer PCV13) in animals after immunization were significantly different according to the serotype. Overall, the antibody detection level after immunization with Preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) was significantly higher than that of Preparation C (Pfizer PCV13). Among them, for the 13 pneumococcal polysaccharide serotypes contained in Preparations C and D, from D14 ( Figure 5 ) showed that, except for serotype 7F, the IgG antibody levels of the other 12 serotypes 1, 3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A, 19F, and 23F polysaccharides were all higher in preparation D than in preparation C; from D28 ( Figure 6 ) and D35 ( Figure 7 ) showed that, except for serotypes 18C and 19A, the IgG antibody levels of the other 11 serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 19F and 23F polysaccharides were all higher in preparation D than in preparation C; from D42 ( Figure 8) It can be seen that, except for serotype 19A, the IgG antibody levels of the other 12 serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F and 23F polysaccharides were all higher in preparation D than in preparation C.

[0029] On the other hand, the immunogenicity of the gE protein used as a carrier in the immunogenic composition prepared by the present invention is also enhanced, stimulating a stronger specific immune response and inducing the production of serum with higher protective titers. As shown in Example 11, compared to Preparation E (control PBS), the IgG antibody titers against gE protein in rabbit immune antisera from Preparations F (gE protein) and D (29-valent polysaccharide-gE protein conjugate vaccine) were significantly enhanced, with significant increases in total IgG, IgG1, and IgG2a antibody titers. Furthermore, the protein antibody titers in rabbit immune sera from Preparation D (29-valent polysaccharide-gE protein conjugate vaccine) were the highest, measured for total IgG, IgG1, and IgG2a antibodies, significantly higher than those from the unconjugated gE recombinant protein Preparation F.

[0030] Accordingly, Example 12 also shows that, compared to Preparation E (control PBS), both Preparation F (gE protein) and Preparation D (29-valent polysaccharide-gE protein conjugate vaccine) significantly stimulated immune cells in the spleen of animals, such as T cells (Th1 subset) and natural killer cells (NK cells), to secrete the IFN-γ cytokine after immunization with Preparation D. In particular, spleen cells from mice immunized with Preparation D, stimulated by either gE antigen or VZV virus, resulted in higher levels of IFN-γ secretion by sensitized immune cells. Furthermore, the amount of IFN-γ produced by splenic immune cells sensitized with Preparation D was significantly higher than that produced by Preparation F. This demonstrates that, in addition to the excellent cellular immune effects of the gE recombinant protein itself, the 29-valent pneumococcal polysaccharide-gE recombinant protein conjugate prepared using it as a carrier protein has a stronger ability to induce cellular immune responses in mice, further enhancing the ability to regulate immune responses and enhance defense against pathogens.

[0031] (4) The immunogenic composition containing a polyvalent pneumococcal polysaccharide-protein conjugate prepared by the present invention can cover higher-order pneumococcal serotypes, expand the scope of vaccine protection, and also ensure a good immune effect. As can be seen from Example 9, on D14, D28, D35, and D42 after immunization (see Figure 5-Figure 8) The IgG antibody levels for the newly added serotypes 2, 8, 9N, 10A, 11A, 12F, 15A, 15B, 17F, 20, 22A, 22F, 24F, 33F, 34, and 35B polysaccharides in preparation D of the present invention were generally high and significantly higher than the overall average level of IgG antibody titers for the 13 serotype polysaccharides in preparation C; especially for serotypes 2, 9N, 10A, 12F, 15A, 17F, 22A, and 33F polysaccharides. Among all 29 serotype polysaccharides, the IgG antibody titers for the newly added serotypes 2, 9N, 10A, 12F, 15A, 17F, 22A, and 33F polysaccharides in the present invention were always at a significantly high antibody level.

[0032] (5) The present invention also provides a method for preparing a polyvalent pneumococcal polysaccharide-protein conjugate composition. The core of this method is to use a VZV gE protein modified by genetic recombination and amino acid mutation as a polysaccharide carrier. During the preparation process, it is chemically coupled with polysaccharides of pneumococcal capsular polysaccharides of multiple serotypes and formulated with a new adjuvant to obtain the immunogenic composition. Animal experiments have confirmed that the immunogenic composition can induce a dual immune response: its polysaccharide antigen portion can stimulate the body to produce specific antibodies against 29 serotypes of pneumococci; and the gE carrier protein portion can effectively activate humoral immunity and cellular immunity against varicella-zoster virus. Therefore, a single vaccination of this preparation can prevent infection with both pneumococcus and herpes zoster virus, achieving the innovative goal of "one vaccine, two protections". BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a temperature stability test of gE protein before and after modification (0.06 ug / mL).

[0034] Figure 2 This is a temperature stability test of gE protein before and after modification (2.0 ug / mL).

[0035] Figure 3 To detect the gE protein antibody titer in the immune sera of mice prepared with preparations A and B.

[0036] Figure 4 Detection of IgG antibodies to 29 serotypes of pneumococcal polysaccharide in rabbit immune sera from preparations C and D (D0).

[0037] Figure 5 To detect IgG antibodies to 29 serotypes of pneumococcal polysaccharide in rabbit immune sera from preparations C and D (D14).

[0038] Figure 6 To detect IgG antibodies to 29 serotypes of pneumococcal polysaccharide in rabbit immune sera from preparations C and D (D28).

[0039] Figure 7 To detect IgG antibodies to 29 serotypes of pneumococcal polysaccharide in rabbit immune sera from preparations C and D (D35).

[0040] Figure 8 To detect IgG antibodies to 29 serotypes of pneumococcal polysaccharide in rabbit immune sera from preparations C and D (D42). DETAILED DESCRIPTION

[0041] 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.

[0042] Example 1: Preparation of Pneumococcal Capsular Polysaccharide (1) Preparation of master seeds and working seeds 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, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34, and 35B by the following method: Pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34, and 35B were obtained from the American Type Culture Collection. The strains 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, freeze-dried, and stored at 2–8°C as master seeds.

[0043] The strains from the master seed freeze-dried tubes of pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34, and 35B were inoculated into 5 mL of yeast-acid hydrolyzed casein culture medium and cultured at 36°C ± 2°C for 18 hours. When the bacteria grew to an OD600 reading of 1.0, the 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 for the serotypes.

[0044] (2) Bacterial fermentation 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.

[0045] (3) Capsular polysaccharide purification 1. Add phosphoric acid to adjust the pH of the fermentation liquid to between 3-5 and stir for 1 hour; 2. Centrifuge in a disc centrifuge at 9600 rpm, collect the supernatant and discard the residue; 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; 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; 5. Use a 50kD membrane package to further filter the polysaccharide solution for 10 sample volumes to concentrate the polysaccharide sample solution.

[0046] 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.

[0047] Example 2: Preparation of carrier protein Varicella zoster virus (VZV) is a member of the alphaherpesvirus subfamily of the Herpesviridae genus, namely Human herpesvirus 3.

[0048] 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.

[0049] (1) Protein construction The full-length sequence of the VZV gE protein mentioned in the present invention refers to 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).

[0050] 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.

[0051] 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.

[0052] (1) Amino acid sequence design 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: 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 W at position 200 of the VZV gE protein was mutated to C, and the A at position 246 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.

[0053] The present invention also relates to a VZV pre-modified gE recombinant protein, the amino acid sequence of which is designed as follows: The transmembrane region and intracellular region in the full-length sequence of VZV gE protein were deleted to obtain a truncated gE protein sequence as shown in SEQ ID NO. 2. At the same time, Throm and 6his sequences were connected to its C-terminus to obtain the full-length sequence of VZV gE recombinant protein before modification, whose amino acid sequence is shown in SEQ ID NO. 3.

[0054] SEQ ID NO. 1 to SEQ ID NO. 4 are shown below: SEQ ID NO. 1: > VZV gE 1-623 (NP_040190.1 / 623aa / envelope glycoprotein E [Humanalphaherpesvirus 3] / strain="Dumas") 1 MGTVNKPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHT DEDKLDTNSV YEPYYHSDHA 61ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG VYNQGRGIDS GERLMQPTQM 121 SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD QRQYGDVFKG DLNPKPQGQR LIEVSVEENH 181 PFTLRAPIQR IYGVRYTETW SFLPSLTCTG DAAPAIQHIC LKHTTCFQDV VVDVDCAENT 241 KEDQLAEISY RFQGKKEADQ PWIVVNTSTL FDELELDPPE IEPGVLKVLR TEKQYLGVYI 301 WNMRGSDGTS TYATFLVTWK GDEKTRNPTP AVTPQPRGAE FHMWNYHSHV FSVGDTFSLA 361 MHLQYKIHEA PFDLLLEWLY VPIDPTCQPM RLYSTCLYHP NAPQCLSHMN SGCTFTSPHL 421 AQRVASTVYQ NCEHADNYTA YCLGISHMEP SFGLILHDGG TTLKFVDTPE SLSGLYVFVV 481 YFNGHVEAVA YTVVSTVDHF VNAIEERGFP PTAGQPPATT KPKEITPVNP GTSPLLRYAA 541 WTGGLAAVVL LCLVIFLICT AKRMRVKAYR VDKSPYNQSM YYAGLPVDDF EDSESTDTEE 601 EFGNAIGGSH GGSSYTVYID KTR SEQ ID NO.2: > VZV gE 1-546 MGTVNKPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHT DEDKLDTNSV YEPYYHSDHA ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG VYNQGRGIDS GERLMQPTQM SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD QRQYGDVFKG DLNPKPQGQR LIEVSVEENH PFTLRAPIQR IYGVRYTETW SFLPSLTCTG DAAPAIQHIC LKHTTCFQDV VVDVDCAENT KEDQLAEISY RFQGKKEADQ PWIVVNTSTL FDELELDPPE IEPGVLKVLR TEKQYLGVYI WNMRGSDGTS TYATFLVTWK GDEKTRNPTP AVTPQPRGAE FHMWNYHSHV FSVGDTFSLA MHLQYKIHEA PFDLLLEWLY VPIDPTCQPM RLYSTCLYHP NAPQCLSHMN SGCTFTSPHL AQRVASTVYQ NCEHADNYTA YCLGISHMEP SFGLILHDGG TTLKFVDTPE SLSGLYVFVV YFNGHVEAVA YTVVSTVDHF VNAIEERGFP PTAGQPPATT KPKEITPVNP GTSPLLRYAA WTGGLA SEQ ID NO.3: >VZV modified gE MGTVNKPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHT DEDKLDTNSV YEPYYHSDHA ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG VYNQGRGIDS GERLMQPTQM SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD QRQYGDVFKG DLNPKPQGQR LIEVSVEENH PFTLRAPIQR IYGVRYTETW SFLPSLTCTG DAAPAIQHIC LKHTTCFQDV VVDVDCAENT KEDQLAEISY RFQGKKEADQ PWIVVNTSTL FDELELDPPE IEPGVLKVLR TEKQYLGVYI WNMRGSDGTS TYATFLVTWK GDEKTRNPTP AVTPQPRGAE FHMWNYHSHV FSVGDTFSLA MHLQYKIHEA PFDLLLEWLY VPIDPTCQPM RLYSTCLYHP NAPQCLSHMN SGCTFTSPHL AQRVASTVYQ NCEHADNYTA YCLGISHMEP SFGLILHDGG TTLKFVDTPE SLSGLYVFVV YFNGHVEAVA YTVVSTVDHF VNAIEERGFP PTAGQPPATT KPKEITPVNP GTSPLLRYAA WTGGLA GGLVPRGSHHHHHH SEQ ID NO .4: >VZV 修饰后gE W200C A246C MGTVNKPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHT DEDKLDTNSV YEPYYHSDHA ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG VYNQGRGIDS GERLMQPTQM SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD QRQYGDVFKG DLNPKPQGQR LIEVSVEENH PFTLRAPIQR IYGVRYTETC SFLPSLTCTG DAAPAIQHIC LKHTTCFQDV VVDVDCAENT KEDQLCEISY RFQGKKEADQ PWIVVNTSTL FDELELDPPE IEPGVLKVLR TEKQYLGVYI WNMRGSDGTS TYATFLVTWK GDEKTRNPTP AVTPQPRGAE FHMWNYHSHV FSVGDTFSLA MHLQYKIHEA PFDLLLEWLY VPIDPTCQPM RLYSTCLYHP NAPQCLSHMN SGCTFTSPHL AQRVASTVYQ NCEHADNYTA YCLGISHMEP SFGLILHDGG TTLKFVDTPE SLSGLYVFVV YFNGHVEAVA YTVVSTVDHF VNAIEERGFP PTAGQPPATT KPKEITPVNP GTSPLLRYAA WTGGLA GGLVPRGSHHHHHH。

[0055] (2)VZV gE蛋白目的基因的合成 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.

[0056] (3) Plasmid amplification and target gene extraction 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.

[0057] (4) Construction of eukaryotic expression vector 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 and culture, and the amplified plasmid was extracted using the TaKaRa MidiBEST Endo-free Plasmid Purification Kit, and was named VZVgE.

[0058] (II) Expression of gE protein in CHO cells and cloning screening 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 9.6 cm cells / well 2Wells were filled with Iscove's optimized DMEM medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (IMEM+FBS) (Gibco). Cells were incubated in a humidified incubator with 5% CO₂ and 37°C. 4 μg of pcDNAVZVE vector was added to each well. DNA was mixed with Lipofectamine 2000 (Sigma-Aldrich) and added to two wells. Lipofectamine 2000 alone was added to the third well as a negative control. After 48 hours, the medium was removed, the cells were centrifuged at 200 × g for 5 minutes, and the supernatant was stored at -20°C. IMDM+FBS medium and 10 μg / mL blasticidin-HCl (Invitrogen) were added to one well of transfected cells. The other well was washed with PBS, and the cells were lysed with 50 mM Tris-HCl, pH 8, 150 mM NaCl, and 1% (v / v) Triton X-100 containing complete, EDA-free protease inhibitor cocktail (Roche Diagnostics). The cells were centrifuged at 16,000 × g for 10 minutes at 4°C, and the lysate was stored at -20°C. The supernatant and lysate were assayed for the presence of recombinant protein by Western blot. After 5 days of culture in selective medium, the cells were eluted with trypsin (Invitrogen) and seeded onto 9 cm Petri dishes. Serial dilutions were performed to isolate single clones. Over the next 7–11 days, 42 single clones were selected and transferred to wells of a 96-well plate. Western blot was used to detect the culture supernatant and screen for highly expressed gE protein. The clones secreting the highest amount of protein were selected for the next round of screening. Finally, the cells were expanded and 30 new clones were selected and stored.

[0059] The selected clones were expanded into three T175 flasks (NETS). Trypsin was added for digestion, washed with PBS, and resuspended in 100 mL of ProCHO4 (Lonza) in a 250 mL spinner flask with 1 × ProHT, 4 mM L-glutamine and 2% FBS (Lonza). Culture was carried out in a humidified incubator at 37 ° C, 5% CO2, with a stirring speed of 90 rpm 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.

[0060] 3. Production of gE protein in bioreactors 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.

[0061] 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 sample was filtered through a 0.45 μm membrane and then concentrated by ultrafiltration using a 10 kDa membrane pack. The sample solution was then washed with ultrafiltration buffer and 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.

[0062] (IV) Purification of gE protein 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 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, loaded onto a Butyl-Sepharose (GE Bioscience) column, and washed with phosphate buffered saline (PBS, 6mM Na2HPO4, 1.5mM KH2PO4, 0.15M sodium chloride pH 6.8) with 800mM ammonium sulfate added to the column. 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.

[0063] VZV-modified gE protein and VZV-modified gE protein were prepared respectively using the above method.

[0064] Example 3: Stability test of the carrier protein obtained in Example 2 1) Dilute the test protein (pre-modified gE protein prepared in Reference Example 2 and modified gE protein prepared in Example 2) to 20 μg / mL with 1×PBS pH 7.4 buffer and place in a 1.5 mL centrifuge tube to a total volume of 1 mL.

[0065] 2) Incubate at different temperatures according to the table below.

[0066]

[0067] 3) After incubation, store the samples at 4°C.

[0068] 4) Preparation of detection antibodies (E5-G6) BALB / c mice were immunized with a gE protein vaccine (GSK) at a dose of 100 µg / mouse. After two immunizations, B lymphocytes were extracted from the spleens of BALB / c mice. Monoclonal antibodies (mAbs) against VZV-gE were then prepared and cloned using hybridoma cell technology (numbers: E5-G6).

[0069] 5) Perform ELISA test as follows: Samples stored at 4°C were diluted to 1 μg / mL with 1x PBS, pH 7.4, and 100 μL / well was added to an ELISA plate (NUNC442404) and coated 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 a plate washer according to 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.

[0070] The results of protein temperature stability test before and after modification are as follows Figure 1-2 As shown in the figure, significant differences in antibody binding activity between the modified and unmodified gE proteins were observed 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 antibody (0.06 μg / mL E5-G6 and 2.0 μg / mL E5-G6) were significantly higher than those of the unmodified gE protein. This indicates that the modified gE protein prepared by the present invention can maintain high antigen-binding activity after treatment at different temperatures, indicating that the stability of the modified gE protein prepared by the present invention is further enhanced compared to the unmodified gE protein.

[0071] Example 4: Detection of protein antibody titer in mouse immune serum 0.1 mg / mL of the VZV pre-modified gE protein / modified gE protein obtained by the method of Example 2 was added to a phosphate buffer solution at pH 5.8, sterilized by filtration using a 0.22 μm membrane, aseptically packaged in 0.8 mL / bottle, and stored at 4°C for immunization, namely, Preparation A (pre-modified gE protein) and Preparation B (modified gE protein).

[0072] Female BALB / c mice aged 4-6 weeks were randomly divided into two groups and immunized with preparation A and preparation B, respectively, subcutaneously once every two weeks with 0.1 mL each time, for a total of two immunizations. Blood was collected 35 days after immunization, and the blood was then placed 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.

[0073] 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, wash three times with 300 μL per well of plate wash buffer, and store at 4°C.

[0074] Dilute the corresponding test serum obtained from immunized mice to prepare 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 two-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.

[0075] Figure 3 The test results showed that there were differences in the antibody titers of mice immunized with the gE protein before and after modification. The antibody titers of mice immunized with the amino acid-modified gE protein (Preparation B) were significantly higher than those of the unmodified gE protein (Preparation A). Moreover, after the serum of the immunized mice was diluted at different multiples, the antibody titers of the amino acid-modified gE protein (Preparation B) were still higher than those of the unmodified gE protein (Preparation A) at all concentrations. Moreover, as the serum dilution multiple gradually increased, the antibody titers of both the unmodified and modified gE proteins showed a downward trend. This indicates that compared with the unmodified gE protein, the immunogenicity of the amino acid-modified gE protein antigen prepared by the present invention has been improved.

[0076] Example 5: Preparation of Pneumococcal 29 serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34 and 35B polysaccharide-VZV recombinant protein conjugates (CDAP) 1) Weigh 17.5 mg of purified capsular polysaccharide of the corresponding serotype and dissolve it in 4 mL of sodium phosphate buffer; 2) Add 12 mg of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) (Sigma-Aldrich) to the polysaccharide solution, stir, and react at room temperature for 1 hour; 3) Add 3 Eqm cystamine and react at room temperature for 1 hour; 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; 5) Add 8 Eqm of 3 (2-chloroethyl) phosphate to the polysaccharide solution to reduce the disulfide bonds in the polysaccharide; 6) Transfer the activated polysaccharide solution to a dialysis bag and dialyze against phosphate buffered saline at 4°C, changing the buffer four times; 7) Weigh 30 mg of carrier protein and dissolve it in phosphate buffer to a protein concentration of 10 mg / mL; 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 buffered saline at 4°C, changing the buffer four times. 9) Mix 4 mL of activated polysaccharide solution with 4 mL of activated protein solution and react at room temperature for 4 hours; 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. 11) Transfer the polysaccharide conjugate reaction solution to a dialysis bag and dialyze against phosphate buffer at 4°C. 12) The sample solution was loaded onto Sepharose CL-4B, purified, and the bound product in the external water volume was collected.

[0077] 13) After filtering with a 0.22 μm filter membrane, store the product at 2-8°C.

[0078] 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.

[0079] Example 6: Preparation of Pneumococcal 29 serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34 and 35B polysaccharide-VZV recombinant protein conjugates (reductive amine method) 1) Weigh 500 mg of purified capsular polysaccharide of the corresponding serotype and dissolve it in 500 mL of purified water; 2) Degrade the product by high-pressure homogenization at 600 bar for three cycles, add 0.12 eqm sodium periodate (Sigma-Aldrich), and react in the dark for 18 hours; 3) Use ultrafiltration and filtration with a 50Kd membrane to filter the purified water, and then concentrate and freeze-dry; 4) Weigh 18 mg of activated polysaccharide, add 4 mL of DMSO, and stir to dissolve completely; 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; 6) After quenching the reaction by adding 2 EqM sodium borohydride (Sigma-Aldrich) for 4 h, the synthesis reaction solution was transferred to a dialysis bag and dialyzed against the buffer solution, changing the buffer four times; 7) Load the sample solution onto Sepharose CL4B and collect the bound fraction in the void volume.

[0080] 8) Filter through a 0.22 μm filter membrane and store at 4°C for preparation.

[0081] 9) Take samples to detect the molecular weight of the conjugate, polysaccharide-protein concentration and ratio.

[0082] 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.

[0083] Example 7: Preparation of immune preparations C, D, E, and F (1) Formulation C (Pfizer PCV13): Prevenar 13 is a 13-valent pneumococcal polysaccharide conjugate vaccine (PCV13) developed by Pfizer. It has been approved by the FDA and the EU, and officially launched in China in November 2016. It is the world's first 13-valent pneumococcal polysaccharide conjugate vaccine. This vaccine utilizes pneumococcal capsular polysaccharide conjugate protein (CRM197) technology to protect against invasive diseases caused by 13 pneumococcal serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F), such as bacteremic pneumonia and meningitis. Prevenar 13 is the most widely used pneumococcal conjugate vaccine globally and is currently included in immunization programs in many countries and regions around the world and in the Asia-Pacific region.

[0084] (2) Preparation D (29-valent pneumococcal polysaccharide-VZV recombinant protein conjugate vaccine): The polysaccharide concentration in the monovalent conjugate was detected by measuring the VZV recombinant protein conjugate solution equivalent to 2.2 μg of polysaccharide (the conjugate preparation method is shown in Example 5 or 6, and the conjugate prepared in Example 5 was used in this example), including Pn1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F , 24F, 33F, 34 and 35B into a sterile container, sample for detection of protein content of about 99 μg, add CpG (Genscript), the final amount is 0.1 mg, add phosphate buffer pH 5.8 buffer, sterile filter with a 0.22 μm membrane; add sterile aluminum phosphate gel (Benetag), the final aluminum ion amount is 0.125 mg, stir at 4 ° C for 1 hour, aseptically divide into 0.5 mL / bottle, and store at 4 ° C until immunization.

[0085] (3) Preparation E (PBS control) The protein sample 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 using 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. The mixture is aseptically dispensed into 0.5 mL / bottle and stored at 4°C for immunization.

[0086] (4) Preparation F (gE protein) The modified gE protein obtained in Example 2 was sampled at a content of 66 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 using 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, and aseptically dispensed into 0.5 mL / bottles and stored at 4°C for immunization.

[0087] Example 8: Immunization of rabbits with preparations C and D and blood collection Ten 2.5-3.5 kg New Zealand white rabbits were collected, divided into groups of five. One group received immunization preparation D (prepared in Example 7) and the other received immunization preparation C (Pfizer PCV13). Each rabbit was injected with 0.5 mL of the vaccine each time, every two weeks, for a total of three immunizations. Blood samples were collected on days 0, 14, 28, 35, and 42 (i.e., D0, D14, D28, D35, and D42). 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, then centrifuged at 10,000 rpm at room temperature. The supernatant serum was collected and stored at -70°C for testing.

[0088] Example 9: Detection of IgG Antibodies to 29 Serotypes of Pneumococcal Polysaccharide in Rabbit Immune Serum Prepare pneumococcal polysaccharides of different serotypes (in 1× PBS) and store in a refrigerator at 4°C. Dilute the pneumococcal polysaccharide of the serotype to be tested to 4 μg / mL. Add 100 μL of coating solution to each well of the ELISA plate and incubate overnight at room temperature. Wash the plate four times with plate wash buffer, add 100 μL of blocking buffer, incubate at room temperature for 2 hours, wash the plate four times with plate wash buffer, and store at 4°C.

[0089] The test serum prepared in Example 8 was diluted 1:10 to prepare the working sample serum. The serum was added to the first row of wells of the ELISA plate in a total volume of 200 μL. Two-fold serial dilutions were performed starting from the first row and proceeding downwards. The plate was incubated at room temperature for 2 hours. The plate was washed four times with plate wash buffer. 100 μL of alkaline phosphatase-conjugated goat anti-rabbit antibody (1:2000 dilution) was added and the plate was incubated at room temperature for 4 hours. The plate was washed four times with plate wash buffer. 100 μL of 4-nitrophenyl phosphate disodium salt substrate solution (Sigma-Aldrich) was added and the plate was read at 405 nm.

[0090] The results of the detection of IgG antibodies to 29 serotypes of pneumococcal polysaccharide in rabbit immune antiserum of pneumococcal polysaccharide-protein conjugate vaccine showed that (see Figure 4-Figure 8 ), D0 before preparation immunization (see Figure 4), the levels of polysaccharide IgG antibodies of various serotypes of pneumococcus originally contained in the rabbit serum were low, and there was no significant difference in the test results; after the first injection (D14), the second injection (D28), and the third injection (D35, D42) of the preparation, the preparation D (29-valent pneumococcal polysaccharide gE protein conjugate vaccine) stimulated the animals to produce the corresponding 29 serotypes of polysaccharide IgG antibodies, and the titers showed a significant upward trend. The immune effect after the third injection (D35) was significantly better than that after the first injection (D14) and the second injection (D28), that is, as the number of immunizations increased, the IgG antibody titer increased significantly; and after the completion of the immunization, blood tests continued to show that on D42 (see Figure 8 ) Although the titer of IgG antibody to pneumonia polysaccharide was higher than that of D35 (see Figure 7 ) test results decreased slightly, but the titers of IgG antibodies to 29 serotypes of pneumococcal polysaccharide remained at a stable and high level.

[0091] At the same time, in addition to the pre-immunization D0 (see Figure 4 ), D14, D28, D35, D42 after immunization (see Figure 5-Figure 8 ), Preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) and Preparation C (Pfizer PCV13) stimulated animals to produce corresponding serotype polysaccharide IgG antibody titers, which varied significantly depending on the serotype. Overall, the antibody detection level after immunization with Preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) was significantly higher than that with Preparation C (Pfizer PCV13).

[0092] Among them, on the one hand, for the 13 pneumococcal polysaccharide serotypes contained in both preparations C and D, from D14 ( Figure 5 ) showed that, except for serotype 7F, the IgG antibody levels of the other 12 serotypes 1, 3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A, 19F, and 23F polysaccharides were all higher in preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) than in preparation C (Pfizer PCV13); from D28 ( Figure 6 ) and D35 ( Figure 7 ) showed that, except for serotypes 18C and 19A, the IgG antibody levels of the other 11 serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 19F and 23F polysaccharides were all higher in preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) than in preparation C (Pfizer PCV13); from D42 ( Figure 8) It can be seen that, except for serotype 19A, the IgG antibody levels of the remaining 12 serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F, and 23F polysaccharides are all higher in Preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) than in Preparation C (Pfizer PCV13). This shows that after immunization of animals with Preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) prepared by the present invention, the IgG antibody level in the animal body is effectively increased, and the serum protection titer is higher; and compared with the pre-immunization period, the antibody effect is significantly improved. At the same time, Preparation D of the present invention is even better than the immunization effect of Preparation C (Pfizer PCV13) using CRM197 as the carrier protein. This also shows that the gE recombinant protein carrier in the polysaccharide-protein conjugate vaccine prepared by the present invention has a good enhancing and promoting effect on the antigenicity of 29 serotypes of pneumococcal polysaccharides.

[0093] On the other hand, for the newly added serotype 2, 8, 9N, 10A, 11A, 12F, 15A, 15B, 17F, 20, 22A, 22F, 24F, 33F, 34 and 35B polysaccharides in the preparation D of the present invention, from D14, D28, D35, D42 (see Figure 5-Figure 8 ) shows that the IgG antibody levels for the 16 newly added serotype polysaccharides in the present invention are generally high and significantly higher than the overall average level of IgG antibody titers for the 13 serotype polysaccharides in formulation C (Pfizer PCV13). Among them, the IgG antibody titers for serotypes 2, 9N, 10A, 12F, 15A, 17F, 22A, and 33F polysaccharides are particularly high among all 29 serotype polysaccharides. This further demonstrates that the 29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine prepared by the present invention can cover higher-valent pneumococcal serotypes, expand the scope of immune protection, and also ensure good immune efficacy.

[0094] Example 10: Immunization of rabbits with preparations D, E, and F and blood collection Nine New Zealand white rabbits weighing 2.5-3.5 kg were divided into three groups of three. Each group was immunized with Formulation E, Formulation F, and Formulation D prepared in Example 7, respectively. Each rabbit was subcutaneously immunized once every two weeks with 0.5 mL injected per time for a total of two immunizations. Blood was collected one week after immunization, and the collected blood was 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.

[0095] Example 11: Detection of protein antibody titer in rabbit immune serum Prepare a 1 mg / mL stock solution of purified gE protein in 1× PBS and store in a refrigerator at 4°C. Dilute the protein stock solution to 4 μg / mL in coating buffer. Coat the ELISA plate with 100 μL of coating solution per well 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.

[0096] The test serum prepared in Example 10 was diluted 1:10 to prepare the working sample serum and added to the first row of wells of the ELISA plate in a total volume of 200 μL. Two-fold serial dilutions were performed starting from the first row downwards and incubated at room temperature for 2 hours. The plate was washed four times with plate wash buffer, and 100 μL of alkaline phosphatase-labeled goat anti-rabbit antibody (1:2000 dilution) was added. The plate was incubated at room temperature for 4 hours. The plate was washed four times with plate wash buffer, and 100 μL of 4-nitrophenyl phosphate disodium salt substrate solution was added. The plate was read at 405 nm.

[0097]

[0098] Note: In Table 2, the values ​​outside the brackets are the geometric means, and the values ​​inside the brackets are the specific numerical ranges of the samples.

[0099] The test results in Table 2 show that compared to Preparation E (control PBS), the IgG antibody titers against gE protein in rabbit immune antisera from Preparations F (gE protein) and D (29-valent polysaccharide-gE protein conjugate vaccine) were significantly enhanced, with significant increases in total IgG, IgG1, and IgG2a antibody titers. Among them, the protein antibody titers in rabbit immune serum from Preparation D (29-valent polysaccharide-gE protein conjugate vaccine) were the highest, significantly higher than those from Preparation F (gE protein). This demonstrates that the gE recombinant protein prepared in this invention, in addition to enhancing the antigenic immune efficacy of the gE protein itself, can also be used as a carrier protein in the preparation of 29-valent pneumococcal polysaccharide-gE recombinant protein conjugate preparations, resulting in sera with higher protective titers and superior immune efficacy.

[0100] Example 12: IFN-γ cytokine detection Six-week-old female BALB / c mice were randomly divided into three groups of eight mice each. Each group was immunized with Formulation E, Formulation F, or Formulation D prepared in Example 7, respectively, subcutaneously once every two weeks with 0.1 mL each time, for a total of two immunizations. The spleens were collected one week after immunization.

[0101] 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 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, 5% CO2 for 24 hours. The number of IFN-γ-secreting cells was determined using an ELISpot kit (MabTech), and positive spots were counted using a CTL ImmunoSpot S5UV Micro analyzer.

[0102]

[0103] Note: In Table 3, the values ​​outside the brackets are the geometric means, and the values ​​inside the brackets are the specific numerical ranges of the samples.

[0104] The test results in Table 3 show that, compared to Preparation E (control PBS), both Preparation F (gE protein) and Preparation D (29-valent polysaccharide-gE protein conjugate vaccine) significantly stimulated immune cells in the spleen of mice, such as T cells (Th1 subset) and natural killer cells (NK cells), to secrete the cytokine IFN-γ. In particular, spleen cells sensitized with Preparation D (29-valent polysaccharide-gE protein conjugate vaccine) in response to gE antigen or VZV virus stimulation resulted in higher levels of IFN-γ secretion. Furthermore, the amount of IFN-γ produced by spleen cells sensitized with Preparation D (29-valent polysaccharide-gE protein conjugate vaccine) was significantly higher than that produced by Preparation F (gE protein). This demonstrates that the gE recombinant protein prepared in this invention, in addition to its own excellent cellular immune effects, also demonstrates that the 29-valent pneumococcal polysaccharide-gE recombinant protein conjugate prepared using it as a carrier protein has a stronger ability to induce cellular immune responses in mice, further enhancing the ability to regulate immune responses and enhance defense against pathogens.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be included within the scope of protection of the claims of the present invention to be approved.

Claims

1. An immunogenic composition, characterized in that Include: at least one pneumococcal polysaccharide-protein conjugate, wherein the pneumococcal polysaccharide is covalently linked to a carrier protein; The carrier protein is varicella-zoster virus glycoprotein E, namely VZV gE recombinant protein, whose amino acid sequence is shown in SEQ ID NO: 3 or SEQ ID NO:

4.

2. The immunogenic composition according to claim 1, characterized in that The pneumococcal polysaccharide is selected from at least two of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34, and 35B.

3. The immunogenic composition according to claim 2, characterized in that The composition comprises a 29-valent pneumococcal polysaccharide-protein conjugate, wherein the pneumococcal polysaccharide comprises 29 serotypes, namely serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22A, 22F, 23F, 24F, 33F, 34 and 35B.

4. The immunogenic composition according to claim 3, characterized in that The amino acid sequence of the carrier protein is shown in SEQ ID NO:

3.

5. The immunogenic composition according to claim 3, characterized in that The amino acid sequence of the carrier protein is shown in SEQ ID NO:

4.

6. The immunogenic composition according to claim 1, characterized in that The composition also includes an adjuvant.

7. The immunogenic composition according to claim 6, characterized in that The adjuvant is selected from one or more of aluminum salt adjuvants, emulsion adjuvants, immunostimulatory complexes, Toll-like receptor agonists, saponin adjuvants or cytokines.

8. A method for preparing the immunogenic composition according to any one of claims 1 to 7, characterized in that: The following steps are involved: The pneumococcal polysaccharide and the VZV gE recombinant protein are coupled under appropriate reaction conditions to form a covalently linked pneumococcal polysaccharide-protein conjugate.

9. The method according to claim 8, characterized in that The method further comprises one or more of the following steps: activating the pneumococcal polysaccharide; expressing and purifying the VZV gE recombinant protein; The coupling reaction product is purified to obtain the pneumococcal polysaccharide-protein conjugate.

10. The method according to claim 9, characterized in that The activation treatment of pneumococcal polysaccharide includes degradation and / or chemical activation of the polysaccharide.

11. The method according to claim 10, characterized in that The degradation method is selected from high-pressure homogenization, acid hydrolysis or enzyme digestion; and the chemical activation method is selected from activation using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate CDAP or cyanogen bromide CNBr.

12. The method according to claim 8, characterized in that The coupling reaction is selected from the reductive amine method, the carbodiimide method, or the adipic acid dihydrazide (ADH)-mediated coupling method.

13. Use of the immunogenic composition according to any one of claims 1 to 7 in the preparation of a medicament for use in a subject: Preventing or treating illness caused by Streptococcus pneumoniae infection; and / or To prevent or treat illness caused by varicella-zoster virus (VZV) infection.

14. The use according to claim 13, characterized in that The diseases caused by Streptococcus pneumoniae infection include pneumonia, bacteremia, meningitis or otitis media; the diseases caused by VZV infection include chickenpox, herpes zoster or postherpetic neuralgia.

15. An isolated nucleic acid molecule encoding a VZV gE recombinant protein having an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO:

4. A recombinant expression vector comprising the nucleic acid molecule according to claim 15 .

17. A host cell comprising the recombinant expression vector according to claim 16 or a host cell in which the nucleic acid molecule according to claim 15 is integrated into its genome.

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