Immunogenic compositions comprising multivalent pneumococcal polysaccharide-protein conjugates, methods of making and using the same

Abstract

The present application relates to the technical field of biotechnology, in particular to a kind of multivalent pneumococcal polysaccharide-protein conjugate immunogenic composition for preventing pneumococcus and varicella-zoster virus infection and its preparation method and application.The immunogenic composition of the present application comprises:at least one pneumococcal polysaccharide-protein conjugate, wherein the pneumococcal polysaccharide is connected with carrier protein by covalent bond;The carrier protein is varicella-zoster virus glycoprotein E, i.e.VZV gE recombinant protein, and its amino acid sequence is shown as SEQ ID NO:3 or SEQ ID NO:4.The immunogenic composition containing multivalent pneumococcal polysaccharide-protein conjugate provided by the present application shows significant synergistic effect, realizes "double enhancement" effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a multivalent pneumococcal polysaccharide-protein conjugate immunogenic composition for preventing pneumococcal and varicella-zoster virus infection, and a preparation method and application thereof. BACKGROUND

[0002] Streptococcus pneumoniae, also known as pneumococcus, is a gram-positive diplococcus, and the outer membrane of the bacterium is wrapped by a layer of capsular polysaccharide. Based on the differences in composition and structure of capsular polysaccharide, nearly 100 serotypes can be distinguished, and the capsular polysaccharide is not only the molecular basis of serotyping, but also the core virulence factor that mediates immune escape. Pneumococcus normally parasitizes in the nasopharynx of healthy people and generally does not cause clinical symptoms. When the parasitic environment changes, such as decreased body resistance, respiratory virus infection such as measles and influenza, malnutrition or old age, it can invade the mucosal defense system and cause invasive infection. According to the site of pneumococcal infection, invasive pneumococcal disease (IPD) including meningitis, bacteremia and bacteremia pneumonia, and non-invasive pneumococcal disease (NIPD) including acute otitis media, sinusitis and non-bacteremia pneumonia can be distinguished.

[0003] Pneumococcal disease is a major challenge in the field of global public health, and its disease burden is showing a trend of continuous aggravation. According to the global bacterial infectious disease burden research in 2019, among the 33 major bacterial pathogens that caused 7.7 million deaths, Streptococcus pneumoniae caused 829,000 deaths (ranking third). If the life loss year (YLL) is used as an evaluation index, its harmfulness is the first. And the death cases of this disease show a significant age stratification feature, mainly distributed in children under 5 years old and the elderly over 60 years old. On the one hand, for infants and young children, especially those under 2 years old, they are extremely susceptible to pneumococcal infection and have a high mortality rate. About 75% of invasive pneumococcal diseases and 83% of pneumococcal meningitis occur in this age group (WHO, 2019). On the other hand, the elderly population also faces the severe threat of pneumococcal disease. According to relevant statistics, pneumonia is the fourth leading cause of death among the elderly in China, with an average annual death toll of about 125,000 people. The regional infection rate varies significantly (28.0%-71.5%), and the incidence rate increases sharply with the age of the elderly. It is worth noting that under the background of the accelerated aging of the global population, the United Nations World Social Report 2023 pointed out that the global population aged 65 and above was 761 million in 2021, and this number will increase to 1.6 billion by 2050, and the population aged 80 and above is growing even faster. Therefore, the trend of population aging is becoming increasingly intense, and the base of high-risk groups of pneumococcal infection will continue to expand in the future, and the disease prevention and control pressure will also be further increased.

[0004] The common treatment for pneumococcal disease is mainly based on antibacterial drugs, but the widespread use and abuse of antibiotics has led to the problem of pneumococcal drug resistance becoming more and more serious. Years of clinical practice have proved that vaccination against pneumococcal vaccine is the most economical and effective way to prevent pneumococcal disease. At present, the vaccines on the market are pneumococcal polysaccharide vaccine (PPV) and pneumococcal conjugate vaccine (PCV). In the 1980s, the 23-valent pneumococcal polysaccharide vaccine (PPV23) produced by Wyeth (acquired by Pfizer in 2009) achieved about 90% global coverage of the dominant serotypes, but still had limitations. Studies have found that the bacterial capsular polysaccharide antigen is a T cell-independent antigen that can stimulate mature B lymphocytes but not T lymphocytes. The immune response mediated by this antigen lasts for a short time and cannot produce immune memory. Because the immune function of children under the age of 2 is not yet fully developed, their response to T cell-independent antigens is poor, so polysaccharide vaccine (PPV) cannot induce protective immune responses in infants. Further research and development have shown that the combination of polysaccharide and carrier protein can convert non-T cell-dependent antigens into T cell-dependent antigens, activating T cell-dependent immune responses. Pneumococcal polysaccharide protein conjugate vaccine (PCV) prepared using polysaccharide protein conjugate technology can be administered to infants at 2 months of age, can promote a strong immune response and memory response, and is an advantageous immunization method that can more quickly and earlier prevent pneumococcal disease. The first PCV to be approved for marketing by the FDA in 2000 was PCV7 (Prevnar 7) produced by Wyeth. In 2010, Pfizer introduced PCV13 (Prevnar 13), which covers more serotypes, replacing PCV7 (Prevnar 7) for routine vaccination of children. There are currently three WHO pre-qualified PCVs: GSK's PCV10 (Synflorix), Pfizer's PCV13 (Prevenar 13), and India Serum Institute's PCV10 (PNEUMOSIL). In 2021, the FDA approved the marketing of higher-valence PCVs, including Pfizer's PCV20 (Prevnar 20) and Merck's PCV15 (VAXNEUVANCE). Studies have shown that conjugate vaccines (PCV) can induce more durable and more effective immune responses in all age groups, and will gradually replace polysaccharide vaccines (PPV) as the mainstream product for preventing pneumococcal infection.

[0005] With the global vaccination of pneumococcal conjugate vaccine (PCV), vaccine pressure selection has led to significant changes in serotype epidemiology, and the incidence of some non-vaccine coverage serotypes has shown a clear upward trend, prompting the development of higher-priced pneumococcal conjugate vaccines. At the same time, the current market conjugate vaccine products have a common shortcoming that the carrier protein is not endowed with protective function of immunogenicity; that is, although the conjugate vaccine carrier can stimulate the body to produce antibodies, vaccine designers have not been able to use the antibodies produced by the carrier protein to prevent disease. The widely used carriers such as tetanus toxoid (TT), diphtheria toxoid (DT) and diphtheria non-toxic mutant toxin (CRM197) can effectively enhance the immunogenicity of polysaccharide antigens and are safe and reliable, but the antibodies induced do not have direct disease prevention function. In addition, tetanus toxoid and diphtheria toxoid are already two components of the diphtheria-pertussis-tetanus triple vaccine, which has been used for routine vaccination, so whether the carrier protein in the pneumococcal conjugate vaccine can stimulate the body to produce protective antibodies is not important. Therefore, in the process of promoting the development of multivalent vaccines to expand the coverage of serotypes, the optimization of carrier proteins is also an important consideration factor.

[0006] Varicella zoster virus (VZV) belongs to the alpha herpesvirus subfamily of the herpesvirus family. Its properties are oval particles with a diameter of about 150-200 nm, and a linear double-stranded DNA molecule as genetic material is covered by a 20-sided nuclear envelope. The outer part of the nuclear envelope has a lipid protein envelope, and the envelope mainly has six glycoproteins, which are now designated as gB, gC, gE, gH, gI and gL. These glycoproteins are related to the processes of virus infection, replication, assembly and intercellular transmission. In infected cells, the gE protein is the most abundant envelope glycoprotein, which is adsorbed to gI in the form of non-covalent bond, can be adsorbed to the Fc fragment of antibody G (IgG), and is a very important antigen protein and an important target of cellular and humoral immune responses.

[0007] VZV virus is a highly contagious and globally distributed pathogen, only one serotype has been found so far and humans are the only natural host. The primary infection caused by the virus manifests as the common childhood symptoms of varicella, and the virus can remain latent in the host sensory neurons after infection. Notably, studies have found that the Oka strain live attenuated vaccine approved by FDA for the prevention of varicella is similar to the wild-type virus and can also establish latent infection. When the cellular immune response is weakened due to age growth or immune function decline, the latent virus may be reactivated and cause herpes zoster (HZ), which is more common in the adult and elderly population. The clinical manifestations of this disease are unilateral vesicular skin rash, often accompanied by fever, fatigue and other symptoms, and the affected skin will appear red and swollen with significant burning sensation and neuralgia. Studies have shown that about 9%-34% of herpes zoster patients will develop post-herpetic neuralgia (PHN), and the pain level can reach the severe standard of 7 or more, causing serious distress to patients' daily life.

[0008] With the increasing trend of global population aging, the incidence and burden of herpes zoster are also showing a significant upward trend. Among them, the incidence of herpes zoster in the Asia-Pacific region is about 1%, and the incidence increases with age, with a herpes zoster incidence rate of more than 5% after the age of 50. And with the further continuous growth of VZV infection every year, the infection incidence is expected to increase by 35%-100%. According to statistics, by the end of 2023, the number of people aged 60 and above in China reached nearly 300 million, accounting for 21.1% of the total population, of which the population aged 65 and above accounted for 15.4%. The number of herpes zoster patients in China each year is 2.77 million, of which the number of new herpes zoster cases in people aged 50 and above each year exceeds 1.5 million, with a disease cost of about 1.3 billion yuan. In 2010, there were about 9 million herpes zoster patients in China's population aged 50 and above, with an economic burden of more than 7.7 billion yuan. However, there is no specific treatment for herpes zoster and post-herpetic neuralgia at present, and most of the clinical treatment uses broad-spectrum antiviral drugs such as acyclovir, and some anesthetic or non-anesthetic analgesics, anticonvulsants, and antidepressants are also used to relieve the severe neuralgia caused by herpes zoster. Even so, even if the cure is still likely to recur, drug treatment cannot solve the problem of virus exclusion and prevention of hidden infection, therefore, vaccination is the most effective means of preventing and controlling herpes zoster and its complications.

[0009] Currently, there are two VZV vaccines approved by the FDA in the United States, namely Zostavax from Merck and Shingrix from GSK. Zostavax is a live attenuated vaccine based on the Oka strain. In the 60-69 age group, a single subcutaneous injection of Zostavax can achieve a preventive efficacy of 64%. However, the efficacy of Zostavax decreases with age, and in adults over 60 years old, the vaccine's effectiveness 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 of VZV, containing potential neutralizing epitopes and T cell epitopes, and plays a key role in viral propagation and transmission between ganglion cells. This vaccine is administered by intramuscular injection and shows a vaccine efficacy of 97.4% in preventing HZ in individuals aged 60-69 years, with an efficacy of >90% in all age groups tested, including individuals ≥80 years of age, which is superior to Merck's live attenuated vaccine Zostavax. Although the Shingrix recombinant protein vaccine is significantly more effective than the live attenuated vaccine Zostavax, the vaccine formulation is complex, the production cost is high, and the price is expensive, so it is necessary to develop a self-innovated vaccine product that can prevent VZV infection in China.

[0010] The core characteristic of a vaccine product is its ability to prevent a specific pathogen, such as the pneumococcal polysaccharide vaccine, which can effectively prevent pneumonia, meningitis, and otitis media caused by pneumococcus, while the VZV vaccine can prevent the reactivation of latent virus leading to herpes zoster. To achieve the goal of preventing multiple diseases with a single formulation, current technologies mainly use combination vaccines, such as the DPT-Hib-IPV pentavalent vaccine for children, the DPT-Hib-IPV-Hib-Hepatitis B hexavalent vaccine, and others. It is worth noting that although these combination vaccines are prepared by mixing single vaccines, clinical evaluations have shown that the preventive effect of some single vaccines in combination formulations is not good. Currently, children of the appropriate age in China need to be vaccinated with more than 15 types of vaccines, and the actual number of vaccinations is much higher than the number of vaccine types. With the intensification of the trend of population aging (as of 2024, China's population over 60 years old has reached 310 million, accounting for 22% of the total population), the development of vaccines for the elderly population has also become a key public health priority. Therefore, the development of multi- combination multi-valent combination vaccines to replace single vaccines and achieve full population protection coverage will undoubtedly become an important strategic direction in the field of vaccine research and development.

[0011] However, in the development of new vaccines, the choice of carrier protein directly affects the immune effect, especially as the vaccine dose increases, polysaccharide protein conjugate vaccines face the dual challenges of enhancing polysaccharide immunogenicity and avoiding carrier protein inhibition. In addition, how to design a stable and high expression carrier protein, and combine it with immunogenic substances such as capsular polysaccharide to develop an innovative product with dual immunogenicity, "one vaccine to prevent two diseases", these are the key challenges in the process of vaccine design and development. SUMMARY

[0012] In order to solve the problems in the prior art, the present application provides an immunogenic composition containing a multivalent pneumococcal polysaccharide-protein conjugate. The immunogenic composition uses polysaccharide-protein conjugate technology, and creatively designs a genetically engineered VZV gE protein with enhanced stability, and combines the protein as a carrier protein with 29 serotypes of pneumococcal polysaccharide. Thus, the immunogenic composition of the present application can not only induce protective immunity against 29 serotypes of pneumococcal, but also simultaneously stimulate immune response against varicella-zoster virus, showing excellent dual immunogenicity. The immunogenic composition provided by the present application can significantly reduce the number of inoculations, reduce the cost and complexity of immunization programs, and greatly broaden the breadth and effectiveness of immunoprotection, and also provides a more efficient and more economical solution for public health prevention strategies.

[0013] The technical solution of the present application to solve the technical problem is as follows:

[0014] In the first aspect of the present application, an immunogenic composition is provided, comprising:

[0015] at least one pneumococcal polysaccharide-protein conjugate, wherein the pneumococcal polysaccharide is connected to a carrier protein by a covalent bond;

[0016] The carrier protein is a varicella-zoster virus glycoprotein E, i.e. VZV gE recombinant protein, and its amino acid sequence is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0017] Further, 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.

[0018] In one preferred embodiment of the present application, the composition comprises a 29-valent pneumococcal polysaccharide-protein conjugate, wherein the pneumococcal polysaccharide comprises 29 serotypes, i.e. 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.

[0019] In one preferred embodiment of the present application, the composition comprises a 29-valent pneumococcal polysaccharide-protein conjugate, wherein the pneumococcal polysaccharide comprises 29 serotypes, i.e. 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.

[0020] Further, the composition further comprises an adjuvant. Preferably, the adjuvant is selected from one or more of an aluminum salt adjuvant, an emulsion-type adjuvant, an immunostimulatory complex, a Toll-like receptor agonist, a saponin-based adjuvant, or a cytokine.

[0021] In a second aspect of the present application, a method for preparing the immunogenic composition of the first aspect is provided, comprising the step of: coupling a pneumococcal polysaccharide to a VZV gE recombinant protein under suitable reaction conditions to form a covalently linked pneumococcal polysaccharide-protein conjugate.

[0022] Further, the method further comprises one or more of the following steps: activating the pneumococcal polysaccharide; expressing and purifying the VZV gE recombinant protein; purifying the coupling reaction product to obtain the pneumococcal polysaccharide-protein conjugate. Preferably, the activating the pneumococcal polysaccharide comprises degrading and / or chemically activating the polysaccharide. The degrading method is selected from high pressure homogenization, acid hydrolysis, or enzymatic digestion; the chemical activation method is selected from using 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) or cyanogen bromide (CNBr) for activation. The coupling reaction is selected from reductive amination, carbodiimide, or adipic acid dihydrazide (ADH)-mediated coupling.

[0023] In a third aspect of the present application, the immunogenic composition of the first aspect is provided for use in the preparation of a medicament for: preventing or treating a disease caused by Streptococcus pneumoniae infection; and / or preventing or treating a disease caused by Varicella-zoster virus (VZV) infection in a subject.

[0024] Further, the disease caused by the infection of Streptococcus pneumoniae includes pneumonia, bacteremia, meningitis or otitis media; the disease caused by the infection of VZV includes chickenpox, shingles or post-herpetic neuralgia.

[0025] In a fourth aspect of the present application, an isolated nucleic acid molecule encoding a VZV gE recombinant protein with an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4 is provided.

[0026] In a fifth aspect of the present application, a recombinant expression vector comprising the nucleic acid molecule of the fourth aspect is provided.

[0027] In a sixth aspect of the present application, a host cell comprising the recombinant expression vector of the fifth aspect or having the nucleic acid molecule of the fourth aspect integrated into its genome is provided.

[0028] The present application has the following technical effects:

[0029] (1) The present application provides an immunogenic composition containing a multivalent Streptococcus pneumoniae polysaccharide-protein conjugate, which is designed based on in-depth analysis of the epidemiological characteristics of Streptococcus pneumoniae in China. In order to achieve the best protection effect, 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 are selected and combined. The immunogenic composition of the present application can effectively prevent invasive Streptococcus pneumoniae diseases caused by the above-mentioned 29 serotypes, and can induce high and balanced immune responses to each serotype.

[0030] (2) Based on the immunogenicity research of Streptococcus pneumoniae polysaccharide and carrier protein combination, a new type of carrier protein (VZV gE recombinant protein) is innovatively selected and designed. Through specific amino acid mutation modification of the gE protein, on the one hand, the stability of the carrier protein is significantly improved, so that it can maintain the integrity of the high-level structure and the function of the key antigen cluster under various temperature conditions and in the subsequent chemical reaction process, thereby ensuring its own immunogenicity; on the other hand, the modified gE protein as a carrier can significantly enhance the antigenicity of the 29 serotype Streptococcus pneumoniae polysaccharide combined therewith, thereby further optimizing the overall immune efficacy of the vaccine.

[0031] (3) The immunogenic composition containing a multivalent Streptococcus pneumoniae polysaccharide-protein conjugate provided by the present application exhibits a significant synergistic effect, achieving a "double enhancement" effect:

[0032] In one aspect, the immunogenic composition prepared by the present application uses an optimized gE recombinant protein as a novel carrier, which can significantly enhance the antigenicity of 29 serotypes of pneumococcal polysaccharide. As can be seen from Example 9, after immunizing animals with the preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) prepared by the present application, the IgG antibody level in the animals can be effectively improved, and the serum protection titer is higher; and compared with before immunization, the antibody effect is significantly improved, and the preparation D of the present application is further superior to the immunization effect of the preparation C (Pfizer PCV13) using CRM197 as a carrier protein. Among them, the IgG antibody level of each serotype of pneumococcal polysaccharide originally contained in the rabbit serum before preparation immunization D0 (see Figure 4 ), after the first needle (D14), the second needle (D28), and the third needle (D35, D42) of the preparation, the preparation D (29-valent pneumococcal polysaccharide-gE protein conjugate vaccine) stimulates the animals to produce a significant upward trend in the IgG antibody titer of the corresponding 29 serotypes of pneumococcal polysaccharide, and the immunization effect after the third needle (D35) is obviously superior to that of the first needle (D14) and the second needle (D28), that is, with the increase of the number of immunization, the IgG antibody titer is significantly enhanced; and the blood detection after the completion of immunization shows that the IgG antibody titer of pneumococcal polysaccharide at D42 (see Figure 8 ) is slightly lower than that at D35 (see Figure 7 ), but the IgG antibody titer of each of the 29 serotypes of pneumococcal polysaccharide still maintains a stable and high level.

[0033] At the same time, after D14, D28, D35, and D42, the preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) and the preparation C (Pfizer PCV13) stimulate the animals to produce the IgG antibody titer of the corresponding serotype polysaccharide, which is obviously different according to the serotype. Overall, the antibody detection level after immunization of the preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) is obviously higher than that of the preparation C (Pfizer PCV13). Among them, for the 13 pneumococcal polysaccharide serotypes commonly contained in the preparation C and the preparation D, it can be seen from D14 ( Figure 5 ) that the IgG antibody level of the remaining 12 serotypes 1, 3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A, 19F, and 23F polysaccharide is higher in the preparation D than in the preparation C; it can be seen from D28 ( Figure 6 ) and D35 ( Figure 7 ) that the IgG antibody level of the remaining 11 serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 19F, and 23F polysaccharide is higher in the preparation D than in the preparation C; and it can be seen from D42 ( Figure 8As can be seen in Table 2, the IgG antibody levels for the 12 serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F and 23F polysaccharides were all higher for Formulation D than for Formulation C, except for serotype 19A.

[0034] On the other hand, the immunogenicity of the gE protein itself as a carrier protein is also enhanced in the immunogenic composition of the present application, which can stimulate a stronger specific immune response and induce the body to produce serum with a higher protective titer. As can be seen from Example 11, the IgG antibody titers of the gE protein in the rabbit immune antisera of Formulation F (gE protein) and Formulation D (29-valent polysaccharide-gE protein conjugate vaccine) are significantly enhanced compared to Formulation E (control PBS), and the total IgG, IgG1 and IgG2a antibody titers are all significantly improved. The protein antibody titers of Formulation D (29-valent polysaccharide-gE protein conjugate vaccine) are the highest among the total IgG, IgG1 and IgG2a antibodies, which are significantly higher than those of the non-conjugate gE recombinant protein of Formulation F.

[0035] Correspondingly, as can be seen from Example 12, compared to Formulation E (control PBS), Formulation F (gE protein) and Formulation D (29-valent polysaccharide-gE protein conjugate vaccine) after immunization can significantly stimulate the immune cells in the spleen of the animal, such as T cells (Th1 subpopulation), natural killer cells (NK cells), etc., to secrete IFN-γ cytokines. The sensitized immune cells of the mice immunized with Formulation D can secrete higher IFN-γ under the stimulation of the gE antigen or VZV virus, and the amount of IFN-γ produced by the spleen immune cells sensitized by Formulation D is significantly higher than that of Formulation F. It is indicated that the gE recombinant protein prepared by the present application has a good cellular immune effect, and the 29-valent pneumococcal polysaccharide-gE recombinant protein conjugate prepared by using the gE recombinant protein as a carrier protein has a stronger ability to induce cellular immune response in mice, and the regulation of immune response and the enhancement of the defense ability against pathogens are further improved.

[0036] (4) The immunogenic composition containing the multivalent pneumococcal polysaccharide-protein conjugate prepared by the present application can cover more valence pneumococcal serotypes, expand the protection range of the vaccine, and also ensure a good immune effect. As can be seen from Example 9, the antibody titers of the 29-valent pneumococcal polysaccharide-gE protein conjugate vaccine (Formulation D) are the highest among the total IgG, IgG1 and IgG2a antibodies at D14, D28, D35 and D42 (see Table 3). Figures 5-8The IgG antibody levels of the newly added serotypes 2, 8, 9N, 10A, 11A, 12F, 15A, 15B, 17F, 20, 22A, 22F, 24F, 33F, 34 and 35B polysaccharides in the preparation D are higher as a whole, and are obviously higher than the total average IgG antibody titers of the 13 serotypes polysaccharides in the preparation C; among them, especially the serotypes 2, 9N, 10A, 12F, 15A, 17F, 22A, 33F polysaccharides, and in all the 29 serotypes polysaccharides, the IgG antibody titers of the newly added serotypes 2, 9N, 10A, 12F, 15A, 17F, 22A, 33F polysaccharides in the preparation are always at a significantly higher antibody level.

[0037] (5) The application further provides a preparation method of the multivalent pneumococcal polysaccharide-protein conjugate composition. The core of the method is to use a VZV gE protein modified by genetic recombination and amino acid mutation as a polysaccharide carrier, and in the preparation process, the gE protein is chemically coupled with a plurality of serotype pneumococcal capsular polysaccharides and formulated with a new adjuvant, so that the immunogenic composition is obtained. Animal experiments prove that the immunogenic composition can induce a double immune response: the polysaccharide antigen part can stimulate the body to produce specific antibodies against 29 serotypes of pneumococcus; and the gE carrier protein part can effectively activate the humoral and cellular immunity against varicella-zoster virus. Therefore, a single vaccination of the preparation can simultaneously prevent pneumococcal and varicella-zoster virus infections, and achieve the innovative goal of "one vaccine for double prevention". BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Temperature stability test of gE protein before and after modification (0.06 ug / mL).

[0039] Figure 2 Temperature stability test of gE protein before and after modification (2.0 ug / mL).

[0040] Figure 3 Detection of gE protein antibody titers in mouse immune serum of preparations A and B.

[0041] Figure 4 Detection of 29 serotype pneumococcal polysaccharide IgG antibodies in rabbit immune serum of preparations C and D (D0).

[0042] Figure 5 Detection of 29 serotype pneumococcal polysaccharide IgG antibodies in rabbit immune serum of preparations C and D (D14).

[0043] Figure 6 Detection of 29 serotype pneumococcal polysaccharide IgG antibodies in rabbit immune serum of preparations C and D (D28).

[0044] Figure 7 Detection of IgG antibodies to 29 serotypes of pneumococcal polysaccharides in rabbit immune sera for Formulations C, D (D35).

[0045] Figure 8 Detection of IgG antibodies to 29 serotypes of pneumococcal polysaccharides in rabbit immune sera for Formulations C, D (D42). DETAILED DESCRIPTION

[0046] In order to make the technical solutions, objectives and advantages of the present application more comprehensible, the technical solutions of the present application are described in detail below in conjunction with specific examples and drawings. The test methods used in the following examples are conventional methods unless otherwise specified; the instruments, reagents and materials used are conventional commercial products unless otherwise specified.

[0047] Example 1: Preparation of pneumococcal capsular polysaccharides

[0048] (1) Preparation of master seed and working seed

[0049] Purification of capsular polysaccharides from 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 was carried out as follows:

[0050] Purification of capsular polysaccharides from 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 was carried out as follows:

[0051] The main seed of Streptococcus pneumoniae serotype 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 inoculated into 5 mL of yeast-acid hydrolyzed casein culture solution, and cultured at 36°C ± 2°C for 18 hours. When the bacteria grew to an OD600 reading of 1.0, the bacterial solution was transferred and inoculated into 150 mL of fresh yeast-acid hydrolyzed casein culture solution, and cultured at 36°C ± 2°C for 8 hours to the exponential growth phase. The culture was stopped, aliquoted, and stored at 4°C as a working seed for the serotype.

[0052] (2) Bacterial fermentation

[0053] The seed tube was taken from the working seed bank and inoculated into 5 mL of yeast-acid hydrolyzed casein culture solution and cultured at 36°C ± 2°C until the bacteria grew to the exponential growth phase. The bacterial solution was transferred and inoculated into 150 mL of fresh yeast-acid hydrolyzed casein culture solution and cultured at 36°C ± 2°C for 5-10 hours to the exponential growth phase. 50 mL of the bacterial solution was transferred and inoculated into 2 L of yeast-acid hydrolyzed casein culture solution and cultured at 36°C ± 2°C until the bacteria grew to the middle exponential growth phase to prepare a fermentation seed solution. The fermentation seed solution was inoculated into a 50 L fermenter containing 30 liters of yeast-acid hydrolyzed casein culture solution. Sodium hydroxide was used to maintain the pH of the fermentation solution at 6.8 ± 0.2, and the bacteria were allowed to grow to the late exponential phase.

[0054] (3) Capsular polysaccharide purification

[0055] 1. The pH of the fermentation solution was adjusted to between 3-5 by adding phosphoric acid, and stirred for 1 hour.

[0056] 2. Centrifugation was performed using a disc centrifuge at a speed of 9600 rpm, and the supernatant was collected and the residue was discarded.

[0057] 3. The centrifuged solution was microfiltered using a microfiltration membrane to remove residual cell debris and insoluble small particulate matter. The fermentation supernatant was microfiltered using a 0.22 μm membrane, and the filtrate was collected.

[0058] 4. The microfiltrate was concentrated and diafiltered using a 100 kD membrane pack to obtain a crude bacterial capsular polysaccharide solution. The solution was diafiltered using a 30 Kd membrane to 15 sample volumes.

[0059] 5. The polysaccharide solution was further diafiltered using a 50 kD membrane to 10 sample volumes, and the polysaccharide sample solution was concentrated.

[0060] 6. The purified polysaccharide solution was collected in a freeze-drying bottle and freeze-dried on a vacuum freeze-dryer, and stored at -70°C.

[0061] Example 2: Preparation of carrier protein

[0062] Varicella zoster virus (VZV) is a member of the alpha herpesvirus subfamily of the Herpesviridae family, i.e., Human herpesvirus 3.

[0063] gE (glycoprotein E, gE) is encoded by the ORF68 gene, located in the short segment region of the VZV genome, and the encoded gE contains 623 amino acids, and the gE protein molecule is mainly composed of a hydrophilic extracellular region (containing a signal peptide) composed of amino acids 1-546, a hydrophobic transmembrane region composed of amino acids 547-623, and an intracellular tail.

[0064] (I) Construction of the protein

[0065] The full-length sequence of the VZV gE protein mentioned in the present application refers to NCBI Reference Sequence: NP_040190.1, and the specific sequence is shown in SEQ ID NO. 1 (envelope glycoprotein E [Human alpha herpesvirus 3] / strain="Dumas" / 623aa).

[0066] The VZV gE protein used in the present application is different from the original full-length protein, but is a truncated protein, and the present application selects a conserved truncated gE protein amino acid sequence as a design optimization template. In order to improve the expression efficiency and protein structure stability, only the sequence of the signal peptide region and the mature antigen region is selected, and the specific sequence is shown in SEQ ID NO. 2.

[0067] The VZV gE protein mutant provided by the present application mainly mutates and modifies the mature antigen region amino acid sequence of the selected truncated protein, optimizes the protein stability and immunogenicity, and the mutant is different from the amino acid sequence of the VZV gE protein included in the NCBI database, and the specific sequence is shown in SEQ ID NO. 4. In addition, the specific sequence of the VZV gE protein before modification provided by the present application is shown in SEQ ID NO. 3.

[0068] (1) Amino acid sequence design

[0069] The present application relates to a VZV gE recombinant protein with enhanced stability and immunogenicity obtained by amino acid mutation modification, and the amino acid sequence design method is as follows:

[0070] The transmembrane region and intracellular region in the full-length sequence of VZV gE protein are deleted to obtain a truncated gE protein sequence as shown in SEQ ID NO. 2, and on this basis, point mutation of amino acids is further carried out, and the specific mutation mode is as follows: the W at the 200th position of the VZV gE protein is mutated to C, the A at the 246th position is mutated to C, and the Throm and 6his sequences are connected at the C-terminal end, and then a VZV gE protein full-length mutant sequence is obtained, and the amino acid sequence is shown in SEQ ID NO. 4.

[0071] The present application also relates to a VZV modified pre-gE recombinant protein, and the design mode of the amino acid sequence is as follows:

[0072] The transmembrane region and intracellular region in the full-length sequence of VZV gE protein are deleted to obtain a truncated gE protein sequence as shown in SEQ ID NO. 2, and on this basis, point mutation of amino acids is further carried out, and the specific mutation mode is as follows: the W at the 200th position of the VZV gE protein is mutated to C, the A at the 246th position is mutated to C, and the Throm and 6his sequences are connected at the C-terminal end, and then a VZV gE protein full-length mutant sequence is obtained, and the amino acid sequence is shown in SEQ ID NO. 4.

[0073] The sequences of SEQ ID NO. 1-SEQ ID NO. 4 are as follows:

[0074] SEQ ID NO. 1:

[0075] > VZV gE 1-623 (NP_040190.1 / 623aa / envelope glycoprotein E [Human alphaherpesvirus 3] / strain="Dumas")

[0076] 1 MGTVNKPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHT DEDKLDTNSV YEPYYHSDHA

[0077] 61 ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG VYNQGRGIDS GERLMQPTQM

[0078] 121 SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD QRQYGDVFKG DLNPKPQGQR LIEVSVEENH

[0079] 181 PFTLRAPIQR IYGVRYTETW SFLPSLTCTG DAAPAIQHIC LKHTTCFQDV VVDVDCAENT

[0080] 241 KEDQLAEISY RFQGKKEADQ PWIVVNTSTL FDELELDPPE IEPGVLKVLR TEKQYLGVYI

[0081] 301 WNMRGSDGTS TYATFLVTWK GDEKTRNPTP AVTPQPRGAE FHMWNYHSHV FSVGDTFSLA

[0082] 361 MHLQYKIHEA PFDLLLEWLY VPIDPTCQPM RLYSTCLYHP NAPQCLSHMN SGCTFTSPHL

[0083] 421 AQRVASTVYQ NCEHADNYTA YCLGISHMEP SFGLILHDGG TTLKFVDTPE SLSGLYVFVV

[0084] 481 YFNGHVEAVA YTVVSTVDHF VNAIEERGFP PTAGQPPATT KPKEITPVNP GTSPLLRYAA

[0085] 541 WTGGLAAVVL LCLVIFLICT AKRMRVKAYR VDKSPYNQSM YYAGLPVDDF EDSESTDTEE

[0086] 601 EFGNAIGGSH GGSSYTVYID KTR

[0087] SEQ ID NO. 2:

[0088] > VZV gE 1-546

[0089] MGTVNKPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHT DEDKLDTNSV YEPYYHSDHA

[0090] ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG VYNQGRGIDS GERLMQPTQM

[0091] SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD QRQYGDVFKG DLNPKPQGQR LIEVSVEENH

[0092] PFTLRAPIQR IYGVRYTETW SFLPSLTCTG DAAPAIQHIC LKHTTCFQDV VVDVDCAENT

[0093] KEDQLAEISY RFQGKKEADQ PWIVVNTSTL FDELELDPPE IEPGVLKVLR TEKQYLGVYI

[0094] WNMRGSDGTS TYATFLVTWK GDEKTRNPTP AVTPQPRGAE FHMWNYHSHV FSVGDTFSLA

[0095] MHLQYKIHEA PFDLLLEWLY VPIDPTCQPM RLYSTCLYHP NAPQCLSHMN SGCTFTSPHL

[0096] AQRVASTVYQ NCEHADNYTA YCLGISHMEP SFGLILHDGG TTLKFVDTPE SLSGLYVFVV

[0097] YFNGHVEAVA YTVVSTVDHF VNAIEERGFP PTAGQPPATT KPKEITPVNP GTSPLLRYAA

[0098] WTGGLA

[0099] SEQ ID NO.3:

[0100] >VZV modified gE

[0101] MGTVNKPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHT DEDKLDTNSV YEPYYHSDHA

[0102] ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG VYNQGRGIDS GERLMQPTQM

[0103] SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD QRQYGDVFKG DLNPKPQGQR LIEVSVEENH

[0104] PFTLRAPIQR IYGVRYTETW SFLPSLTCTG DAAPAIQHIC LKHTTCFQDV VVDVDCAENT

[0105] KEDQLAEISY RFQGKKEADQ PWIVVNTSTL FDELELDPPE IEPGVLKVLR TEKQYLGVYI

[0106] WNMRGSDGTS TYATFLVTWK GDEKTRNPTP AVTPQPRGAE FHMWNYHSHV FSVGDTFSLA

[0107] MHLQYKIHEA PFDLLLEWLY VPIDPTCQPM RLYSTCLYHP NAPQCLSHMN SGCTFTSPHL

[0108] AQRVASTVYQ NCEHADNYTA YCLGISHMEP SFGLILHDGG TTLKFVDTPE SLSGLYVFVV

[0109] YFNGHVEAVA YTVVSTVDHF VNAIEERGFP PTAGQPPATT KPKEITPVNP GTSPLLRYAA

[0110] WTGGLA GGLVPRGSHHHHHH

[0111] SEQ ID NO.4:

[0112] >VZV modified gE W200C A246C

[0113] MGTVNKPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHT DEDKLDTNSV YEPYYHSDHA

[0114] ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG VYNQGRGIDS GERLMQPTQM

[0115] SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD QRQYGDVFKG DLNPKPQGQR LIEVSVEENH

[0116] PFTLRAPIQR IYGVRYTETC SFLPSLTCTG DAAPAIQHIC LKHTTCFQDV VVDVDCAENT

[0117] KEDQLCEISY RFQGKKEADQ PWIVVNTSTL FDELELDPPE IEPGVLKVLR TEKQYLGVYI

[0118] WNMRGSDGTS TYATFLVTWK GDEKTRNPTP AVTPQPRGAE FHMWNYHSHV FSVGDTFSLA

[0119] MHLQYKIHEA PFDLLLEWLY VPIDPTCQPM RLYSTCLYHP NAPQCLSHMN SGCTFTSPHL

[0120] AQRVASTVYQ NCEHADNYTA YCLGISHMEP SFGLILHDGG TTLKFVDTPE SLSGLYVFVV

[0121] YFNGHVEAVA YTVVSTVDHF VNAIEERGFP PTAGQPPATT KPKEITPVNP GTSPLLRYAA

[0122] WTGGLA GGLVPRGSHHHHHH.

[0123] (2) Synthesis of the VZV gE protein gene of interest

[0124] According to the VZV gE protein amino acid sequences SEQ ID NO. 3, SEQ ID NO. 4 designed above and the codon preference of the host cell, the corresponding gene coding sequence is determined, and the restriction endonuclease EcoRI sequence is added at the C terminal of the segment gene, and the restriction endonuclease XbaI sequence is added at the N terminal. The designed nucleotide sequence is chemically synthesized.

[0125] (3) Plasmid amplification and extraction of the gene of interest

[0126] The plasmid vector pUC19 is double digested by EcoRI and XbaI restriction enzymes, and then connected with the above synthesized gene, introduced into the amplification host DH5a, and screened for single clones using LB(Amp+) agar solid medium; the single clone containing the gene of interest is inoculated in LB(Amp+) liquid medium, cultured and amplified at 37°C, 200 rpm, and the Sigma-Aldrich GenEluteTM HP plasmid medium preparation kit is used to extract the plasmid pUC19-gE; the extracted plasmid is double digested by EcoRI and XbaI restriction enzymes, and the TaKaRa MiniBest Agarose Gel Extraction Kit is used to recover the gene fragment of interest.

[0127] (4) Construction of eukaryotic expression vector

[0128] The mammalian cell expression plasmid pGN-M, which contains a CMV promoter and a dihydrofolate reductase (DHFR) gene, was double-digested with EcoRI and XbaI restriction enzymes, and the vector DNA fragment was recovered using a TaKaRa MiniBEST DNA Fragment Purification Kit Ver. 4.0. The vector DNA fragment and the target gene fragment were ligated by cohesive end method and introduced into a DH5a amplification host, and a single colony containing the eukaryotic expression plasmid pGN-M_gE was obtained by screening. The single colony was inoculated in LB (Amp+) for amplification culture, and the amplified plasmid was extracted using a no-endotoxin plasmid extraction kit, TaKaRa MidiBEST Endo-free Plasmid Purification Kit, and the plasmid was named VZVgE.

[0129] (B) Expression of gE protein in CHO cells and clone screening

[0130] CHO K1 (ATCC) cells were used as host cells, and after the cells were recovered, they were cultured in a 10% newborn calf serum DMEM medium (Sigma-Aldrich), and subcultured once every 3 days. After subculturing for 2 generations, it was observed that the cells grew well, and then the CHO K1 cells were inoculated at a concentration of 0.75 x 10 6 cells / well in three 9.6 cm 2The cells were incubated in a humidified incubator at 5% C02 and 37°C with 4 μg of pcDNAVZVE vector in each well of the well, with IMDM + FBS (Gibco) added to two of the wells after mixing the DNA with Lipofectamine 2000 (Sigma-Aldrich) and Lipofectamine 2000 alone added to the third well as a negative control. After 48 hours, the medium was removed and centrifuged at 200 x g for 5 minutes and the supernatant stored at -20°C. IMDM + FBS medium and 10 μg / mL Blasticidin-HCl (Invitrogen) was added to one of the wells of transfected cells and the other well of transfected cells was washed with PBS and then the cells were lysed with 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% (v / v) Triton X-100 containing complete, EDA-free protease inhibitor cocktail (Roche Diagnostics). The lysate was centrifuged at 16000 x g for 10 minutes at 4°C and stored at -20°C. Western blotting was used to detect the presence of recombinant protein in the supernatant and lysate. After 5 days in selective medium, the cells were detached with trypsin (Invitrogen) and then seeded onto 9 cm Petri dishes for isolation of single clones by serial dilution. Forty-two single clones were selected and transferred to wells of a 96 well plate over the following 7-11 days. The culture supernatant was tested by Western blotting to select for high protein gE producers. The clone that secreted the highest amount of protein was selected for the next round of screening and the cells were expanded and 30 new clones were selected for storage.

[0131] The selected clones were expanded into three T175 flasks (NETS). Trypsin was added, the cells were washed with PBS and resuspended in 100 mL of ProCHO4 (Lonza) containing 1 x ProHT, 4 mM L-glutamine and 2% FBS (Lonza) in a 250 mL spinner flask. The cells were incubated at 37°C in a humidified incubator at 5% C02 with the lid slightly ajar to ensure air diffusion at 90 rpm agitation. Samples were taken daily, stained with trypan blue (Sigma-Aldrich) and the cells counted and passaged every 3-5 days. When the viable cell concentration was greater than 0.3 x 106 cells / mL and the viable cell number was greater than 90% at plateau phase, the FBS was gradually removed and the cells were considered fully adapted to serum-free suspension growth.

[0132] (III) Production of gE protein in bioreactor

[0133] A 1.5 liter perfusion culture was set up in a bioreactor with a spin filter (10 μm) separator. The culture parameters were set as follows: temperature was controlled at 37°C by a heating blanket, pH was adjusted at 6.9 by CO2or 0.3 M sodium hydroxide, agitation was set at 200-300 RPM, and dissolved oxygen (dO2) was adjusted to 40% of saturated air with a mixture of N2and O2at a maximum flow rate of 200 mL / min. The perfusion rate was set at 0.3 to 0.8 V dilution per day, and cell counts were performed daily by sampling the culture broth. Trypan blue staining was used, and the glucose and lactate concentrations in the supernatant were measured off-line.

[0134] A total of 12.5 liters of cell-free broth was collected, centrifuged at 8000 x g for 30 minutes at 4°C, filtered through a 0.45 μm membrane, and concentrated by ultrafiltration using a 10 kDa membrane cassettes. The sample solution was concentrated to 0.5 liter, and 0.5 liter of PBS was added. The solution was concentrated to 0.5 liter again. The above steps were repeated five times.

[0135] (IV) Purification of gE protein

[0136] The sample solution was loaded onto a Q-Sepharose fast flow (GE Bioscience) column, and the column was washed with 20 mM Tris-HCl pH 7.5. The column was then washed with 20 mM Tris-HCl pH 7.5 with 200 mM sodium chloride added, to further remove adsorbed protein impurities. The gE protein was eluted with a solution in which the concentration of sodium chloride was increased to 300 mM. Ammonium sulfate was added to the combined eluate 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) with 800 mM ammonium sulfate added, and then with PBS containing 400 mM ammonium sulfate. Finally, the gE protein was eluted with purified water. The final sample was loaded onto a Sephacryl S-400 HR (GE Bioscience) column, which was washed with PBS. The protein peak was collected, and a cosolvent was added. The sample was lyophilized in a vacuum lyophilizer and stored at -70°C until use.

[0137] Both the VZV-modified pre-gE protein and the VZV-modified gE protein were prepared using the above-described method.

[0138] Example 3: Stability test of the carrier protein obtained in Example 2

[0139] 1) The proteins to be tested (modified gE protein prepared in Reference Example 2 and modified gE protein prepared in Example 2) were diluted to 20 ug / mL with 1*PBS pH7.4 buffer and placed in 1.5 mL centrifuge tubes, with a total volume of 1 mL.

[0140] 2) Incubation at different temperatures was performed according to the following table.

[0141]

[0142] 3) After incubation, each sample was placed at 4°C for temporary storage.

[0143] 4) Preparation of detection antibody (E5-G6)

[0144] BALB / c mice were immunized with gE protein vaccine (GSK) at a dose of 100 ug per mouse, and the mice were immunized twice. B lymphocytes were extracted from the spleen of the BALB / c mice, and a monoclonal antibody mAb (number: E5-G6) against VZV-gE was prepared and cloned by hybridoma cell technology.

[0145] 5) ELISA detection was performed according to the following steps:

[0146] The samples stored at 4°C were diluted to 1 ug / mL with 1*PBS pH7.4, and 100 uL / well was added to the enzyme-labeled plate (NUNC442404) for coating at 4°C overnight. The enzyme-labeled plate was spun dry, 1% BSA-PBS was added at 150 uL / well, and incubation was performed at 37°C for 1 hour. The plate washer was programmed to wash the plate 3 times, and different concentrations of detection antibody (primary antibody) were added according to the design: 0.06 ug / mL E5-G6, 2.0 ug / mL E5-G6, 100 uL / well, and incubation was performed at 37°C for 2 hours. The plate washer was programmed to wash the plate 3 times, and anti-mouse secondary antibody was added at 1:2000 dilution, 100 uL / well, and incubation was performed at 37°C for 1 hour. The plate washer was programmed to wash the plate 3 times, pNPP substrate solution was added at 100 uL / well, and the enzyme-labeled instrument was set at a wavelength of 405 nm for reading.

[0147] The results of the temperature stability detection of the proteins before and after modification are shown in Figures 1-2As shown in the figure, the gE protein before and after modification has obvious difference in antibody binding activity at different temperatures. The OD value of the modified gE protein detected by using different concentrations of detection antibody (0.06 ug / mL E5-G6, 2.0 ug / mL E5-G6) at 4℃, 37℃, 60℃ and 80℃ is obviously higher than that of the gE protein before modification. It is indicated that the modified gE protein prepared in the application can still maintain high antigen binding activity after treatment at different temperatures, that is, compared with the gE protein before modification, the stability of the modified gE protein prepared in the application is further enhanced.

[0148] Example 4: Detection of protein antibody titer in mouse immune serum

[0149] The VZV gE protein before modification / modified gE protein 0.1 mg / mL obtained by the method of Example 2 is added with phosphate buffer pH 5.8 buffer, filtered with a 0.22 μm membrane, sterilely packaged 0.8 mL / bottle, and stored at 4℃, ready for use, that is, preparation A (gE protein before modification) and preparation B (modified gE protein).

[0150] 4-6 week old female BALB / c mice are taken and randomly divided into two groups, and immunized with preparation A and preparation B respectively, 0.1 mL each time, subcutaneously immunized once every two weeks, a total of two times, and blood is collected after 35 days of immunization. The blood is then placed at room temperature for 4 hours, centrifuged at 10,000 RPM at room temperature, and the supernatant serum is collected and stored at -70℃ for detection.

[0151] Prepare a purified gE protein stock solution of 1 μg / mL (1×PBS solution) and store it in a 4℃ refrigerator. Dilute the protein stock solution to 4 μg / mL in coating buffer, add 100 μL of coating solution to each well of the ELISA plate, and incubate at room temperature overnight. Wash 3 times with plate washing buffer, add 150 μL of blocking buffer, and incubate at 37℃ for 1 hour. Wash each well 3 times with 300 μl of plate washing buffer, and store at 4℃.

[0152] Dilute the corresponding serum to be tested obtained after immunization of the mouse into a working sample serum, dilute it by an appropriate multiple, and add it to the first row of holes of the ELISA plate, 100 μl per hole. Start from the first row and perform 2-fold serial dilution downward, and incubate at 37℃ for 2 hours. Wash each hole 3 times with 300 μl of plate washing buffer, dilute the AP-labeled goat anti-mouse secondary antibody by 1:1000, add 100 uL per hole, and incubate at 37℃ for 1 hour. Wash the plate 3 times according to the program, add 100 uL of pNPP substrate solution per hole, set the wavelength of the enzyme label instrument to 405 nm, and read the data.

[0153] Figure 3The detection results show that the antibody titers of the mice immunized with the gE protein before and after modification are different, the antibody titer of the mouse immunized with the gE protein after amino acid modification (preparation B) is obviously higher than that of the gE protein before modification (preparation A); and the serum of the immunized mouse is diluted by different times, at each concentration, the antibody titer of the gE protein after amino acid modification (preparation B) is still higher than that of the gE protein before modification (preparation A), and with the gradual increase of the serum dilution times, the antibody titers of the gE proteins before and after modification all show a decreasing trend. It is shown that compared with the gE protein before modification, the antigen immunization effect of the gE protein after amino acid modification prepared by the application is improved.

[0154] Example 5: Preparation of a 29-serotype pneumococcal 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 conjugate (CDAP)

[0155] 1) 17.5 mg of the corresponding serotype purified capsular polysaccharide was weighed and dissolved in 4 mL of sodium phosphate buffer;

[0156] 2) 12 mg of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) (Sigma-Aldrich) was added to the polysaccharide solution, stirred, and reacted at room temperature for 1 hour;

[0157] 3) 3 Eqm of cystamine was added, and reacted at room temperature for 1 hour;

[0158] 4) 0.3 mL of 1M lysine (Sigma-Aldrich) solution was added to quench the reaction, and reacted at room temperature for 1-2 hours;

[0159] 5) 8 Eqm of 3 (2-chloroethyl) phosphate was added to the polysaccharide solution to reduce the disulfide bond in the polysaccharide;

[0160] 6) The activated polysaccharide solution was transferred to a dialysis bag, and dialyzed against phosphate buffer at 4°C, with four changes of solution;

[0161] 7) 30 mg of carrier protein was weighed and dissolved in phosphate buffer, and the protein concentration was 10 mg / mL;

[0162] 8) 8 mg of bromoacetic acid N-hydroxysuccinimidyl ester (BAANS) (Sigma-Aldrich) was added to the carrier protein solution, and reacted at room temperature for 2 hours, and the activated protein solution was transferred to a dialysis bag (Thermo Scientific), and dialyzed against phosphate buffer at 4°C, with four changes of solution;

[0163] 9) Mix 4 mL of activated polysaccharide solution with 4 mL of activated protein solution, and react at room temperature for 4 hours;

[0164] 10) Add 4 Eqm of N-acetyl-L-cysteine (Sigma-Aldrich), and react at 2-8°C for 4 hours, and then add 12 Eqm of iodoacetamide (Sigma-Aldrich), and react at 2-8°C for 4 hours;

[0165] 11) Transfer the polysaccharide conjugate reaction solution to a dialysis bag, and dialyze against phosphate buffer at 4°C;

[0166] 12) Load the sample solution onto Sepharose CL-4B, and purify by column, and collect the outer water volume conjugate.

[0167] 13) After filtration with a 0.22 μm filter, store at 2-8°C until formulation.

[0168] The carrier protein used in Step 7) is a modified gE protein, and a non-modified gE protein can also be used as a carrier protein.

[0169] Example 6: Preparation of a Pneumococcal 29 serotype 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 conjugate (reductive amination method)

[0170] 1) Weigh 500 mg of each corresponding serotype purified capsular polysaccharide, and dissolve in 500 mL of purified water;

[0171] 2) Degradate by a high-pressure homogenizer at a pressure of 600 bar for three cycles, add 0.12 eqm of sodium periodate (Sigma-Aldrich), and react in the dark for 18 hours;

[0172] 3) Perform ultrafiltration washing with a 50 Kd membrane pack, and perform ultrafiltration washing and concentration of purified water for lyophilization;

[0173] 4) Weigh 18 mg of activated polysaccharide, and add 4 mL of DMSO, and stir until dissolved completely;

[0174] 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;

[0175] 6) After 4 hours of quenching reaction by adding 2 Eqm of sodium borohydride (Sigma-Aldrich), the synthesis reaction solution was transferred to a dialysis bag and dialyzed against buffer solution for four times.

[0176] 7) The sample solution was loaded on Sepharose CL4B, and the bound fraction was collected from the outer water volume.

[0177] 8) After filtration with 0.22 μm filter membrane, the sample was stored at 4°C for preparation.

[0178] 9) The molecular weight of the conjugate, the polysaccharide-protein concentration and the ratio were detected by sampling.

[0179] The carrier protein used in step 5) is the modified gE protein, and the unmodified gE protein can also be used as a carrier protein.

[0180] Example 7: Preparation of immunological preparations C, D, E and F

[0181] (1) Preparation C (Pfizer PCV13):

[0182] Prevenar 13 (Pneumococcal Conjugate Vaccine 13) is a 13-valent pneumococcal polysaccharide conjugate vaccine (PCV13) developed by Pfizer, which has been approved for marketing by FDA and EU, and officially approved for marketing in China in November 2016. It is the first 13-valent pneumococcal polysaccharide conjugate vaccine in the world. The vaccine uses pneumococcal capsular polysaccharide conjugate protein carrier (CRM197) technology to prevent related invasive diseases caused by 13 pneumococcal serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F), such as bacteremia pneumonia, meningitis, etc. Prevenar 13 is the most widely used pneumococcal conjugate vaccine product in the world, and has been included in the immunization program in many countries or regions in the world and Asia-Pacific region.

[0183] (2) Preparation D (29-valent pneumococcal polysaccharide-VZV recombinant protein conjugate vaccine):

[0184] Detection of polysaccharide concentration in monovalent conjugate, respectively, 2.2 μg polysaccharide amount of VZV recombinant protein conjugate solution (conjugate preparation method see example 5 or 6, this example uses the conjugate prepared in example 5), 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 to a sterile container, sample detection protein content is about 99 μg, add CpG (Genscript), the final amount is 0.1 mg, add phosphate buffer pH 5.8 buffer, filter sterilization with 0.22 μm membrane; add sterile aluminum phosphate gel (Benetag), the final aluminum ion amount is 0.125 mg, stir at 4 ℃ for 1 h, sterilely divide 0.5 mL / bottle, store at 4 ℃, wait for immunization.

[0185] (3) preparation E (control PBS)

[0186] Protein sample content is 0 μg, add CpG (Genscript), the final amount is 0.1 mg, add phosphate buffer pH 5.8 buffer, filter sterilization with 0.22 μm membrane; add sterile aluminum phosphate gel (Benetag), the final aluminum ion amount is 0.125 mg, stir at 4 ℃ for 1 h, sterilely divide 0.5 mL / bottle, store at 4 ℃, wait for immunization.

[0187] (4) preparation F (gE protein)

[0188] Use the modified gE protein obtained in example 2, the sample content is 66 μg, add CpG (Genscript), the final amount is 0.1 mg, add phosphate buffer pH 5.8 buffer, filter sterilization with 0.22 μm membrane; add sterile aluminum phosphate gel (Benetag), the final aluminum ion amount is 0.125 mg, stir at 4 ℃ for 1 h, sterilely divide 0.5 mL / bottle, store at 4 ℃, wait for immunization.

[0189] Example 8: preparation C, D immunization of rabbits and blood collection

[0190] Take 2.5-3.5 kg of New Zealand white rabbits 10, 5 groups, one group of immune agent D (prepared in Example 7), another group of immune agent C (Pfizer PCV13), each injection 0.5 mL / time, every two weeks immunization, a total of three times; blood collection time is 0 days, 14 days, 28 days, 35 days, 42 days (i.e. D0, D14, D28, D35, D42). Part of the blood is prepared into PBMC, frozen on dry ice and stored at low temperature, and the other part of the blood is placed at room temperature for 4 hours, centrifuged at 10000 RPM at room temperature, and the supernatant serum is collected and stored at -70℃ for detection.

[0191] Example 9: Detection of IgG antibodies of 29 serotypes of pneumococcal polysaccharide in rabbit immune serum

[0192] Prepare different serotypes of pneumococcal polysaccharide (1xPBS solution) respectively and store in a 4℃ refrigerator. Dilute the detected serotypes of pneumococcal polysaccharide to 4 μg / mL, add 100 μL coating solution to each well to coat the ELISA plate, and incubate at room temperature overnight. Wash 4 times with plate washing buffer, add 100 μL blocking buffer, incubate at room temperature for 2 hours, and wash 4 times with plate washing buffer and store at 4℃.

[0193] Dilute the serum to be tested prepared in Example 8 to 1:10 to prepare a working sample serum, add it to the first row of holes of the ELISA plate, and the total volume is 200 μL. Start from the first row and proceed to the next row in a two-fold serial dilution, and incubate at room temperature for 2 hours. Wash 4 times with plate washing buffer, add 100 μL of alkaline phosphatase-labeled goat anti-rabbit antibody (1:2000 dilution), and incubate at room temperature for 4 hours. Wash 4 times with plate washing buffer, add 100 μL of phospho-4-nitrophenyl phosphate disodium salt substrate (Sigma-Aldrich) solution, and read the plate at 405 nm.

[0194] The results of the detection of IgG antibodies of 29 serotypes of pneumococcal polysaccharide in the immune antiserum of the pneumococcal polysaccharide-protein conjugate vaccine rabbit are shown (see Figures 4-8 ), D0 before immunization (see Figure 4 ), the original IgG antibody level of each serotype of pneumococcal polysaccharide in the rabbit serum is low, and there is no significant difference in the detection 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) stimulates the animal to produce corresponding 29 serotypes of pneumococcal polysaccharide IgG antibody titers, which shows a clear upward trend, and the immune effect after the third injection (D35) is obviously better than that after the first injection (D14) and the second injection (D28), that is, with the increase of the number of immunizations, the IgG antibody titer is significantly enhanced; and the continued blood collection after the completion of the immunization shows that the pneumococcal polysaccharide IgG antibody titer at D42 (see Figure 8 ) is higher than that at D35 (see Figure 7The detection results slightly decreased, but the IgG antibody titers of the 29 serotypes of pneumococcal polysaccharide were still maintained at a stable and high level.

[0195] Meanwhile, in addition to the pre-immune D0 (see Figure 4 ), the post-immune D14, D28, D35, D42 (see Figures 5-8 ), the IgG antibody titers of the corresponding serotypes of polysaccharide stimulated by the preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) and the preparation C (Pfizer PCV13) were obviously different according to the serotypes. Overall, the antibody detection level after the immunization of the preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) was obviously higher than that of the preparation C (Pfizer PCV13).

[0196] Among them, on the one hand, for the 13 serotypes of pneumococcal polysaccharide that are commonly contained in the preparation C and the preparation D, it can be seen from the D14 ( Figure 5 ) that the IgG antibody levels of the 12 serotypes 1, 3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A, 19F and 23F polysaccharide were higher in the preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) than in the preparation C (Pfizer PCV13) except for the serotype 7F; it can be seen from the D28 ( Figure 6 ) and the D35 ( Figure 7 ) that the IgG antibody levels of the 11 serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 19F and 23F polysaccharide were higher in the preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) than in the preparation C (Pfizer PCV13) except for the serotypes 18C and 19A; it can be seen from the D42 ( Figure 8 ) that the IgG antibody levels of the 12 serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F and 23F polysaccharide were higher in the preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) than in the preparation C (Pfizer PCV13) except for the serotype 19A. It can be seen that the preparation D (29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine) prepared by the present application can effectively improve the IgG antibody level in the animal body after the immunization of the animal, and the serum protection titer is higher; and compared with the pre-immune, the antibody effect is significantly improved, and the preparation D of the present application is further superior to the immunization effect of the preparation C (Pfizer PCV13) with CRM197 as the carrier protein, which also shows that the gE recombinant protein carrier in the polysaccharide protein conjugate vaccine prepared by the present application has a good enhancement and promotion effect on the antigenicity of the 29 serotypes of pneumococcal polysaccharide.

[0197] On the other hand, for the newly added serotypes 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 application, it can be seen from the D14, D28, D35 and D42 (see Figures 5-8 ) that the IgG antibody levels of the 16 newly added polysaccharide serotypes in the present application are generally high, and are significantly higher than the total average level of the IgG antibody titers of the 13 polysaccharide serotypes of the preparation C (Pfizer PCV13); among them, especially the serotypes 2, 9N, 10A, 12F, 15A, 17F, 22A and 33F polysaccharides, among all the 29 polysaccharide serotypes, the IgG antibody titers of the newly added serotypes 2, 9N, 10A, 12F, 15A, 17F, 22A and 33F polysaccharides are always at a significantly higher antibody level. This further illustrates that the 29-valent pneumococcal polysaccharide-gE recombinant protein conjugate vaccine prepared in the present application, while covering higher-valent pneumococcal serotypes and expanding the immune protection range, can also ensure good immune effect.

[0198] Example 10: Immunization of rabbits and blood collection with preparation D, E and F

[0199] Take 9 New Zealand white rabbits of 2.5-3.5 kg, 3 rabbits in a group, and divide them into 3 groups. Each group is immunized with the preparation E, preparation F and preparation D prepared in Example 7, respectively. Immunize subcutaneously every two weeks, inject 0.5 mL per rabbit each time, a total of two times, collect blood one week after immunization, and place the collected blood at room temperature for 4 hours. Centrifuge at 10,000 RPM at room temperature, and aspirate the supernatant serum. Store at -70°C for detection.

[0200] Example 11: Detection of protein antibody titers in rabbit immune serum

[0201] Prepare a purified gE protein stock solution of 1 mg / mL (1xPBS solution) and store it in a 4°C refrigerator. Dilute the protein stock solution to 4 μg / mL in coating buffer, add 100 μL of coating solution to each well to coat the ELISA plate, and incubate at room temperature overnight. Wash 4 times with plate washing buffer, add 100 μL of blocking buffer, incubate at room temperature for 2 hours, wash 4 times with plate washing buffer, and store at 4°C for one week.

[0202] Dilute the serum to be tested prepared in Example 10 to 1:10 to prepare a working sample serum, and add it to the first row of wells of the ELISA plate, with a total volume of 200 μL. Start from the first row and proceed to two-fold serial dilution, and incubate at room temperature for 2 hours. Wash 4 times with plate washing buffer, add 100 μL of alkaline phosphatase-labeled goat anti-rabbit antibody (1:2000 dilution), and incubate at room temperature for 4 hours. Wash 4 times with plate washing buffer, add 100 μL of phosphoric acid-4-nitrophenyl phosphate disodium salt substrate solution, and read the plate at 405 nm.

[0203]

[0204] Note: In Table 2, the values outside the brackets are the geometric mean values, and the values inside the brackets are the specific numerical ranges of the samples.

[0205] The detection results in Table 2 show that, compared with the preparation E (control PBS), the IgG antibody titers of the gE protein in the rabbit immune antisera of the preparation F (gE protein) and the preparation D (29-valent polysaccharide-gE protein conjugate vaccine) are significantly enhanced, and the total IgG, IgG1 and IgG2a antibody titers are significantly improved. Among them, whether it is total IgG, IgG1 or IgG2a antibody, the detection value of the protein antibody titer in the rabbit immune serum of the preparation D (29-valent polysaccharide-gE protein conjugate vaccine) is the highest, which is significantly higher than that of the preparation F (gE protein). It is proved that the gE recombinant protein prepared by the present application can obtain serum with higher protection titer and better immune effect, in addition to improving the antigen immune effect of the gE protein itself.

[0206] Example 12: IFN-γ cytokine detection

[0207] Six-week-old female BALB / c mice were randomly divided into groups, 8 mice in each group, and divided into 3 groups, each of which was immunized with the preparation E, the preparation F and the preparation D prepared in Example 7, respectively, subcutaneously every two weeks, 0.1 mL each time, a total of two times, and the spleen was collected one week after immunization.

[0208] After the collected spleen was ground, PBS pH 7.4 was added, and after suspension, gradient dilution was performed, and the obtained single cell suspension was added to the ELISpot plate, which was pre-coated with capture antibody. 5 μg / mL of gE protein, VZV (100 pfu / mL), concanavalin A (1 μg / mL, positive control) (Sigma-Aldrich) were added, and incubated at 37°C, 5% CO2 for 24 hours. The number of IFN-γ cells was detected by ELISpot kit (MabTech), and the positive spots were determined by CTL ImmunoSpot S5UV Micro Analyzer.

[0209]

[0210] Note: In Table 3, the values outside the brackets are the geometric mean values, and the values inside the brackets are the specific numerical ranges of the samples.

[0211] The detection results in Table 3 show that, compared with preparation E (control PBS), after immunization of preparation F (gE protein) and preparation D (29-valent polysaccharide-gE protein conjugate vaccine), the immune cells in the spleen of the animals, such as T cells (Th1 subgroup), natural killer cells (NK cells), etc., can obviously secrete IFN-γ cytokines. Among them, the spleen cells of the mice immunized by preparation D (29-valent polysaccharide-gE protein conjugate vaccine) can secrete higher IFN-γ under the stimulation of gE antigen or VZV virus; and the amount of IFN-γ produced by the spleen immune cells sensitized by preparation D (29-valent polysaccharide-gE protein conjugate vaccine) is obviously higher than that of preparation F (gE protein). It is indicated that the gE recombinant protein prepared in the application has better cell immune effect except for the protein preparation itself, and the 29-valent pneumococcal polysaccharide-gE recombinant protein conjugate preparation prepared by using the gE recombinant protein as a carrier protein has stronger ability to induce cell immune response of mice, and further improves the regulation of immune response and the enhancement of the defense ability against pathogenic bacteria.

[0212] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it. Although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that the specific embodiments of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application. Any modification or equivalent replacement without departing from the spirit and scope of the present application should be covered in the protection scope of the claims of the application.

Claims

1. An immunogenic composition, comprising: the composition comprises 29-valent pneumococcal polysaccharide-protein conjugates, wherein the pneumococcal polysaccharides are covalently linked to carrier proteins; the pneumococcal polysaccharides comprise 29 serotypes, i.e., 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 carrier proteins are varicella-zoster virus glycoprotein E (VZV gE) recombinant proteins, the amino acid sequence of which is set forth in SEQ ID NO:

4.

2. The immunogenic composition of claim 1, wherein, the composition further comprises an adjuvant.

3. The immunogenic composition of claim 2, wherein, the adjuvant is selected from one or more of an aluminum salt adjuvant, an emulsion adjuvant, an immunostimulatory complex, a Toll-like receptor agonist, a saponin adjuvant, or a cytokine.

4. A method of preparing an immunogenic composition as claimed in any one of claims 1 to 3, characterized in that, comprising the steps of: coupling the pneumococcal polysaccharides to the VZV gE recombinant proteins under suitable reaction conditions to form the covalently linked pneumococcal polysaccharide-protein conjugates.

5. The method of claim 4, wherein, the method further comprises one or more of the steps of: activating the pneumococcal polysaccharides; expressing and purifying the VZV gE recombinant proteins; purifying the coupling reaction product to obtain the pneumococcal polysaccharide-protein conjugates.

6. The method of claim 5, wherein, the activation comprises degrading and / or chemically activating the polysaccharides.

7. The method of claim 6, wherein, the degrading method is selected from high pressure homogenization, acid hydrolysis, or enzymatic digestion; the chemical activation method is selected from using 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) or cyanogen bromide (CNBr).

8. The method of claim 5, wherein, the coupling reaction is selected from reductive amination, carbodiimide, or adipic acid dihydrazide (ADH)-mediated coupling.

9. Use of the immunogenic composition of any one of claims 1-3 in the manufacture of a medicament for: preventing or treating a disease caused by Streptococcus pneumoniae infection; and / or preventing or treating a disease caused by varicella-zoster virus (VZV) infection.

10. Use according to claim 9, characterized in that, the disease caused by Streptococcus pneumoniae infection comprises pneumonia, bacteremia, meningitis, or otitis media; the disease caused by VZV infection comprises chickenpox, shingles, or post-herpetic neuralgia.

11. An isolated nucleic acid molecule encoding a VZV gE recombinant protein having the amino acid sequence set forth in SEQ ID NO:

4.

12. A recombinant expression vector comprising the nucleic acid molecule of claim 11.

13. A host cell comprising the recombinant expression vector of claim 12 or having integrated into its genome the nucleic acid molecule of claim 11.

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