Recombinant human rotavirus vaccine and preparation method thereof
By preparing a recombinant human rotavirus vaccine containing recombinant human rotavirus VP8 fusion protein, the problems of insufficient safety and effectiveness of existing vaccines have been solved, and a more efficient immune protection effect has been achieved, which is suitable for the immunization needs of many countries.
Patent Information
- Application Number
- CN202510929063.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-07
- Publication Date
- 2025-09-19
AI Technical Summary
Existing rotavirus vaccines have problems with intussusception risk and differences in effectiveness, especially in developing countries where the protection effect is poor. Existing vaccines also have problems with safety and effectiveness in preventing viral diseases.
A recombinant human rotavirus vaccine containing recombinant rotavirus VP8 fusion protein of P[4], P[6] and P[8] antigens was used as the antigen. Aluminum hydroxide adjuvant and 0.85% sodium chloride, 10mM HEPES solution were used as the buffer solution. The vaccine was prepared through high-pressure homogenization, anion chromatography, hydrophobic chromatography and other steps to ensure that the particle size, adsorption rate and pH value were within the appropriate range.
The safety and effectiveness of the vaccine have been improved, and its immunogenicity has been enhanced, especially showing better immune effects in P[8] type reactions, making it suitable for the immunization needs of different countries.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of vaccines, and in particular to a recombinant human rotavirus vaccine and a preparation method thereof. Background Art
[0002] Rotavirus (RV) belongs to the Reoviridae family and is an icosahedral RNA virus approximately 70 nm in diameter. It lacks an envelope and consists of a three-layered structure (outer capsid, inner capsid, and core), encapsidated by a double-stranded RNA gene containing 11 segments. These genes encode six structural proteins and six nonstructural proteins (VP1 to VP4, VP6, VP7, and NsP1 to NSP6). The structural proteins VP4 and VP7 each harbor specific antigenic determinants, eliciting serum-specific neutralizing antibodies and inducing a serum-specific protective immune response in vivo. VP6, the most abundant structural protein, possesses group-specific antigenic determinants; the nonstructural protein NSP4 acts as an enterotoxin.
[0003] Based on serological classification, identified rotavirus strains can be divided into 10 groups, A through J. Group A rotaviruses primarily cause diarrhea in infants and young animals, and group A rotaviruses are further divided into serotypes P and G. Recent studies indicate that among the serotypes identified that can infect humans and animals, there are 32 G serotypes and 47 P serotypes, with G1P, G2P, G3P, G4P, and G9P being the most prevalent strains.
[0004] There is currently no specific treatment for rotavirus-induced diarrhea. Given the high morbidity and mortality rates associated with rotavirus, the World Health Organization (WHO) recommends rotavirus vaccination to prevent rotavirus diarrhea. Currently, five rotavirus vaccines are commercially available domestically and internationally, all of which are oral, live attenuated vaccines. Nearly 100 countries worldwide have incorporated rotavirus vaccines into their national immunization programs, and their widespread use has significantly reduced the number of severe diarrheal illnesses and child deaths caused by rotavirus.
[0005] Studies have found that live attenuated vaccines have certain risk factors when preventing viral diseases. Regarding the currently available rotavirus vaccines, a large amount of clinical trial data and post-marketing pharmacovigilance monitoring data have demonstrated that they have two major flaws: (1) Risk of intussusception: As early as 1998, Wyeth launched an oral rotavirus vaccine (Rotashield). After one year of use, it was found that children who received the vaccine had an increased risk of intussusception, so the product had to be withdrawn from the market. Subsequently, Merck and GSK launched oral rotavirus live vaccines, which were found to increase the risk of intussusception after vaccination in many countries.
[0006] (2) Vaccine effectiveness differences: The protective effect and duration of vaccines in developing countries are significantly lower than those in developed countries. Rotarix and RotaTeq have an effectiveness of 80% to 90% in Europe and the United States, but only 60% to 70% in Asia and 30% to 50% in Africa.
[0007] Given the defects of the RV vaccines currently on the market, there is an urgent need for a new vaccine that is safe, effective, quality-controlled, and easy to immune recognize. Summary of the Invention
[0008] To address the deficiencies in the prior art, the present invention provides a recombinant human rotavirus vaccine and a preparation method thereof. The recombinant human rotavirus vaccine is effective and safe.
[0009] In order to achieve the purpose of the present invention, the following scheme is proposed: A recombinant human rotavirus vaccine comprising: ① Antigen: 25 μg to 65 μg each of P[4], P[6], and P[8] antigens. The amino acid sequences of these antigens are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively. The active ingredient of each type of antigen is a recombinant rotavirus VP8 fusion protein, which consists of a first polypeptide, a second polypeptide, and a third polypeptide. The first polypeptide is a truncated human rotavirus VP8 protein, which has 64 amino acids truncated at the N-terminus compared to the wild-type rotavirus VP8 protein; the second polypeptide is the amino acid sequence of the Fc fragment of immunoglobulin IgG4; the third polypeptide is uncharged or negatively charged, and the sequence is GGGGSGGGGSGGGGS; ②Adjuvant: aluminum hydroxide adjuvant 4.0mg~6.0mg; ③Buffer solution: sodium chloride and HEPES solution.
[0010] Furthermore, the specification is 0.5 ml / dose.
[0011] Furthermore, the physicochemical properties of the vaccine are as follows: Osmotic pressure: 240~320; Particle size: 3μm~6μm; Adsorption rate: greater than 95%; pH: 6.3~6.8.
[0012] Furthermore, the properties of the human rotavirus VP8 protein corresponding to the three types of antigens, P[4], P[6], and P[8], are: The molecular weight is 44864.87 and the isoelectric point is 5.49; The molecular weight is 45196.60 and the isoelectric point is 5.67; The molecular weight is 44976.06 and the isoelectric point is 5.82.
[0013] Furthermore, the buffer solution selected was 0.85% sodium chloride and 10 mM HEPES solution.
[0014] The method for preparing the above-mentioned recombinant human rotavirus vaccine comprises the following steps: S100, preparation of VP8 P[4], P[6], P[8] antigen stock solutions: S101, resuscitating the working seeds, culturing and inducing expression to obtain a culture solution, and collecting Escherichia coli cells by centrifugation; S102, setting the pressure of the high-pressure homogenizer to 800 bar, breaking the diluted E. coli through the homogenizer, and centrifuging to obtain a broken supernatant; S103, purifying by anion chromatography and hydrophobic chromatography in sequence; S104, ultrafiltration replacement was performed using 0.85% sodium chloride, 10 mM HEPES, pH 7.5 buffer, and sterile filtration was performed to obtain the stock solution; S200, preparation of aluminum adjuvant: S201, preparing solution: preparing aluminum chloride salt solution and sodium hydroxide salt solution; S201, adding aluminum chloride salt solution into the reactor, stirring and heating; S201, after the temperature is constant, slowly add sodium hydroxide salt solution and measure the pH value; S201, after reaching the predetermined pH value, stop adding the sodium hydroxide solution and continue stirring for 1 hour to 1.5 hours; S205. After the reaction is completed, 4 parts of water for injection are added to 1 part of the reaction solution, and after standing for 3 to 24 hours, 3 parts of the supernatant are removed; 3 parts of water for injection are added, mixed and allowed to stand until clarified, and 4 parts of the supernatant are removed to complete the liquid replacement; S206, sterilize at 121℃ for 20 to 45 minutes.
[0015] The properties of the prepared aluminum adjuvant are: D50 particle size of 3μm~7μm, pH of 6.0~6.6, and adsorption rate greater than 95%.
[0016] S300. Use P[4], P[6], and P[8] antigen stock solutions with aluminum adjuvant and HEPES buffer to prepare monovalent vaccines respectively. After overnight adsorption, mix the three monovalent vaccines and package them separately to obtain the recombinant human rotavirus vaccine.
[0017] The beneficial effects of the present invention are: using a truncated human rotavirus VP8 protein and an IgG4 Fc segment sequence to construct a soluble recombinant fusion protein as an antigen, wherein the IgG4 Fc segment is used as a leader sequence to construct the VP8 fusion protein, thereby increasing the expression level of the soluble protein and the molecular weight of the VP8 fusion protein, thereby enhancing immunogenicity. A trivalent vaccine containing three antigen types, P[4], P[6], and P[8], was prepared. Experimental results showed that an antigen content of 30 μg to 60 μg per dose had a good immune effect, and had a better effect on the most predominant P[8] type reaction. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 The pET-30a(+) plasmid map is shown; Figure 2 The P[4]-Fc_pET-30a(+) plasmid map is shown; Figure 3 The P[6]-Fc_pET-30a(+) plasmid map is shown; Figure 4 The P[8]-Fc_pET-30a(+) plasmid map is shown; Figure 5 Shown is the electrophoresis diagram of P[4]-Fc_pET-30a(+) plasmid enzyme digestion; Figure 6 Shown is the electrophoresis diagram of P[6]-Fc_pET-30a(+) plasmid enzyme digestion; Figure 7 Shown is the electrophoresis diagram of P[8]-Fc_pET-30a(+) plasmid enzyme digestion; Figure 8 shows the elution profile during anion chromatography; Figure 9 shows the elution profile during hydrophobic chromatography; Figure 10 A graph showing the results of rotavirus vaccine antigen dose study B2-D42 IgG antibody testing; Figure 11 A graph showing the results of neutralizing antibody testing for rotavirus vaccine antigen dose study B2-D42 is shown; Figure 12 A graph showing rotavirus vaccine immunogenicity versus B2-D42 IgG antibody test results is shown; Figure 13 Shown is a graph of rotavirus vaccine immunogenicity versus B2-D42 neutralizing antibody test results. DETAILED DESCRIPTION
[0019] Example 1 This example provides a method for preparing VP8-Fc protein, which is as follows: When expressed in E. coli, the full-length VP8 gene typically forms inclusion bodies. N-terminally truncated VP8 proteins produce very low yields, making them unsuitable for industrial production. Researchers have discovered that using the IgG4 Fc fragment as a leader sequence to construct a VP8 fusion protein can increase soluble protein expression, increase the molecular weight of the VP8 fusion protein, and enhance immunogenicity. Furthermore, to ensure that the higher-order structure of the protein is not affected, a flexible linker was added between the leader sequence and the VP8 encoding gene. Because the VP8 protein lacks glycosylation sites, the E. coli protein expression system was chosen.
[0020] pET-30a(+) is used as the prokaryotic expression vector for VP8 fusion protein. It contains a kanamycin resistance gene that can be used for screening positive transformed bacteria. It also contains regulatory elements such as the T7 promoter and the lactose operon for recombinant expression. During the culture process, the inducer (IPTG) is added to induce and initiate efficient and specific expression of the target gene. The map of pET-30a(+) is shown below. Figure 1 As shown, the cloning site selected in the pET-30a(+) plasmid is accessible to restriction endonucleases NdeI and HindIII, and provides the prokaryotic expression start codon ATG (encoding methionine), allowing direct insertion of the target gene fragment reading frame. Furthermore, two stop codons, TAA and TGA, were added during gene synthesis to ensure proper translation termination.
[0021] The synthesized target gene and pET-30a(+) vector were connected by double enzyme digestion with NdeI and HindIII to form a recombinant insertion of NdeI-target protein-stop codon-HindIII. The structures of the constructed plasmids P[4]-Fc_pET-30a(+), P[6]-Fc_pET-30a(+), and P[8]-Fc_pET-30a(+) correspond to Figure 2-Figure 4 .
[0022] On this basis, a preliminary study was conducted on the number of truncated amino acids in the N segment of the VP8 protein. VP8 protein amino acid sequences aa26-223, aa45-223, and aa64-223 were selected to construct VP8 fusion proteins. Plasmids were synthesized and transfected into Escherichia coli (SHuffle® T7 Express, BL21) for expression testing. The results are shown in Table 1: Table 1 Expression level records of three VP8 protein amino acid sequences The results showed that aa64~223 had better soluble expression, and the strain BL21 was better than SHuffle® T7 Express.
[0023] Through the above strategy, the target gene was inserted and transformed into competent cells of E. coli. The positive single colonies were selected by plating on LB plate medium containing kanamycin. The plasmids were extracted from the positive clones and sequenced to confirm that the inserted sequence was consistent with the designed sequence. P[4]-Fc_pET-30a(+), P[6]-Fc_pET-30a(+), and P[8]-Fc_pET-30a(+) were constructed. The electrophoresis patterns of the plasmids of the three proteins are shown as follows: Figure 5-Figure 7 shown.
[0024] Escherichia coli BL21 strain was selected as the expression strain. This strain contains a highly active T7 RNA polymerase gene and a LacI inhibitory gene, which can regulate the expression of the target gene together with the T7 promoter and lactose operator element on the pET-30a(+) vector.
[0025] The constructed plasmid was transformed into E. coli, and positive clones were selected from each E. coli BL21 strain as primary seeds. Working seed batches were established by reviving and culturing the primary seeds.
[0026] Example 2 This example provides a method for producing an antigen, which is as follows: To resuscitate glycerol bacteria, inoculate 500µl to 50ml of LB liquid culture medium containing 50µg / ml kanamycin sulfate at a ratio of 1%, and culture at 36℃-38℃, shaking at 200rpm for 4-5 hours, and stop culturing when the OD600 of the bacterial liquid reaches 1.0-2.5; then, after culturing in a seed tank, transfer the culture to a large tank for culturing. When the OD600 is 10-14, add an aqueous solution containing 6g IPTG, adjust the temperature to 16℃-20℃, feed supplementary culture medium at a rate of 5ml / L, and terminate the culture after 16-19 hours; harvest the fermentation broth and obtain the bacterial cells by centrifugation.
[0027] Resuspend the cells in a 1:40 (w / v) buffer solution and disrupt them once using a high-pressure homogenizer at 800 bar. Centrifuge at 12,500–16,000 g until the turbidity is less than 190 NTU. Filter through a 1.0 μm + 0.45 μm filter to obtain the disrupted supernatant.
[0028] Anion chromatography: Equilibrate the column with 20mM Tris-Cl pH7.5 solution at a flow rate of 1.5ml / min. Take the supernatant from the bacteria and directly load it onto the anion chromatography column. Elute linearly with 1M NaCl 20mM Tris-Cl pH7.5 as the eluent. Collect the eluate to obtain the anion chromatography protein. The elution profile is shown below. Figure 8 shown.
[0029] Hydrophobic chromatography: Use 0.5M ammonium sulfate 20mM Tris-Cl pH 7.5 as the equilibration solution at a flow rate of 1.5ml / min to equilibrate the column; take the anionic chromatography protein, add 3.6M ammonium sulfate mother solution to adjust the sample conductivity, and then load it onto the hydrophobic chromatography medium. Use 20mM Tris-Cl pH 7.5 solution as the eluent for linear elution, and collect the eluate to obtain the hydrophobic chromatography protein. The hydrophobic chromatography spectrum is shown as follows: Figure 9 shown.
[0030] Ultrafiltration was performed with 0.85% sodium chloride and 10 mM HEPES at pH 7.5, and sterile filtration was performed to obtain the stock solution.
[0031] Example 3 This example provides a buffer screening process, which is as follows: Phosphate, histidine, and HEPES were selected as buffers for vaccine preparation and screening. The buffer composition is shown in Table 2 below, and the results are shown in Table 3 below: Table 2 Composition of different buffers Table 3 Test results of vaccines prepared with different buffer compositions The results showed that the vaccine prepared with HEPES buffer had better particle size uniformity, so 10mM HEPES+0.85% sodium chloride was selected as the buffer.
[0032] Example 4 This embodiment provides an aluminum hydroxide adjuvant production process, which is as follows: Weigh appropriate amounts of sodium chloride and aluminum chloride to prepare an aluminum chloride salt solution; weigh appropriate amounts of sodium hydroxide and sodium chloride to prepare a sodium hydroxide salt solution; Add aluminum chloride solution into the reactor, set the stirring speed to 700 rpm~1200 rpm, and set the heating temperature to 60℃~80℃; When the temperature rises to the set temperature, add sodium hydroxide solution to the reactor at a rate of 20ml / min~70ml / min and check the pH value; When the pH value is between 6.8 and 7.5, stop adding the sodium hydroxide solution and continue the reaction for 1 to 1.5 hours while maintaining the original temperature and stirring speed. After the reaction is completed, the aluminum adjuvant is replaced by natural sedimentation. Taking 1L reaction liquid as an example, 4 parts of water for injection are added to 1 part of reaction liquid, mixed and allowed to stand for 3 hours to 24 hours, and 3 parts of supernatant are removed to complete the first liquid replacement; then the same amount of water for injection is added, mixed and allowed to stand, and 4 parts of supernatant are removed to complete the second liquid replacement, and then sterilized at 121℃ for 20 minutes to 45 minutes.
[0033] The properties of multiple batches of aluminum adjuvants prepared according to the above process are shown in Table 4: Table 4 Record of properties of multiple batches of aluminum adjuvants Example 5 This embodiment provides a vaccine preparation method, which is as follows: There are two options for preparing semi-finished products: ① After mixing the three monovalent stock solutions, add aluminum hydroxide adjuvant for adsorption and then package; ② The monovalent stock solution is mixed with aluminum hydroxide adjuvant to prepare a monovalent vaccine, which is then mixed and packaged; Considering that in scale-up production, the second process can separately detect the adsorption rate and identification of the three monovalent vaccines, and can better ensure the quality of the vaccine, the second process was chosen.
[0034] Specifically, P[4], P[6], and P[8] antigens were used in separate monovalent vaccines with aluminum hydroxide adjuvant and HEPES buffer, and adsorbed overnight. The three monovalent vaccines were then mixed and packaged to form the finished product. The test results for the three batches of finished products are shown in Table 5 below: Table 5 Test results of three batches of finished vaccines Example 6 This example provides a research experiment on recombinant human rotavirus vaccine, which is as follows: Experiment 1: Antigen dosage study Prepare the vaccine for immunization according to Table 6 below: Table 6 Record of vaccines of different concentrations Note: After the vaccine is diluted 3-fold, 300 μl is injected; D42 blood was collected and centrifuged to separate the serum for testing. The IgG antibody test results were as follows: Figure 10 The results of the neutralization test are shown in Figure 11 As shown, the results showed that the antigen content in the range of 30μg~60μg / dose had a better immune effect.
[0035] Experiment 2: Vaccine Application Prepare the vaccine according to Table 7 below: Table 7 Record of vaccines prepared with different types of proteins Animal experiments were conducted according to Table 8 below: Table 8 Experimental method record table Note: After the vaccine is diluted 3-fold, 300 μl is injected; D42 blood was collected and centrifuged to separate the serum for testing. The IgG antibody test results were as follows: Figure 12 The results of the neutralization test are shown in Figure 13 As shown, the results show that the vaccine provided by the present invention has a better effect in the most important P[8] type reaction.
[0036] The above embodiments are only used to illustrate the technical ideas and features of the present invention and are not intended to be exclusive or limit the present invention. It should be understood by those skilled in the art that various changes or equivalent substitutions made to the present invention without departing from the scope of the present invention are within the scope of protection of the present invention.
Claims
1. A recombinant human rotavirus vaccine, characterized in that: include: ① Antigen: 25 μg to 65 μg each of P[4], P[6], and P[8] antigens. The amino acid sequences of these antigens are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively. The active ingredient of each type of antigen is a recombinant rotavirus VP8 fusion protein, which consists of a first polypeptide, a second polypeptide, and a third polypeptide. The first polypeptide is a truncated human rotavirus VP8 protein, which has 64 amino acids truncated at the N-terminus compared to the wild-type rotavirus VP8 protein; the second polypeptide is the amino acid sequence of the Fc fragment of immunoglobulin IgG4; the third polypeptide is uncharged or negatively charged, and the sequence is GGGGSGGGGSGGGGS; ②Adjuvant: aluminum hydroxide adjuvant 4.0mg~6.0mg; ③Buffer solution: sodium chloride and HEPES solution.
2. The recombinant human rotavirus vaccine according to claim 1, characterized in that The specification is 0.5ml / dose.
3. The recombinant human rotavirus vaccine according to claim 1, characterized in that The physicochemical properties of the vaccine are as follows: Osmotic pressure: 240~320; Particle size: 3μm~6μm; Adsorption rate: greater than 95%; pH: 6.3~6.
8.
4. The recombinant human rotavirus vaccine according to claim 1, characterized in that The properties of the corresponding human rotavirus VP8 proteins of the three types of antigens, P[4], P[6], and P[8], are: The molecular weight is 44864.87 and the isoelectric point is 5.49; The molecular weight is 45196.60 and the isoelectric point is 5.67; The molecular weight is 44976.06 and the isoelectric point is 5.
82.
5. The recombinant human rotavirus vaccine according to claim 1, characterized in that The buffer solution used was 0.85% sodium chloride and 10 mM HEPES solution.
6. The method for preparing the recombinant human rotavirus vaccine according to claim 1, characterized in that: The following steps are involved: S100, preparation of VP8 P[4], P[6], P[8] type antigen stock solution: S101, resuscitating the working seeds, culturing and inducing expression to obtain a culture solution, and collecting Escherichia coli cells by centrifugation; S102, setting the pressure of the high-pressure homogenizer to 800 bar, breaking the diluted E. coli through the homogenizer, and centrifuging to obtain a broken supernatant; S103, purifying by anion chromatography and hydrophobic chromatography in sequence; S104, ultrafiltration replacement was performed using 0.85% sodium chloride, 10 mM HEPES, pH 7.5 buffer, and sterile filtration was performed to obtain the stock solution; S200, preparing aluminum adjuvant; S300. Use P[4], P[6], and P[8] antigen stock solutions with aluminum adjuvant and HEPES buffer to prepare monovalent vaccines respectively. After overnight adsorption, mix the three monovalent vaccines and package them separately to obtain the recombinant human rotavirus vaccine.
7. The preparation method according to claim 6, wherein step S200 specifically comprises: S201, preparing solution: preparing aluminum chloride salt solution and sodium hydroxide salt solution; S201, adding aluminum chloride salt solution into the reactor, stirring and heating; S201, after the temperature is constant, slowly add sodium hydroxide salt solution and measure the pH value; S201, after reaching the predetermined pH value, stop adding the sodium hydroxide solution and continue stirring for 1 hour to 1.5 hours; S205. After the reaction is completed, 4 parts of water for injection are added to 1 part of the reaction solution, and after standing for 3 to 24 hours, 3 parts of the supernatant are removed; 3 parts of water for injection are added, mixed and allowed to stand until clarified, and 4 parts of the supernatant are removed to complete the liquid replacement; S206, sterilize at 121℃ for 20 to 45 minutes.
8. The preparation method according to claim 7, wherein the properties of the aluminum adjuvant are: D50 particle size of 3 μm to 7 μm, pH of 6.0 to 6.6, and adsorption rate greater than 95%.