Rotavirus VP8-mRNA vaccine and preparation method thereof

Abstract

The invention belongs to the technical field of mRNA vaccine preparation, particularly discloses a rotavirus VP8-mRNA vaccine and a preparation method thereof, and provides a protein for preparing the rotavirus vaccine, and the amino acid sequence of the protein is shown as SEQ ID NO.1. According to the invention, a recombined VP8 gene is used as an antigen gene to prepare an LNP / mRNA vaccine, and the LNP / mRNA vaccine is used for preparing the rotavirus VP8-mRNA vaccine. Experimental immune evaluation is carried out in an adult female Balb / c mouse, and results show that the vaccine VP8-mRNA-LNP provided by the invention can stimulate a body to generate a relatively high antibody level, not only can stimulate the mouse to generate relatively good humoral immunity, but also can stimulate the mouse to generate cellular immunity, and can simultaneously prevent and treat G1P [8] and G9P [8] type rotaviruses in a broad spectrum.

Description

Technical Field

[0001] The present invention belongs to the technical field of mRNA vaccine preparation, and particularly relates to a rotavirus VP8-mRNA vaccine and a preparation method thereof. Background Art

[0002] Rotavirus (RV for short) is the most common pathogen causing severe diarrhea in infants and young children worldwide and is also one of the main causes of infant death. Approximately 1.3 million deaths are related to rotavirus infection every year, mainly affecting children under 5 years old. Rotavirus belongs to the genus Rotavirus of the family Reoviridae and is an unenveloped double-stranded RNA virus. The genome is discontinuous and consists of 11 segments of double-stranded RNA, encoding 6 structural proteins (VP1, VP2, VP3, VP4, VP6, VP7) and 6 non-structural proteins (NSP1, NSP2, NSP3, NSP4, NSP5, and NSP6). The outer capsid proteins VP4 and VP7 of rotavirus respectively constitute the outermost surface antigens of rotavirus and determine the serotype of rotavirus. Among them, the rotavirus outer capsid protein VP7 (glycoprotein) determines the G serotype, and the outer capsid protein VP4 (protease-sensitive protein) determines the P serotype. VP4 is a spike-like protein on the outer capsid of RV, has hemagglutinin function, is sensitive to proteolytic enzymes, and can be cleaved into VP5* (AA 248-776) and VP8* (AA 1-231). Among them, VP5* constitutes the main body of the spike, and VP8* is responsible for interacting with the host receptor to mediate virus attachment and has been proven to be an effective vaccine target. So far, 42 G types and 58 P types have been identified from humans and various animal species. Globally, six G types (i.e., G1, G2, G3, G4, G9, and G12) and three P types (i.e., P[4], P[6], and P[8]) are dominant. In addition, six strains of group A rotavirus, G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8], usually account for more than 90% of the globally prevalent group A rotavirus species.

[0003] The currently marketed vaccines are mainly live attenuated rotavirus vaccines, which can prevent rotavirus infection to a certain extent. However, due to differences in economic conditions, medical levels, etc. in different regions of the world, the efficacy of attenuated vaccines varies, and the development of rotavirus vaccines still faces huge challenges. To reduce the harm caused by rotavirus, various methods have been developed, including vaccines and antiviral drugs. In terms of vaccines, although the existing two live attenuated rotavirus vaccines can effectively protect infants and young children from rotavirus infection, they also have the risk of side effects such as reverse mutation and intussusception. At the same time, the two currently marketed vaccines mainly target the rotavirus serotypes in European and American countries and have little effect on the epidemic rotavirus serotypes in Asian and African countries. Therefore, there is an urgent need to develop a universal rotavirus vaccine that is safe, induces broad-spectrum and long-term immunity. Summary of the Invention

[0004] The main object of the present invention is to provide a rotavirus VP8-mRNA vaccine and its preparation method to solve the problems in the background technology and provide a new technical means for the prevention and treatment of rotavirus. Specifically, the present invention provides the following technical solutions: The present invention provides a protein for preparing a rotavirus vaccine, and the amino acid sequence of the protein is as shown in SEQ ID NO:1.

[0005] In one embodiment, the present invention provides a recombinant polynucleotide, and the polynucleotide encodes the protein; the polynucleotide sequence is as shown in SEQ ID NO:2.

[0006] In one embodiment, the present invention provides a recombinant mRNA, and the recombinant mRNA is an mRNA encoding the protein, and the mRNA further contains a 5`-UTR, a 3`-UTR and a polyA sequence.

[0007] In one embodiment, the present invention provides a recombinant mRNA, and the recombinant mRNA is obtained by transcription from the polynucleotide sequence as shown in SEQ ID NO:2.

[0008] In one embodiment, the present invention provides a recombinant vector, and the vector contains the polynucleotide or the recombinant mRNA.

[0009] In one embodiment, the present invention provides an engineered cell, and the cell contains the recombinant vector.

[0010] In one embodiment, the present invention provides a rotavirus mRNA vaccine, including the recombinant mRNA and a pharmaceutically acceptable delivery vector.

[0011] In one embodiment, the present invention provides a pharmaceutical composition comprising the rotavirus mRNA vaccine.

[0012] In one embodiment, the present invention provides a kit comprising the recombinant polynucleotide, or the recombinant mRNA, or the recombinant vector, or the engineered cell, or the rotavirus mRNA vaccine.

[0013] In one embodiment, the present invention provides the use of the protein, the recombinant polynucleotide, the recombinant mRNA, the recombinant vector, the engineered cell, and the rotavirus mRNA vaccine in the preparation of a drug for preventing and controlling rotavirus infection.

[0014] Technical effects achieved by the present invention: The present invention selects the rotavirus ZTR-68 strain with a high prevalence rate and easy variability as a model to carry out the research on mRNA vaccines, and uses the recombinant VP8 gene as the antigen gene to prepare the LNP / mRNA vaccine. Experimental immunological evaluation is carried out in adult female Balb / c mice. The results show that the vaccine VP8-mRNA-LNP provided by the present invention can stimulate the body to produce a relatively high antibody level, not only can stimulate good humoral immunity in mice, but also can stimulate cellular immunity in mice. In terms of broad-spectrum, it can prevent and control G1P[8] and G9P[8] rotaviruses simultaneously. Description of the drawings

[0015] Figure 1 Construction strategy diagram of the VP8-mRNA vaccine; Figure 2 . Flow chart of animal experiments; Figure 3 Single enzyme digestion reaction diagram of pUC57-VP8; M: DNA Marker; 1: Single enzyme digestion product of pUC57-VP8; Figure 4 Western blot identification result diagram of VP8-mRNA; M: Protein Marker; 1: VP8-mRNA (+); 2: VP8-mRNA (-); 3: Virus positive control; Figure 5 Physicochemical property determination diagram of the VP8-mRNA vaccine; Note: A: RNA capillary electrophoresis diagram after in vitro transcription of the plasmid; B: Particle size detection using a Malvern laser particle size analyzer; C: Encapsulation efficiency detection; D: Polymer dispersity index detection.

[0016] Figure 6 Transmission electron microscopy result diagram of VP8-mRNA; Figure 7 Statistical chart of specific IgG antibody levels in mouse serum; Figure 8 Statistical chart of neutralizing antibody titers in mouse serum; Figure 9 Detection of lymphocyte subsets in mouse spleen cells and detection of cytokine IFN-γ after immunization; (A) Percentage of CD3+CD8+ T lymphocyte count; (B) Percentage of CD3+CD4+ T lymphocyte count; (C) Number of IFN-γ spots produced in different groups; (D) Statistical count of increased spots in the vaccine group and the control group. Detailed implementation manners

[0017] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. For purchased commodities in the test methods, if the specific conditions are not indicated, they shall be carried out according to the conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not indicated, they can all be obtained as conventional products through commercial purchase.

[0018] Example 1 I. Materials (1) HEK293 cells (human embryonic kidney cells), preserved by the Molecular Biology Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College; (2) Sodium chloride (NaCl) (10 g / L), tryptone (10 g / L), and yeast extract (5 g / L) were all purchased from Beijing Tiangen Biotechnology Co., Ltd.; (3) Rotavirus ZTR-68 strain, preserved by the Molecular Biology Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, and passaged and cultured from MA104 cells; (4) PBS solution (for washing cells in the sterile room): Prepared by the Fourth Room of the Biological Products Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, and provided for use after passing the verification, and preserved in the Molecular Biology Laboratory; (5) DMEM medium: Purchased from Gibco, USA; (6) Double antibody (penicillin + streptomycin): Prepared by the Fourth Room of the Biological Products Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, and provided for use after passing the verification, and preserved in the Molecular Biology Laboratory; (7) 1% trypsin (for digesting cells): Prepared by the Fourth Room of the Biological Products Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, and provided for use after passing the verification, and preserved in the Molecular Biology Laboratory; (8) Fetal bovine serum (FBS): Purchased from Gibco; (9) pUC57-VP8 plasmid was synthesized by Suzhou Genewiz Co., Ltd., and amplified and extracted by shaking bacteria and preserved by the Molecular Biology Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College; (10)Restriction endonuclease BspQⅠ was purchased from Nanjing Novoprotein Co., Ltd.; (11)T7 High Yield RNA Transcription Kit was purchased from Nanjing Novoprotein Co., Ltd. (12)mRNA Cap 2′ -O-Methyltransferase was purchased from Nanjing Novoprotein Co., Ltd. (13)Ionizable lipids and PEG-lipids were purchased from Shanghai Avextar Biotechnology Co., Ltd.; (14)RNA quantification kit Quant-iT RiboGreen RNA Assay Kit was purchased from ThermoFisher Scientific; (15)Mouse lymphocyte separation solution and lymphocyte serum-free medium were both purchased from Beijing Dakewei Co., Ltd.; (16)Flow antibodies PerCP anti-mouse CD3ε, APC anti-mouse CD4, and PE anti-mouse CD8 were purchased from Beijing Dakewei Co., Ltd.; (17)ELISpot Plus: Mouse IFN-γ (ALP kit was purchased from Mab Tech, Inc., USA; (18)Bovine serum albumin (BSA) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; (19)Absolute ethanol was purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.; (20)15mL and 20mL centrifuge tubes were purchased from Corning; (21)1.5mL and 2mL EP tubes were purchased from KIRGEN; (22)Pipette tips were purchased from KIRGEN; II. Methods Design and preparation of VP8-mRNA vaccine 1. Construction strategy of RV mRNA candidate vaccine Based on the recombinant VP8 gene of rotavirus ZTR-68 strain, the RV mRNA vaccine was designed. The amino acid sequence of VP8 protein is shown in SEQ ID NO.1. The basic framework of vaccine design includes 5'-untranslated region, open reading frame, 3'-untranslated region, and poly-A tail, as shown in Figure 1, and the sequence is shown in SEQ ID NO.2.

[0019] 2. Plasmid synthesis and plasmid linearization The plasmid was commissioned to GENEWIZ (Suzhou) Co., Ltd. for synthesis and cloned onto the pUC57-Kan-SapI-free vector to obtain pUC57-VP8. The synthesized gene sequence was determined to be consistent with the target gene sequence by sequencing from GENEWIZ (Suzhou) Co., Ltd. The plasmid was taken for transformation, culture, and single colony identification. After correct identification, it was amplified and cultured in LB medium containing kanamycin sulfate, and the plasmid was extracted for identification and sequence determination. The plasmid was extracted with reference to the instructions of the endotoxin-free large plasmid extraction kit, and its concentration and purity were measured using a NanoDrop 2000 ultra-micro spectrophotometer. The plasmid was linearized with the BspQI restriction endonuclease.

[0020] Table 1: Preparation of plasmid digestion reaction system

[0021] 3. In vitro transcription, capping, and purification The plasmid pUC57-VP8 was extracted using a large plasmid extraction kit (Tiangen, DP117). The extracted plasmid pUC57-VP8 was linearized by single digestion with the restriction endonuclease SapI, and the linearized product was purified using VAHTS DNA Clean Beads. Under the condition of ensuring no nuclease throughout the process, the DNA was transcribed into RNA using the T7 High Yield RNA Transcription Kit (N1-Me-Pseudo UTP) in vitro transcription kit. The integrity of the RNA in the in vitro transcription product was detected by capillary electrophoresis. The transcription product was purified using VAHTS RNA Clean Beads, and after purification, the RNA was capped using a capping kit. After capping, the capped product VP8-mRNA was purified using VAHTS RNA Clean Beads. After purification, the capped product was encapsulated with LNP to obtain the VP8-mRNA-LNP vaccine. The mRNA capping reaction used the kit mRNA Cap2'-O-Methyltransferase. The above operations all referred to the kit instructions.

[0022] Table 2: Preparation of in vitro transcription reaction system

[0023] Table 3: Preparation of mRNA capping reaction system

[0024] 4. Western blot verification of the expression of VP8-mRNA vaccine The Polyplus jetMESSENGER® mRNA transfection reagent, an mRNA transfection kit, was used for the cell transfection experiment of VP8-mRNA. HEK293 cells were seeded in a 12-well plate at a density of 1×10 5 cells / well and cultured until the cell confluence reached nearly 70%-80%; 2 μg of Cap-mRNA and 200 μl of mRNA buffer were mixed evenly, and then 4 μl of jetMESSENGER® mRNA transfection reagent was added and gently mixed. After standing at room temperature for 10 min, the mixture was added dropwise into the wells, and then the plate was placed in an incubator at 37°C and 5% CO2 for further culture. After 48 h, the cell supernatant was collected, concentrated 10-fold using a 3 KDa ultrafiltration tube for cells, and collected into a 1.5 ml EP tube. SDS-PAGE protein loading buffer (5x) was added, and after protein denaturation, 10% SDS-PAGE gel electrophoresis was performed to verify the expression of VP8-mRNA.

[0025] 5. Determination of the particle size, PDI, zeta potential and encapsulation efficiency of mRNA-LNP mRNA was encapsulated in LNP by microfluidic technology. The particle size of LNP was measured by a Malvern laser particle size analyzer, the encapsulation efficiency of LNP-mRNA was detected using the Quant-iT™ RiboGreen™ RNA Reagent and Kit, and the morphology and structure of the encapsulated vaccine were observed by transmission electron microscopy.

[0026] 6. Evaluation of the immune effect in animals To evaluate the immune effect of the mRNA vaccine in animals, an animal immunization experiment was conducted. The experimental animals were healthy adult female Balb / c mice at the SPF level, 6 - 8 weeks old, weighing 18 - 22 g. They were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences. The animal experiment facility license number is SYXK (Yunnan) K2022 - 0006, and the animal experiment production license number is SCXK (Yunnan) K2022 - 0002. The immunization methods were divided into two types: intramuscular injection and subcutaneous injection, and a two - dose immunization method was adopted. The experiment was carried out according to the following groups. There were 4 groups in total, namely the PBS injection group; the group with 10 μg of the vaccine by intramuscular injection, the group with 15 μg of the vaccine by intramuscular injection, and the group with 20 μg of the vaccine by intramuscular injection; to verify the best protective efficacy of the mRNA vaccine. Finally, the IgG antibody level and neutralizing antibody level in the serum samples of the immunized mice were detected by the ELISA method, and the changes of T lymphocytes and cytokines in the spleens of the mice were detected by flow cytometry and ELISpot.

[0027] III. Results 1. Restriction enzyme digestion identification of the recombinant plasmid After a large amount of extraction of the recombinant plasmid pUC57 - VP8, its concentration and purity were measured by an ultra - micro spectrophotometer. Then, the plasmid was linearized by single - enzyme digestion with the restriction enzyme Sap I. Subsequently, the reaction solution was recovered. After purification with a kit, 1 μL was taken for gel electrophoresis verification. The results were as Figure 3 , and the linearized recombinant plasmid showed a single band, with a size of about 5000 bp, which was in line with expectations.

[0028] 2. In vitro expression verification of VP8 - mRNA The mRNA encoding the VP8 gene (VP8 - mRNA) synthesized by in vitro transcription was transfected into HEK293 cells. After 48 h, the cell supernatant was collected and concentrated 10 times, and its expression was identified by Western blot. The concentrated solution of RV was used as a positive control. The results are shown in Figure 4. A positive band of about 23 kD could be detected in the concentrated cell supernatant transfected with VP8 - mRNA, and its size was consistent with that of RV, indicating that the mRNA with a signal peptide could achieve the secretory expression of the target protein.

[0029] 3. Characterization of the VP8 - mRNA - LNP vaccine Capillary electrophoresis was used to detect the integrity of the capped mRNA. The capillary electrophoresis detection results showed that the mRNA vaccine presented a single peak and symmetric distribution, indicating that the integrity of the mRNA vaccine was good ( Figure 5-A). Subsequently, the VP8-mRNA vaccine was encapsulated by a microfluidic device and subjected to quality control analysis. The particle size, polydispersity index (PDI), and zeta potential of the vaccine formulation were characterized using a Malvern Zetasizer Nano ZS90 instrument. The results of the average particle size of the mRNA vaccine showed that the average particle size of VP8-mRNA-LNP was approximately 103 nm, the PDI was 0.0887, and it exhibited a unimodal normal distribution. The encapsulation efficiency and concentration of the mRNA vaccine were detected using the Quant-iT™ RiboGreen RNA Quantification Kit, and the encapsulation rate of VP8-mRNA was 92.3% ( Figure 5 ).

[0030] The encapsulated VP8-mRNA-LNP vaccine was observed to be a uniform sphere with a particle size of approximately 100 nm by transmission electron microscopy, indicating that the LNPs loaded with mRNA had a complete morphology ( Figure 6 ).

[0031] 4. Immunogenicity evaluation of VP8-mRNA-LNP vaccine 4.1 IgG antibody detection After blood was collected and serum was separated at weeks 0, 3, and 6 of the experiment, plates were coated with 0.1 μg / well of VP8 protein, and the specific antibody response of the VP8-mRNA-LNP vaccine was evaluated by indirect ELISA. The IgG antibody titers were shown as Log2 GMT. As shown in Figure 7, on the 21st day after the first dose of immunization, the average IgG antibody titers in the sera of mice in the 10 μg, 15 μg, and 20 μg groups were 10.2, 11.2, and 10.4, respectively. After the second dose of immunization, the antibody levels further increased to 16.6, 17, and 17.2, respectively, showing a certain dose-response relationship, but there was no statistical difference in the IgG antibody levels induced by the 15 μg and 20 μg vaccines. The results indicated that the VP8-mRNA-LNP vaccine could stimulate the body to produce relatively high antibody levels.

[0032] 4.2 Neutralizing antibody detection To evaluate whether the VP8-mRNA-LNP vaccine can induce anti-rotavirus serum neutralizing antibodies in mice, peripheral blood serum of mice was collected for detection of neutralizing antibodies against rotavirus G1P[8] and G9P[8], and the results were presented as Log2 GMT. The neutralizing antibody detection results showed that at 6 weeks after the first immunization (the 42nd day of the experiment), the neutralizing antibody titers of the immune sera in the 10 μg, 15 μg, and 20 μg groups against G1P[8] were 5.2, 7.6, and 7.4 respectively. Among them, the neutralizing antibody level in the 15 μg group was the highest, and the difference in the neutralizing antibody level compared with the 10 μg group was statistically significant (P<0.05). The results of the neutralizing level against RV G9 (G9P[8]) virus showed that the neutralizing antibody titers of the immune sera in the 10 μg, 15 μg, and 20 μg groups were 6, 7.6, and 7.4 respectively, and each group showed good virus neutralizing ability. The results indicated that the VP8-mRNA-LNP vaccine could stimulate the body to produce a certain level of neutralizing antibodies.

[0033] 4.3 T lymphocyte subset analysis and cytokine detection At 6 weeks after the first immunization (the 42nd day of the experiment), flow cytometry was used to detect T lymphocyte subsets in splenocytes. Compared with the negative control group, the proportions of CD4 + cells and CD8 + cells in the mRNA vaccine experimental group increased. The results indicated that the VP8-mRNA vaccine played a stimulating role in the differentiation of CD4+ cells and CD8 + cells (Figure 9). Th1 is involved in cell immunity and mainly secretes IFN-γ. A commercial ELISA Spot Plus: Mouse IFN-γ detection kit was used to detect the INF-γ secreted by splenocytes of mice at 42 days. The results showed that the INF-γ factors produced in the mRNA vaccine group were higher than those in the PBS group. The results indicated that the developed VP8-mRNA-LNP vaccine could not only stimulate mice to produce good humoral immunity but also stimulate mice to produce cell immunity.

[0034] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A protein for preparing a rotavirus vaccine, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO:

1.

2. A recombinant polynucleotide, characterized in that The polynucleotide encodes the protein according to claim 1; the polynucleotide sequence is shown in SEQ ID NO:

2.

3. A recombinant mRNA, characterized in that The recombinant mRNA is an mRNA encoding the protein according to claim 1, and the mRNA further comprises a 5'-UTR, a 3'-UTR and a polyA sequence.

4. A recombinant mRNA, characterized in that The recombinant mRNA is obtained by transcription of the polynucleotide sequence shown in SEQ ID NO:

2.

5. A recombinant vector, characterized in that: The vector contains the polynucleotide according to claim 2, or the recombinant mRNA according to claim 3 or 4.

6. An engineered cell, characterized in that: The cell contains the recombinant vector according to claim 5.

7. A rotavirus mRNA vaccine, characterized in that: Comprising the recombinant mRNA of claim 3 or 4 and a pharmaceutically acceptable delivery carrier.

8. A pharmaceutical composition, characterized in that Containing the rotavirus mRNA vaccine according to claim 7.

9. A kit, characterized in that: The kit contains the recombinant polynucleotide according to claim 2, or the recombinant mRNA according to claim 3 or 4, or the recombinant vector according to claim 5, or the engineered cells according to claim 6, or the rotavirus mRNA vaccine according to claim 7.

10. Use of the protein according to claim 1, the recombinant polynucleotide according to claim 2, the recombinant mRNA according to claim 3 or 4, the recombinant vector according to claim 5, the engineered cell according to claim 6 or the rotavirus mRNA vaccine according to claim 7 in the preparation of a medicament for preventing and controlling rotavirus infection.

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