Rotavirus VP8-mRNA vaccine and preparation method thereof

Abstract

The application belongs to the technical field of mRNA vaccine preparation, and specifically discloses a rotavirus VP8-mRNA vaccine and a preparation method thereof, and provides a protein for preparing a rotavirus vaccine, wherein the amino acid sequence of the protein is shown as SEQ ID NO:1, the application takes the VP8 gene after recombination as an antigen gene, and prepares an LNP / mRNA vaccine, and experiment immunization evaluation is carried out in adult female Balb / c mice, and the results show that the vaccine VP8-mRNA-LNP provided by the application can stimulate the body to produce a higher antibody level, can not only stimulate mice to produce better humoral immunity, but also can stimulate mice to produce cellular immunity, and in the aspect of broad spectrum, can simultaneously prevent and treat G1P[8] and G9P[8] type rotaviruses.

Description

Technical Field

[0001] This invention belongs to the field of mRNA vaccine preparation technology, specifically relating to a rotavirus VP8-mRNA vaccine and its preparation method. Background Technology

[0002] Rotavirus (RV) is the most common pathogen causing severe diarrhea in infants and young children worldwide, and is one of the leading causes of death in these children. Approximately 1.3 million deaths annually are related to rotavirus infection, primarily affecting children under 5 years old. Rotavirus belongs to the genus Rotavirus in the family Reoviridae, and is a non-enveloped, double-stranded RNA virus. The genome is discontinuous, consisting of 11 segmented double-stranded RNAs encoding six structural proteins (VP1, VP2, VP3, VP4, VP6, VP7) and six non-structural proteins (NSP1, NSP2, NSP3, NSP4, NSP5, and NSP6). The rotavirus capsid proteins VP4 and VP7 constitute the outermost surface antigens of the rotavirus, determining its serotype. VP7 (a glycoprotein) determines the G serotype, while VP4 (a protease-sensitive protein) determines the P serotype. VP4 is a spike-like protein of the RV capsid, possessing hemagglutinin function and sensitivity to proteolytic enzymes, cleaving into VP5* (AA 248-776) and VP8* (AA 1-231). VP5* constitutes the main body of the spike, while VP8* is responsible for interacting with the host receptor to mediate viral attachment and has been proven to be an effective vaccine target; to date, 42 VP8* proteins have been identified in humans and various animal species. There are 58 types of rotaviruses, including G and P. Globally, six G types (G1, G2, G3, G4, G9, and G12) and three P types (P[4], P[6], and P[8]) are dominant. In addition, the six strains of A rotavirus, G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8], typically account for more than 90% of the circulating A rotavirus species worldwide.

[0003] Currently available vaccines are mainly live attenuated rotavirus vaccines, which can prevent rotavirus infection to some extent. However, due to differences in economic conditions, medical standards, and other factors across the world, the efficacy of these vaccines varies, and the development of a rotavirus vaccine still faces significant challenges. To mitigate the harm caused by rotavirus, various methods, including vaccines and antiviral drugs, have been developed. Regarding vaccines, although the two existing live attenuated rotavirus vaccines can effectively protect infants and young children from rotavirus infection, they also carry the risk of side effects such as reversion mutations and intussusception. Furthermore, the two currently available vaccines primarily target the rotavirus serotypes prevalent in Europe and the United States, and are less effective against the rotavirus serotypes prevalent in Asia and Africa. Therefore, there is an urgent need to develop a safe, broad-spectrum, and long-lasting universal rotavirus vaccine. Summary of the Invention

[0004] The main objective of this invention is to provide a rotavirus VP8-mRNA vaccine and its preparation method, in order to solve the problems existing in the background art and provide a new technical means for the prevention and control of rotavirus. Specifically, this invention provides the following technical solution: This invention provides a protein for preparing a rotavirus vaccine, the amino acid sequence of which is shown in SEQ ID NO:1.

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

[0006] In one embodiment, the present invention provides a recombinant mRNA that encodes the protein described above, and the mRNA further comprises a 5'-UTR, a 3'-UTR, and a polyA sequence.

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

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

[0009] In one embodiment, the present invention provides an engineered cell containing the recombinant vector described above.

[0010] In one embodiment, the present invention provides a rotavirus mRNA vaccine comprising 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 described above.

[0012] In one embodiment, the present invention provides a kit containing the recombinant polynucleotide, or the recombinant mRNA, or the recombinant vector, or the engineered cells, 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 cells, and the rotavirus mRNA vaccine in the preparation of a medicament for the prevention and control of rotavirus infection.

[0014] The technical effects achieved by this invention are as follows: This invention selects the ZTR-68 strain of rotavirus, which has a high incidence and is prone to mutation, as a model to carry out research on mRNA vaccines, and uses the recombinant VP8 gene as the antigen gene to prepare LNP / mRNA vaccines. Experimental immunization evaluation was carried out in adult female Balb / c mice. The results showed that the vaccine VP8-mRNA-LNP provided by this invention can stimulate the body to produce a high level of antibodies. It can not only stimulate mice to produce good humoral immunity, but also stimulate mice to produce cellular immunity. In terms of broad spectrum, it can simultaneously prevent and treat G1P[8] and G9P[8] rotaviruses. Attached Figure Description

[0015] Figure 1 Diagram of the construction strategy for VP8-mRNA vaccines; Figure 2 Animal experimentation flowchart; Figure 3. pUC57-VP8 single enzyme digestion reaction diagram; M: DNA Marker; 1: pUC57-VP8 single enzyme digestion product; Figure 4. Western blot identification results of VP8-mRNA; M: Protein Marker; 1: VP8-mRNA (+); 2: VP8-mRNA (-); 3: Virus positive control; Figure 5. Physicochemical properties of VP8-mRNA vaccine; Note: A: Capillary electrophoresis of RNA after in vitro transcription of plasmid; B: Particle size detection using a Malvern laser particle size analyzer; C: Encapsulation efficiency detection; D: Polymer dispersion index detection.

[0016] Figure 6. Transmission electron microscopy results of VP8-mRNA; Figure 7. Statistical graph of specific IgG antibody levels in mouse serum; Figure 8. Statistical graph of neutralizing antibody titers in mouse serum; Figure 9. Detection of spleen cell lymphocyte subsets and cytokine IFN-γ in mice after immunization; (A) Percentage of CD3+CD8+ T lymphocytes; (B) Percentage of CD3+CD4+ T lymphocytes; (C) Number of IFN-γ spots generated in different groups; (D) Statistics on the increase in spots in the vaccine group and the control group. Detailed Implementation

[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 the purchased goods in the test methods, if no specific conditions are specified, they shall be carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments used are not specified, they can be conventional products that can be purchased from the market.

[0018] Example 1 I. Materials (1) HEK293 cells (human embryonic kidney cells) were preserved by the Molecular Biology Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences and 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 Biotech Co., Ltd. (3) Rotavirus ZTR-68 strain, preserved by the Molecular Biology Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, was obtained by passage culture in MA104 cells; (4) PBS solution (for washing cells in a sterile room): prepared and tested by the fourth laboratory of the Biological Products Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, and stored in the Molecular Biology Laboratory; (5) DMEM culture medium: purchased from Gibco, USA; (6) Double antibiotics (penicillin + streptomycin): Prepared and tested by the fourth laboratory of the Biological Products Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, and stored in the Molecular Biology Laboratory; (7) 1% trypsin (for cell digestion): Prepared and tested by the fourth laboratory of the Biological Products Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, and stored in the Molecular Biology Laboratory; (8) Fetal bovine serum (FBS): purchased from Gibco; (9) The pUC57-VP8 plasmid was synthesized by Suzhou Genewiz Company and amplified and preserved by the Molecular Biology Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College. (10) Restriction endonuclease BspQⅠ was purchased from Nanjing Novizan Pharmaceutical Co., Ltd.; (11) The T7 High Yield RNA Transcription Kit was purchased from Nanjing Novizan Pharmaceutical Co., Ltd. (12) mRNA Cap 2′-O-Methyltransferase was purchased from Nanjing Novizan Pharmaceutical Co., Ltd. (13) Ionizable lipids and PEG-lipids were purchased from Shanghai Aivito Company; (14) The Quant-iT RiboGreen RNA Assay Kit was purchased from ThermoFisher Scientific. (15) Mouse lymphocyte separation solution and serum-free lymphocyte culture medium were purchased from Beijing Dakwei Co., Ltd. (16) The flow cytometry antibodies PerCP anti-mouse CD3ε, APC anti-mouse CD4 and PE anti-mouse CD8 were purchased from Beijing DaKeWei Company; (17) ELISpot Plus: Mouse IFN-γ (ALP kit purchased from Mab Tech, USA; (18) Bovine serum albumin (BSA) was purchased from Beijing Solarbio Biotechnology Co., Ltd.; (19) Anhydrous ethanol: purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.; (20) 15mL and 20mL centrifuge tubes: purchased from Corning. (21) 1.5mL and 2mL EP tubes: purchased from KIRGEN; (22) Pipette tips: purchased from KIRGEN; II. Methods: Design and preparation of VP8-mRNA vaccine 1. RV mRNA candidate vaccine construction strategy An RV mRNA vaccine was designed based on the recombinant VP8 gene of the rotavirus ZTR-68 strain. The amino acid sequence of the VP8 protein is shown in SEQ ID NO. 1. The basic framework of the vaccine design includes a 5'-untranslated region, an open reading frame, a 3'-untranslated region, and a poly-A tail, as shown in Figure 1. The sequence is shown in SEQ ID NO. 2.

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

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

[0021] 3. In vitro transcription, capping, and purification Plasmid pUC57-VP8 was extracted using a plasmid large-scale extraction kit (Tiangen, DP117). The extracted pUC57-VP8 plasmid was linearized by single-enzyme digestion with restriction endonuclease SapI. The linearized product was then purified using VAHTS DNA Clean Beads. Under nuclease-free conditions, DNA was transcribed into RNA using the T7 High Yield RNA Transcription Kit (N1-Me-Pseudo UTP). The integrity of the RNA was assessed by capillary electrophoresis of the transcribed product. The transcription product was purified using VAHTS RNA Clean Beads, and the RNA was then capped using a capping kit. The capped product, VP8-mRNA, was then 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 was performed with the mRNA Cap2'-O-Methyltransferase kit. All procedures were performed according 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 validation of VP8-mRNA vaccine expression VP8-mRNA transfection was performed using the Polyplus jetMESSENGER® mRNA transfection reagent kit. HEK293 cells were transfected at a concentration of 1×10⁻⁶ mRNA. 5 Cells were seeded at a rate of 10 cells / well in 12-well plates and cultured until cell confluence reached approximately 70%-80%. Following the transfection reagent instructions, 2 μg of Cap-mRNA and 200 μL of mRNA buffer were mixed, 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 to each well and incubated at 37°C in a 5% CO2 incubator. After 48 h, the cell supernatant was collected and concentrated 10-fold using a 3 kDa ultrafiltration tube. The supernatant was then collected in a 1.5 ml EP tube, and SDS-PAGE protein loading buffer (5x) was added. After protein denaturation, 10% SDS-PAGE gel electrophoresis was performed to verify VP8-mRNA expression.

[0025] 5. Determination of mRNA-LNP particle size, PDI, potential, and encapsulation efficiency mRNA was encapsulated in LNPs using microfluidic technology. The particle size of the LNPs was determined using a Malvern laser particle size analyzer, and the encapsulation efficiency of the LNP-mRNA was determined using the Quant-iT™ RiboGreen™ RNAReagent and Kit. The morphology and structure of the encapsulated vaccine were observed using a transmission electron microscope.

[0026] 6. Evaluation of the immunization 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 grouping. There were a total of 4 groups, 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 recombinant plasmid After extracting a large amount of the recombinant plasmid pUC57 - VP8, measuring its concentration and purity by a ultra - micro spectrophotometer, the plasmid was linearized by single - enzyme digestion with the restriction enzyme Sap I. Subsequently, the reaction solution was recovered, purified by a kit, and 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, meeting the expectation.

[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 - fold, 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 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). The VP8-mRNA vaccine was then encapsulated using a microfluidic device for quality control analysis. The particle size, polymer dispersion index (PDI), and zeta potential of the vaccine formulation were characterized using a Malvern Zetasizer Nano ZS90 instrument. The average particle size of the mRNA vaccine showed that the average particle size of VP8-mRNA-LNP was approximately 103 nm, with a PDI of 0.0887, exhibiting a unimodal normal distribution. The encapsulation efficiency and concentration of the mRNA vaccine were determined using the Quant-iT™ RiboGreen RNA Quantitative Kit. The encapsulation efficiency of VP8-mRNA was 92.3% (…). Figure 5 ).

[0030] Transmission electron microscopy revealed that the encapsulated VP8-mRNA-LNP vaccine was a uniform spherical shape with a particle size of approximately 100 nm, indicating that the LNPs loaded with mRNA were morphologically intact. Figure 6 ).

[0031] 4. Immunogenicity evaluation of VP8-mRNA-LNP vaccine 4.1 IgG antibody detection After collecting blood and separating serum 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 to the VP8-mRNA-LNP vaccine was evaluated by indirect ELISA. IgG antibody efficacy was expressed as Log2 GMT. As shown in Figure 7, on day 21 after the first immunization, the mean IgG antibody titers in the serum of mice in the 10 μg, 15 μg, and 20 μg groups were 10.2, 11.2, and 10.4, respectively. Antibody levels further increased after the second immunization, reaching 16.6, 17, and 17.2, respectively, showing a dose-response relationship. However, there was no statistically significant difference in IgG antibody levels induced by the 15 μg and 20 μg vaccines. The results indicate that the VP8-mRNA-LNP vaccine can stimulate the body to produce high antibody levels.

[0032] 4.2 Neutralizing antibody detection To evaluate whether the VP8-mRNA-LNP vaccine could induce mice to produce neutralizing antibodies against rotavirus serum, peripheral blood serum from mice was collected for detection of neutralizing antibodies against G1P[8] and G9P[8] rotaviruses. The results were presented as Log2 GMT. The results of the neutralizing antibody detection showed that at week 6 of the first immunization (day 42 of the experiment), the neutralizing antibody titers against G1P[8] in the 10 μg, 15 μg and 20 μg groups were 5.2, 7.6 and 7.4, respectively. The neutralizing antibody level in the 15 μg group was the highest, and the difference between the 15 μg group and the 10 μg group was statistically significant (P<0.05). The results of the neutralization level of RV G9 (G9P[8]) virus showed that the neutralizing antibody titers in the 10 μg, 15 μg and 20 μg groups were 6, 7.6 and 7.4, respectively, and all groups showed good virus neutralization ability. The results showed 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 Flow cytometry was used to detect T lymphocyte subsets in spleen cells at week 6 of the initial immunization (day 42 of the experiment). Compared with the negative control group, the proportions of CD4+ and CD8+ cells were increased in the mRNA vaccine experimental group, indicating that the VP8-mRNA vaccine stimulated the differentiation of CD4+ and CD8+ cells (Figure 9). Th1 cells are involved in cellular immunity and mainly secrete IFN-γ. The commercially available ELISpot Plus: Mouse IFN-γ detection kit was used to detect IFN-γ secreted by spleen cells of 42-day-old mice. The results showed that the IFN-γ factor produced in the mRNA vaccine group was higher than that in the PBS group. The results indicate that the developed VP8-mRNA-LNP vaccine can not only stimulate good humoral immunity in mice, but also stimulate cellular immunity.

[0034] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to 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 contains a nucleotide encoding the protein of claim 1, and the polynucleotide sequence is shown in SEQ ID NO:

2.

3. A recombinant mRNA, characterized in that, The recombinant mRNA was obtained by transcription of a polynucleotide sequence as shown in SEQ ID NO:

2.

4. A recombinant vector, characterized in that, The vector contains the polynucleotide of claim 2 or the recombinant mRNA of claim 3.

5. An engineered cell, characterized in that, The cells contain the recombinant vector as described in claim 4.

6. A rotavirus mRNA vaccine, characterized in that, This includes the recombinant mRNA as described in claim 3 and a pharmaceutically acceptable delivery vector.

7. A pharmaceutical composition, characterized in that, The rotavirus mRNA vaccine as described in claim 6.

8. A reagent kit, characterized in that, The kit contains the recombinant polynucleotide of claim 2, or the recombinant mRNA of claim 3, or the recombinant vector of claim 4, or the engineered cells of claim 5, or the rotavirus mRNA vaccine of claim 6.

9. The use of the protein of claim 1, the recombinant polynucleotide of claim 2, the recombinant mRNA of claim 3, the recombinant vector of claim 4, the engineered cell of claim 5, or the rotavirus mRNA vaccine of claim 6 in the preparation of a medicament for the prevention and control of rotavirus infection, characterized in that, The rotavirus is a G1P[8] or G9P[8] type rotavirus.

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