Method for preparing BVDV-1 vaccine by cap-dependent mRNA (messenger ribonucleic acid) technology

Abstract

The invention relates to the technical field of vaccine preparation, and discloses a method for preparing a BVDV-1 vaccine by a cap-dependent mRNA (messenger Ribonucleic Acid) technology, which is characterized in that the mRNA vaccine design is systematically optimized, a DNA (Deoxyribose Nucleic Acid) plasmid containing a BVDV-1 complete E2 gene is constructed, the full length is taken as an open reading frame, the integrity of an antigen epitope is ensured, and a foundation is laid for inducing a broad-spectrum neutralizing antibody; optimized 5 'UTR and 3' UTR are integrated on two sides of the E2 gene, and a Kozak sequence is introduced, so that the ribosome binding efficiency is remarkably improved, and the antigen expression level is enhanced; efficient packaging and delivery are realized through a lipid nanoparticle (LNP) delivery system, and mRNA stability and target cell uptake efficiency are ensured; and a dosage scheme adaptive to multiple species gives consideration to safety and immune efficacy.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparation, and specifically to a method for preparing a BVDV-1 vaccine by cap-dependent mRNA technology. Background Art

[0002] Bovine viral diarrhea virus type 1 (BVDV-1) is the main pathogen causing immunosuppression, reproductive disorders and economic losses in cattle. Currently, commercial vaccines are mainly inactivated vaccines and attenuated live vaccines. The former has limited immunogenicity and requires multiple vaccinations; the latter can induce a strong immune response, but there are problems such as virus reversion to virulence, potential pathogenic risks and complex production processes. In recent years, mRNA vaccines have become a research hotspot due to their advantages of rapid development, high safety and efficient expression of target proteins. However, in the existing technology, the antigen-encoding sequence does not completely retain the open reading frame of the E2 gene, which may lead to the deletion of antigenic epitopes and affect the comprehensiveness of the immune response; the design of the 5′UTR and 3′UTR of mRNA is not fully optimized, lacking translation enhancement elements, resulting in low translation efficiency and insufficient antigen expression. These problems limit the immunogenicity and cross-species applicability of the vaccine and restrict its application in multiple species. Summary of the Invention

[0003] Aiming at the deficiencies of the existing technology, the present invention provides a method for preparing a BVDV-1 vaccine by cap-dependent mRNA technology to solve the problems in the background art.

[0004] To achieve the above object, the present invention provides the following technical solution: A method for preparing a BVDV-1 vaccine by cap-dependent mRNA technology, comprising the following steps:

[0005] Step 1, molecular cloning and mRNA synthesis: Construct a DNA plasmid containing the envelope protein E2 gene of BVDV-1. The plasmid uses the entire E2 gene of BVDV-1 as the open reading frame, with 5′UTR and 3′UTR on both sides and a Kozak sequence.

[0006] Step 2, plasmid: Linearize the mRNA prepared in Step 1 with BspQI restriction endonuclease.

[0007] Step 3, in vitro transcription: Use T7 RNA polymerase to perform in vitro transcription on the plasmid prepared in Step 2 to obtain uncapped mRNA.

[0008] Step 4, capping modification: Use vaccinia capping enzyme and mRNA Cap O-methyltransferase to purify and further modify the mRNA in Step 3 with Cap1.

[0009] Step 5, encapsulation of mRNA-LNP: Encapsulate the capped mRNA in lipid nanoparticles (LNP), and the LNP is composed of DHA-1, DSPC, cholesterol, and DMG-PEG2000.

[0010] Preferably, the molar ratio of DHA-1, DSPC, cholesterol, and DMG-PEG2000 in the lipid nanoparticles (LNP) is 50:10:38:1.5.

[0011] Preferably, after Step 3, the residual DNA template is removed by DNase treatment.

[0012] Preferably, the temperature for incubating the DNase with uncapped mRNA is 37 °C, and the incubation time is 15 minutes.

[0013] Preferably, in Step 3, the in vitro transcription temperature is 37 °C, and the transcription time is 2 hours.

[0014] Preferably, before encapsulating the capped mRNA in Step 5, it also includes using spectrophotometric analysis and agarose gel electrophoresis to determine the purity and quality of the RNA.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention systematically optimizes the mRNA vaccine design, constructs a DNA plasmid containing the complete E2 gene of BVDV-1, uses the full length as an open reading frame to ensure the integrity of antigenic epitopes, and lays a foundation for inducing broad-spectrum neutralizing antibodies; integrates optimized 5′UTR and 3′UTR on both sides of the E2 gene and introduces the Kozak sequence to significantly improve the ribosome binding efficiency and enhance the antigen expression level; realizes efficient encapsulation and delivery through the lipid nanoparticle (LNP) delivery system to ensure mRNA stability and target cell uptake efficiency; and the dose regimen adapted to multiple species takes into account both safety and immune efficacy.

[0017] Other features and advantages of the present invention will be described in the following specification, and part of them will become obvious from the specification, or be understood by implementing the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the structures pointed out in the specification, claims, and drawings. Brief Description of the Drawings

[0018] Figure 1 It is a schematic diagram showing the design of cap-dependent (A) and cap-independent (B) mRNA vaccines of the present invention;

[0019] Figure 2 It is a schematic diagram showing the efficacy of the mRNA vaccine of the present invention in a mouse model;

[0020] Figure 3Schematic diagram of the efficacy of the mRNA vaccine of the present invention in a guinea pig model;

[0021] Figure 4 Schematic table of the body weight of mice in the present invention;

[0022] Figure 5 Schematic table of the change of neutralizing antibody titer in mouse serum in the present invention;

[0023] Figure 6 Schematic table of the differential white blood cell count of mice on the 56th day in the present invention;

[0024] Figure 7 Schematic table of the body weight of guinea pigs in the present invention;

[0025] Figure 8 Schematic table of the titer of neutralizing antibody in guinea pig serum in the present invention;

[0026] Figure 9 Schematic table of the differential white blood cell count of guinea pigs on the 56th day in the present invention;

[0027] Figure 10 Schematic table of the body weight of goats in the present invention;

[0028] Figure 11 Schematic table of the rectal temperature of goats in the present invention;

[0029] Figure 12 Schematic table of the blood picture analysis of goats in the present invention;

[0030] Figure 13 Schematic table of the titer of neutralizing antibody in goat serum in the present invention. Detailed implementation manners

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art in the technical field of the present invention without creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0032] Please refer to the figure. A method for preparing a BVDV-1 vaccine by a cap-dependent mRNA technology in the present invention specifically includes the following contents:

[0033] 1. Molecular cloning and plasmid construction

[0034] a. Plasmid construction:

[0035] Design of DNA plasmid: It contains the open reading frame (ORF) of the BVDV-1 envelope protein E2 gene, flanked by 5′UTR and 3′UTR, and the Kozak sequence (GCCACC) is inserted to enhance translation efficiency.

[0036] Plasmid linearization: The plasmid design includes a 3′ polyadenylation sequence located after the 3′UTR. The plasmid is cut with the BspQI restriction endonuclease after the 3′ polyadenylation sequence, and the reaction is carried out at 37°C for 4 hours.

[0037] b. In vitro transcription

[0038] In vitro transcription is carried out using the T7 High Yield RNA Transcription Kit (Vazyme TR101):

[0039] Reaction system: 10 μg of linearized plasmid, 20 μL of T7 RNA polymerase, 40 μL of NTP mixture (25 mM), 50 μL of transcription buffer, and nuclease-free water is added to make up to 200 μL.

[0040] Reaction conditions: Incubate at 37°C for 2 hours, then add DNase I (1 U / μL) to degrade the residual DNA template, and treat at 37°C for 15 minutes.

[0041] C. Capping modification

[0042] Cap1 structure modification is carried out using vaccinia capping enzyme (Vazyme DD4109) and mRNA Cap O-methyltransferase (Vazyme DD4110):

[0043] First step: 100 μg of purified mRNA is mixed with 10 μL of vaccinia capping enzyme and 5 μL of 2 mM SAM, and the reaction is carried out at 37°C for 30 minutes.

[0044] Second step: Add 10 μL of mRNA Cap O-methyltransferase and 5 μL of 2 mM SAM, and continue the reaction at 37°C for 30 minutes.

[0045] 2. Encapsulation of lipid nanoparticles (LNP)

[0046] a. Lipid formulation

[0047] Composition of lipid mixture (molar ratio): DHA-1 (50%), DSPC (10%), cholesterol (38%), DMG-PEG2000 (1.5%).

[0048] b. Dissolution method: Each component is dissolved in ethanol at a final concentration of 10 mM, and sonicated for 10 minutes to ensure homogeneity.

[0049] (2) mRNA encapsulation

[0050] Mixing ratio: The capped mRNA (1 mg / mL) is mixed with the lipid solution at a volume ratio of 1:3.

[0051] Encapsulation process: Use a microfluidic device (flow rate 12 mL / min, aqueous phase: organic phase = 1:3) to self-assemble to form the LNP-mRNA complex.

[0052] Quality control: The particle size measured by dynamic light scattering (DLS) is 80 ± 5 nm, and the polydispersity index (PDI) < 0.2; the encapsulation efficiency measured by the RiboGreen method is ≥ 90%.

[0053] 3. Animal immunization experiments

[0054] Immunization experiments were conducted in mice, guinea pigs, and goats. Mice and guinea pigs were inoculated with 10 μg and 20 μg of the capped mRNA vaccine respectively, and goats were inoculated with 25 μg of the capped mRNA vaccine. After immunization, the body weight, white blood cell count, and neutralizing antibody titer of the animals were monitored.

[0055] 1. Mouse experiment

[0056] Thirty 6-8-week-old female BALB / c mice were divided into two groups and inoculated with 10 μg of the capped mRNA vaccine respectively. Immunization was carried out on day 0 and day 21, with alternate injection into the left and right hind limbs. After immunization, the body weight, white blood cell count, and neutralizing antibody titer of the mice were monitored. As Figure 2 shown, among them, (A) represents the timeline of the mouse experiment design. (B) represents the normalized body weight of the immunized mice (n = 15 per group). The red arrow indicates vaccination (first dose) and boost (second dose). (C) represents the neutralizing antibody titers against BVDV-1 on day 0, day 35, day 42, day 49, and day 56 according to immunofluorescence inhibition (n = 3 per group, and each serum sample was mixed from 5 mice). (D) represents the effect of vaccination on white blood cells (WBC), lymphocytes (LYM), intermediate cells (IMD), and granulocytes (GRA) on day 56.

[0057] 2. Guinea pig experiment

[0058] Six 6-8-week-old female guinea pigs were divided into two groups and inoculated with 20 μg of the capped mRNA vaccine respectively. Immunization was carried out on day 0 and day 21 by intramuscular injection. After immunization, the body weight, white blood cell count, and neutralizing antibody titer of the guinea pigs were monitored. As Figure 3As shown, where (A) represents the experimental design diagram of guinea pigs. (B) represents the standardized body weight of immunized guinea pigs (n = 3 per group). The red arrow indicates the primary immunization and the booster immunization. (C) represents the detection of BVDV-1 specific neutralizing antibody titers in serum based on immunofluorescence inhibition on days 0, 28, 35, 42, 49, and 56 (n = 3 per group). (D) represents the effect of vaccination on white blood cells (WBC), lymphocytes (LYM), intermediate cells (IMD), and granulocytes (GRA) on day 56 (n = 3 per group, represented by black dots).

[0059] 4. Experimental Results

[0060] 1. Efficacy of BVDV-1 mRNA Vaccine in Mice

[0061] The neutralizing antibody titers induced by the capped mRNA vaccine in mice were significantly higher than those of the uncapped mRNA vaccine, reaching 9.4 (expressed as -log2) on day 35. The body weights of the mice remained stable after immunization, and no obvious side effects were observed. The results of white blood cell differential counts showed that the white blood cells, lymphocytes, intermediate cells, and granulocytes of all mice were within the normal range, as Figures 4 - 6 shown.

[0062] 2. Efficacy of BVDV-1 mRNA Vaccine in Guinea Pigs

[0063] The neutralizing antibody titers induced by the capped mRNA vaccine in guinea pigs reached 13.7 (expressed as -log2) on day 35, which was significantly higher than that of the uncapped mRNA vaccine. The body weights of the guinea pigs remained stable after immunization, and no obvious side effects were observed. The results of white blood cell differential counts showed that the white blood cells, lymphocytes, intermediate cells, and granulocytes of all guinea pigs were within the normal range, as Figures 7 - 9 shown.

[0064] 3. Efficacy of BVDV-1 mRNA Vaccine in Goats

[0065] The neutralizing antibody titer induced by the capped mRNA vaccine in goats was 9.1, showing no significant difference compared with the commercial vaccine. The body weight, rectal temperature, and white blood cell count of the goats remained normal, and no obvious side effects were observed, as Figures 10 - 13 shown.

Claims

1. A method for preparing a BVDV-1 vaccine using cap-dependent mRNA technology, characterized in that: The following steps are involved: Step 1, molecular cloning and mRNA synthesis: constructing a DNA plasmid containing the BVDV-1 envelope protein E2 gene, wherein the plasmid uses the entire E2 gene of BVDV-1 as an open reading frame, which is flanked by 5′UTR and 3′UTR and has a Kozak sequence; Step 2, plasmid: linearize the mRNA prepared in step 1 by BspQI restriction endonuclease; Step 3, in vitro transcription: using T7 RNA polymerase to perform in vitro transcription on the plasmid prepared in step 2 to obtain uncapped mRNA; Step 4, capping modification: use vaccinia capping enzyme and mRNA Cap O-methyltransferase to purify and further modify the mRNA Cap1 in step 3; Step 5, lipid nanoparticle (LNP) encapsulation: encapsulate the capped mRNA into lipid nanoparticles (LNP), wherein the LNP is composed of DHA-1, DSPC, cholesterol and DMG-PEG2000.

2. The method for preparing a BVDV-1 vaccine using cap-dependent mRNA technology according to claim 1, characterized in that: The molar ratio of DHA-1, DSPC, cholesterol and DMG-PEG2000 in the lipid nanoparticles (LNP) is 50:10:38:1.

5.

3. The method for preparing a BVDV-1 vaccine using cap-dependent mRNA technology according to claim 1, characterized in that: After step 3, the residual DNA template is removed by DNase treatment.

4. The method for preparing a BVDV-1 vaccine using cap-dependent mRNA technology according to claim 3, characterized in that: The DNase was incubated with non-capped mRNA at 37° C. for 15 minutes.

5. The method for preparing a BVDV-1 vaccine using cap-dependent mRNA technology according to claim 1, characterized in that: In step three, the in vitro transcription temperature is 37° C., and the transcription time is 2 hours.

6. The method for preparing a BVDV-1 vaccine using cap-dependent mRNA technology according to claim 1, characterized in that: Prior to step five of capping the mRNA for packaging, the purity and quality of the RNA is determined by spectrophotometric analysis and agarose gel electrophoresis.

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