Dabie bandavirus mRNA vaccine and preparation method therefor

Abstract

Provided are a Dabie bandavirus mRNA vaccine and a preparation method therefor. Specifically an optimized mRNA molecule is provided, and is cloned into a pGEM-3Zf(+)mRNA vaccine vector; the plasmid is linearized by means of enzyme digestion, and subjected to capping and poly(A) tail addition by means of an in-vitro transcriptase method to prepare an mRNA; the obtained mRNA is transfected into eukaryotic cells, and it is verified by means of an immunoblotting experiment that the mRNA can be expressed in vitro; and the mRNA is encapsulated by a lipid nanoparticle delivery system to obtain the mRNA vaccine, which, after immunizing mice, can induce the generation of a relatively high antibody level in serum. A viral neutralization test further proves that the immune serum can bind to viruses to prevent the viruses from infecting cells.

Description

A Dabiebanda virus mRNA vaccine and its preparation method Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a Dabiebanda virus mRNM vaccine and a preparation method thereof. Background Art

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by infection with the severe fever with thrombocytopenia syndrome virus (SFTSV). This acute infectious disease is caused by a new variant of a bunyavirus, and the disease has been named severe fever with thrombocytopenia syndrome (SFTS). The pathogen has been named fever with thrombocytopenia syndrome virus or new bunyavirus (SFTSV). In 2019, the International Committee on Taxonomy of Viruses renamed it Dabie bandavirus (DBV). In 2010, multiple strains of DBV were isolated from the blood of patients in six provinces: Henan, Hubei, Anhui, Jiangsu, Shandong, and Liaoning. Subsequently, reports have been reported in South Korea, Japan, Vietnam, and Myanmar. Severe cases can develop symptoms such as disseminated intravascular coagulation, multiple organ failure, persistent thrombocytopenia, and elevated inflammatory cytokine levels, and can even lead to death, with a mortality rate ranging from 12% to 30%. In 2017, the World Health Organization designated SFTS as an emerging infectious disease requiring priority research and intervention.

[0003] DBV glycoprotein Gn binds to cell surface receptors, mediating viral entry into host cells. Furthermore, the antigenic determinants contained in Gn interact with host cells, inducing the production of neutralizing antibodies against the glycoprotein and activating cytotoxic T cells, inducing a specific cellular immune response. Specific antibodies against glycoprotein Gn can prevent the entry of pseudoviruses mediated by glycoprotein Gn into HEK293T cells, and this blocking effect is positively correlated with the titer of neutralizing antibodies. These studies suggest that DBV glycoprotein Gn is an important target molecule for inducing the production of neutralizing antibodies.

[0004] To date, there are no specific treatment options or effective vaccines for SFTS, and vaccines play a vital role in the prevention and control of infectious diseases. In recent years, mRNA vaccines have made remarkable progress. Compared with traditional vaccines, mRNA does not integrate into the genome, avoiding concerns about insertional mutations; mRNA vaccines can be manufactured in a cell-free manner, achieving rapid, economical, and efficient production; mRNA vaccines can encode multiple antigens, enhance immune responses against adaptive pathogens, and can target multiple microorganisms or viral variants with a single formulation. At the same time, the development of effective vectors and the control of immunogenicity have facilitated the successful application of mRNA vaccines in the prevention of infectious diseases including the new coronavirus, and mRNA vaccine research and development technology is becoming increasingly mature. mRNA vaccines have become a promising way to prevent and treat a variety of diseases.

[0005] mRNA vaccines have been widely used in the prevention and treatment of infectious diseases. To date, numerous preclinical and clinical trials of mRNA vaccines for inducing antiviral immunity have been conducted against a variety of infectious diseases, such as Zika virus, HIV, and influenza virus. This study, based on this mRNA vaccine strategy, designed and constructed a DBV mRNA vaccine. The vaccine's ability to transcribe in vitro and induce specific neutralizing antibodies in mice was evaluated, potentially providing a candidate preventive strategy for DBV prevention and control.

[0006] Summary of the Invention

[0007] The present invention aims to leverage the advantages of mRNA vaccines over inactivated, subunit, or DNA vaccines. The invention provides an mRNA molecule for the Dabiebanda virus, which has been shown to express the Dabiebanda virus Gn protein through transfection into eukaryotic cells. Furthermore, the prepared mRNA molecule is encapsulated in lipid nanoparticles to produce an mRNA vaccine. After immunization of mice, serum titer and virus neutralization tests were performed, confirming that the Gn mRNA vaccine can induce the production of high levels of specific neutralizing antibodies in mice. Virus neutralization tests also confirmed that the immune serum can bind to the virus and prevent it from infecting cells. The resulting DBV mRNA vaccine provides a candidate vaccine molecule for the prevention of DBV infection.

[0008] In order to solve the above technical problems and achieve the above objectives, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides an mRNA vaccine for the Bepbanda virus, the coding sequence of the mRNA being shown as SEQ ID No: 2.

[0010] Preferably, the mRNA is capped.

[0011] More preferably, the mRNA is an optimized Gn sequence of the glycoprotein of the Bandha virus, which is cloned into the pGEM-3Zf(+)mRNA vaccine vector, and the plasmid is linearized by enzyme digestion, capped by in vitro transcription, and a poly(A) tail is added to obtain the mRNA.

[0012] Among them, the optimized Dabiebandha virus glycoprotein Gn sequence is shown in SEQ ID No: 2.

[0013] Preferably, the amino acid sequence of the Bepanda virus glycoprotein Gn is shown in SEQ ID No: 1.

[0014] In a second aspect, the present invention provides an expression vector, which expresses the mRNA molecule described above.

[0015] Preferably, the expression vector is pGEM-3Zf(+).

[0016] In a third aspect, the present invention provides an mRNA vaccine for the Dabiebanda virus, wherein the mRNA is prepared using the expression vector described above.

[0017] In a fourth aspect, the present invention protects the use of any of the aforementioned mRNA vaccines, or the aforementioned expression vectors, in the preparation of a Dabiebanda virus mRNA vaccine composition.

[0018] In a fifth aspect, the present invention protects a vaccine composition, which comprises any of the mRNA vaccines or expression vectors described above.

[0019] In the sixth aspect, the present invention protects a method for preparing a Dabiebanda virus mRNA vaccine. First, a recombinant plasmid expressing the Dabiebanda virus mRNA vaccine described above is constructed. The mRNA obtained by in vitro transcription is capped and poly(A)-tailed, and then encapsulated by lipid nanoparticles to obtain the mRNA vaccine.

[0020] The present invention also provides a more specific method for preparing a Bebanda virus mRNA vaccine, comprising the following specific steps:

[0021] 1. mRNA plasmid design and template plasmid preparation

[0022] The sequence of the glycoprotein Gn encoded by the M segment of the Dabiebanda virus strain (JS2012-70), GeneBank accession number: KY362350.1, was analyzed. After sequence optimization, the entire gene was synthesized and cloned into the pGEM-3Zf(+) expression vector. Correct plasmid construction was verified by restriction enzyme digestion and gene sequencing.

[0023] 2. mRNA Preparation and In Vitro Expression Verification

[0024] The correctly constructed plasmid was transformed into Escherichia coli for plasmid amplification. The plasmid was purified from 200 ml of bacterial culture using a midi-extraction kit. The plasmid was linearized with BamH I restriction enzyme and recovered using a gel extraction kit. After capping and adding a poly(A) tail, the prepared mRNA was transiently transfected into 293FT cells using Lipofectamine transfection reagent. Gn expression was detected by western blotting 24 hours after transfection.

[0025] 3. Encapsulation of mRNA-LNP

[0026] The prepared mRNA supercoiled plasmid is used to complete mRNA template preparation and lipid nanoparticle (LNP) encapsulation.

[0027] The present invention prepares a large Beppa virus mRNA vaccine through the above technical solution; the vaccine is used for single immunization or sequential immunization with other types of Bunyavirus vaccines.

[0028] The present invention also protects the recombinant plasmid obtained by the above-mentioned preparation method.

[0029] The present invention also protects the Dabiebanda virus vaccine obtained by the above preparation method. Beneficial effects

[0030] The present invention selected the Gn glycoprotein encoded by the M segment of the Dabiebanda virus as an immunogen and successfully constructed a DBV mRNA vaccine; based on the fact that this mRNA vaccine can produce a high level of immune protection against Bunyaviruses including the Dabiebanda virus, and exhibits a high level of humoral immunity, it has broad application prospects in the prevention of Dabiebanda virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] One or more embodiments are exemplarily illustrated by the pictures in the corresponding drawings, and these exemplary illustrations do not constitute a limitation on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments. In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1. Construction diagram of DBV Gn mRNA plasmid and electrophoresis identification results;

[0033] Figure 2 Results of in vitro transcription identification of DBV Gn mRNA;

[0034] Figure 3 Western Blot analysis of mRNA expression in 293FT cells;

[0035] Figure 4 ELISA detection of antibody expression levels in mouse serum at different time points;

[0036] Figure 5 Results of qPCR detection of virus titers in serum neutralization experiments. DETAILED DESCRIPTION

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and comprehensively described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Generally, the solutions of the embodiments of the present invention described and shown in the drawings herein can be designed and implemented in various different ways.

[0038] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort shall fall within the scope of protection of the present invention.

[0039] Example 1 mRNA plasmid design and template plasmid preparation

[0040] The amino acid sequence of the Dabiebanda virus strain (JS2012-70), GeneBank accession number: KY362350.1 (as shown in SEQ ID No: 1) was analyzed, focusing on the sequence information of the glycoprotein Gn encoded by the M segment. After removing the signal peptide and transmembrane region, the whole gene (as shown in SEQ ID No: 2) was synthesized and cloned into the pGEM-3Zf(+) expression vector after sequence optimization. The correct construction of the plasmid was verified by restriction endonuclease digestion and gene sequencing.

[0041] By analyzing the DBV Gn sequence, adding the CD5 signal peptide sequence, and cloning the optimized gene sequence into the pGEM-3Zf(+) expression vector, the vector sequence information is shown in Figure 1A, and the schematic diagram of cloning the Gn sequence into the expression vector is shown in Figure 1B. Agarose gel electrophoresis confirmed that the plasmid clone was correct and the plasmid size was approximately 5172 bp (Figure 1C).

[0042] Example 2 mRNA preparation and in vitro expression verification

[0043] The correctly constructed plasmid was transformed into Escherichia coli for plasmid amplification. The plasmid was purified from 200 ml of bacterial culture using a midi-extraction kit. The plasmid was linearized with BamH I restriction enzyme and recovered using a gel extraction kit. After capping and adding a poly(A) tail, the prepared mRNA was transiently transfected into 293FT cells using Lipofectamine transfection reagent. Gn expression was detected by western blotting 24 hours after transfection.

[0044] The successfully constructed mRNA plasmid template was linearized by BamHI digestion (Figure 2A) and purified using a gel purification kit to obtain the linearized mRNA plasmid template at a concentration of 166.81 ng / μl (Figure 2B). In vitro transcription was performed using T7 polymerase, followed by capping and poly(A) tailing. RNA and mRNA purity was assessed by agarose gel electrophoresis. 12.9 μg of mRNA was obtained from 700 ng of RNA (Figure 2C).

[0045] The obtained 3 μg and 6 μg mRNA were transfected into 293FT cells using Lipofectamine 2000. After 6 hours of transfection, fresh culture medium was replaced. After 24 hours, the cells were collected and lysed using RIPA. Immunoblotting experiments were performed to verify the expression of Gn. The concentration of the primary antibody Gn was 1:1000, and the concentration of the secondary antibody goat anti-mouse was 1:5000. The results showed that the prepared mRNA could be expressed in the eukaryotic cell 293FT (Figure 3).

[0046] Example 3 Encapsulation of mRNA-LNP

[0047] The prepared mRNA supercoiled plasmid was used to prepare the mRNA template and encapsulate it in lipid nanoparticles (LNPs). The mRNA-LNP encapsulation was commissioned to Shanghai Jinan Biotechnology Co., Ltd.

[0048] After testing, the volume of DBV-Gn mRNA-LNP prepared in this batch of the present invention was 1.62 mL; the mass was 0.40 mg; the appearance (visual inspection) was a transparent or ivory suspension; the encapsulation efficiency (fluorescence method) was 97.70%; the mRNA concentration (fluorescence method) was 247.15 ng / μL; the particle size (dynamic light scattering) was 84.65 nm; the polydispersity index (dynamic light scattering) was 0.160; the zeta potential (PALS zeta potential measurement was: -0.60 mV; and the pH value (pH meter) was 7.43.

[0049] Example 4 Immunization of Balb / c mice with mRNA-LNP

[0050] Mice were immunized according to the following immunization scheme. Female BALB / c mice aged 6-8 weeks were divided into three immunization dose groups: low, medium and high (n=5 in each group). The injection doses were 2ug / mouse, 5ug / mouse and 20ug / mouse, respectively. Immunization was performed by intramuscular injection once every 2 weeks. After 4 weeks, the mouse serum was collected for ELISA analysis to evaluate the neutralizing antibody titer induced by the mRNA vaccine in mice.

[0051] Example 5 ELISA detection of mouse serum antibody expression

[0052] The eukaryotic expressed Gn glycoprotein was coated on ELISA plates at 100 ng / well. Blood was collected from the tail vein of mice to collect serum. The serum was diluted with 1:100 as the starting concentration. Three replicate wells were set for each dilution titer. The OD value was 0. 450nm The absorbance is 2.1 times that of the blank control, which is used to judge the mouse serum antibody titer level.

[0053] Mice were immunized as planned, and mouse serum was collected starting at 4 weeks. The mouse serum titer was continuously measured at 6, 8, and 10 weeks. The results showed that at 4 weeks after immunization, the antibody titer of all three immunization dose groups reached 1:640,000 (Figure 4A). As time went on, the antibody titer level gradually decreased. At 10 weeks after immunization, the antibody titer of the high-dose immunization group could still reach 1:160,000 (Figure 4).

[0054] Example 6 Virus Neutralization Experiment

[0055] Vero cells were seeded in 6-well plates, and the virus was inoculated when the cell confluence reached 90%. The virus and mouse serum were diluted with DMEM cell culture medium (dilution ratio of 1:10, 1:100). The virus inoculation amount was MOI = 0.01, and 500 μl of virus solution was mixed with 500 μl of serum diluent and incubated at 37 ° C for 30 minutes. A serum-free blank control was also set up. The culture medium in the 6-well plate was removed, 1 ml of virus and serum mixture was added, and 3 parallel wells were set up for each dilution. After incubation at 37 ° C for 2 hours, the mixture was removed and 2 ml of cell maintenance solution was added. After 24 hours, the virus titer was determined according to the instructions of the Fever with Thrombocytopenia Syndrome Bunyavirus Nucleic Acid Detection Kit (Da'an Gene, DA0340).

[0056] DB virus was incubated with mouse serum at different dilution ratios (1:10 and 1:100) at an MOI of 0.01, then inoculated into Vero cells. Virus titers were measured by qPCR 24 hours after infection. The results showed that serum from immunized mice was able to prevent the virus from infecting cells, indicating that mRNA vaccine immunization can induce specific neutralization in mice.

Claims

1. An mRNA vaccine against Dabie Bandavirus, characterized in that, The coding sequence of the mRNA is shown in SEQ ID No:

2. Preferably, the mRNA is subjected to capping treatment. More preferably, the mRNA is an optimized glycoprotein Gn sequence of Dabie Bandavirus, cloned onto the pGEM-3Zf(+) mRNA vaccine vector, the plasmid is linearized by enzymatic digestion, and the mRNA is obtained by capping with in vitro transcription enzyme and adding a poly(A) tail.

2. The mRNA vaccine according to claim 1, characterized in that, The amino acid sequence of the glycoprotein Gn of Dabie Bandavirus is shown in SEQ ID No:

1.

3. Expression vector, characterized in that, The expression vector expresses the mRNA molecule according to claim 1.

4. The expression vector according to claim 3, wherein The expression vector is pGEM-3Zf(+).

5. An mRNA vaccine against Dabie Bandavirus, characterized in that, The mRNA is prepared using the expression vector according to claim 3 or 4.

6. Use of the mRNA vaccine according to any one of claims 1-2, or the expression vector according to claim 3, in the preparation of a Dabie Bandavirus mRNA vaccine composition.

7. A vaccine composition, characterized in that, The vaccine composition comprises the mRNA vaccine according to any one of claims 1-2 or the expression vector according to claim 3.

8. A method for preparing a Dabie Bandavirus mRNA vaccine, characterized in that, First, a recombinant plasmid expressing the Dabie Bandavirus mRNA vaccine according to claim 1 is constructed. After the in vitro transcribed mRNA is capped with Cap and poly(A) tailed, it is encapsulated with lipid nanoparticles to obtain the mRNA vaccine.

9. The recombinant plasmid obtained by the preparation method according to claim 8.

10. The Dabie Bandavirus vaccine obtained by the preparation method according to claim 8.

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