CN116240223A

CN116240223A - Replication type mRNA vaccine and preparation method thereof

Info

Publication number: CN116240223A
Application number: CN202211611436.5A
Authority: CN
Inventors: 杨永乐; 黄耀伟; 唐建斌
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-12-13
Filing date: 2022-12-13
Publication date: 2023-06-09

Abstract

The invention discloses a replication type mRNA vaccine and a preparation method thereof, and relates to the technical field of vaccines. The invention prepares a replication type mRNA vaccine for expressing a stable trimeric protein before fusion of a novel coronavirus spike protein, constructs a replication type mRNA vector plasmid containing a novel coronavirus S protein, is used for preparing replication type mRNA, is combined with a liposome encapsulation scheme for preparing the mRNA vaccine, and is applied to immunization of the novel coronavirus. The method has application value in coronavirus research and vaccine creation, and can greatly promote the transformation application of the vaccine.

Description

Replication type mRNA vaccine and preparation method thereof

Technical Field

The invention relates to the technical field of vaccines, in particular to a replication type mRNA vaccine, which comprises construction of a synthetic plasmid of the mRNA, synthesis and preparation of the mRNA, and a preparation method of the mRNA vaccine.

Background

The novel coronavirus (SARS-CoV-2) is subject to mutation and variation since the explosion, and the degree of difficulty in transmission, the severity of related diseases, the effectiveness of vaccines and therapeutic drugs, etc. of different variant strains have great differences. With the pandemic of new coronaviruses, and the continued emergence of new viral mutants, some of them, such as Delta or Omicron, have a greater infectivity and a greater immune escape capacity.

Vaccination has proven to be the most effective and powerful method of preventing viral diseases. The technical platforms related to the current research and development include nucleic acid (DNA and RNA), virus-like particles, peptides, virus vectors, recombinant proteins, attenuated live vaccines, inactivated vaccines and other methods. Among them, the novel mRNA-based technology platform has great flexibility in terms of antigen manipulation and speed potential. Compared with the traditional vaccine, the mRNA vaccine is used as a novel vaccine, and has the advantages of simple production process, high development speed, no need of cell culture and low cost. Compared to DNA vaccines, mRNA vaccines do not require entry into the nucleus, there is no risk of integration into the host genome, and half-life can be adjusted by modification. mRNA vaccines can provide a comprehensive stimulus for adaptability and innate immunity, i.e., in situ antigen expression and danger signaling; an "equilibrium" immune response can be induced, including humoral and cellular effectors and immune memory. mRNA vaccines are classified into non-replicating mRNA and replicating mRNA vaccines, with replicating mRNA encoding not only the antigen of interest, but also having a mechanism that enables intracellular RNA amplification and protein expression.

The RNA replicon is an autonomously replicating RNA, the viral replicase gene is reserved, the structural protein gene is deleted and replaced by an exogenous antigen gene, and replicase can control high-level replication of carrier RNA in cytoplasm and high-level expression of the exogenous gene. Venezuelan equine encephalitis Virus (VEE) belongs to the genus alphavirus, has the advantages of fast replication rate, high virus titer, wide host range, and the like, and is an ideal vaccine vector. The replicase gene nsp 1-4 region is the replicase coded by virus, and through simulating the amplification of virus nucleic acid, the expression quantity of antigen gene in cell is increased, on the other hand, the double-stranded RNA formed by replication enhances the immune effect of vaccine through activating the mode recognition receptor mediated signal transduction. Replicon-based vaccines do not produce replicable infectious virions and are unlikely to integrate with the cell genome, but can induce systemic and mucosal immunity as well as mhc i-restricted CTL responses without interference from existing vector antibodies in the body.

At present, a novel coronavirus mRNA vaccine enters clinical trials and applications, but the safety and the effectiveness are yet to be proved, and the risk of failure still exists.

Disclosure of Invention

In view of the above, the invention constructs a replication type novel coronavirus mRNA vaccine based on a VEE replication subsystem, researches and sends out a preparation method of the replication type mRNA vaccine for expressing novel coronavirus proteins, and uses and immunity.

The invention realizes a vaccine based on a VEE virus replicon, designs and constructs a replication type mRNA vaccine based on the VEE for expressing a novel Spike protein pre-fusion stable Trimer protein (Spike primer, S-primer) of the coronavirus, and provides a preparation method and application of the mRNA vaccine for expressing the Spike protein pre-fusion stable Trimer protein for preventing or treating novel coronavirus infection.

A recombinant replicative mRNA synthesis plasmid comprising a plasmid backbone, replicase genes and genes encoding novel coronavirus S protein mutants;

wherein the novel coronavirus S protein mutant comprises the following mutations compared to the wild-type novel coronavirus S protein:

(a) Eliminating or disrupting the mutation of the Furin protease cleavage site at amino acid sequence positions 682 to 685;

(b) Mutations that promote stabilization of the novel coronavirus S protein pre-fusion trimer conformation.

Novel coronaviruses (SARS-CoV-2) are also known as novel coronaviruses, and include viral mutants, such as Delta strains and Omicron BA.1 strains, and the like, having a stronger infectivity and a stronger immune escape ability.

Among them, the Spike protein of the new coronavirus is also called as S protein, which is the largest structural protein Spike of coronavirus, is located on the surface of the virus particle in Spike shape, and plays an important role in the binding of the virus particle to the receptor and the induction of antibody production. In the novel coronavirus S protein, a Furin protease cleavage site exists at the junction of the S1 subunit and the S2 subunit, and Furin protease on the surface of a cell membrane cleaves the cleavage site, so that an S1 domain of the S protein is dissociated and shed, and the rest S2 domain is responsible for the membrane fusion function of a virus membrane and the cell membrane, namely a fused conformation. The S protein of the novel coronavirus presents a trimeric structure on the virus surface, namely, the S protein is composed of three S1 subunits and three S2 subunits, and the S protein trimeric form is the natural form, if the S protein is blocked from binding to a receptor, the novel coronavirus cannot invade cells.

In the present invention, the expression "pre-fusion stable trimer conformation" refers to a mutation comprising the amino acid sequence Furin protease cleavage site of the novel coronavirus S protein and to a fusion comprising the carboxy-terminal junction trimer motif of the S protein.

Wherein the mutation that eliminates or disrupts the Furin protease cleavage site is an addition, deletion, and/or substitution at one or more amino acid positions 682 to 685 of the amino acid sequence; preferably, the mutation is a R682G/R683S/R685S substitution, such that the S protein retains a pre-fusion conformation.

The mutations that promote stabilization of the pre-fusion trimer conformation of the novel coronavirus S protein are K986P/V987P and F817P/A892P/A899P/A942P substitutions.

The novel coronavirus S protein mutant further comprises a mutation that promotes S protein release;

preferably, the mutation promoting release of the S protein comprises deleting amino acids 1209 to 1273 of the amino acid sequence of the S protein of the novel coronavirus to stabilize the trimeric conformation of the S protein and not interfere with the normal folding of the S protein; and adding a motif at the C-terminal end of the novel coronavirus S protein;

more preferably, the motif is a T4 or GCN4 motif.

In order to increase the level of expressed S protein after the VEE-S-Trimer-mRNA vaccine is introduced into the body, the gene sequence encoding the novel coronavirus S protein mutant is codon optimized; preferably optimized for mammalian codons.

Wherein the mRNA sequence size of the expression codon sequence optimized fusion pre-trimer S protein is 11445bp (shown as SEQ ID NO. 2), and the expression codon sequence optimized fusion pre-trimer S protein comprises mutation for stabilizing the conformation of the S protein fusion pre-trimer.

The plasmid backbone comprises a T7 promoter sequence, a 5' utr region, a 3' utr region, and a 3' terminal Poly (a) tail; preferably, the end of the 3' terminal Poly (A) tail comprises a restriction enzyme site for plasmid linearization preparation.

The coding region of the non-structural protein gene is derived from an alphavirus; the replicase gene is a replicase gene 1-4 coding region and comprises a VEE virus replicase gene sequence; preferably, the alphavirus is venezuelan equine encephalitis virus.

The invention also provides application of the recombinant replicative mRNA synthetic plasmid in preparing vaccines for preventing pneumonia caused by novel coronavirus infection.

The invention also provides a replicative mRNA for expressing the novel coronavirus S protein pre-fusion stable trimeric protein, which comprises a 5'UTR region, a 3' terminal PolyA tail, replicase 1-4 coding region sequences, mRNA for encoding the novel coronavirus S protein mutant and a T4 motif.

Preferably, the recombinant replicative mRNA synthesis plasmid is subjected to linearization treatment and transcribed to obtain mRNA of the recombinant replicative mRNA synthesis plasmid.

Preferably, the kit further comprises a 5 'cap structure, wherein the 5' cap structure is a 7-methylguanosine cap structure.

In the present invention, a method for preparing a replicative mRNA expressing a novel coronavirus S protein pre-fusion stable trimeric protein is also disclosed, comprising the steps of:

(1) Performing PCR amplification to obtain a gene fragment containing the novel coronavirus protein;

(2) Replicative mRNA vector plasmid VEE-S-Trimer containing novel coronavirus proteins was constructed by ligation vectors.

(3) Linearizing a recombinant replicative mRNA synthesis plasmid VEE-S-Trimer of an S protein gene in the system by using restriction enzymes;

(4) Carrying out in vitro transcription reaction on the linearized VEE-S-Trimer plasmid by using a T7 promoter sequence, and purifying to obtain VEE-S-Trimer-mRNA;

(5) Cap capping modification reaction is carried out on the obtained VEE-S-Trimer-mRNA by using vaccinia virus capping enzyme and 2' -O-methyltransferase, and Cap-VEE-S-Trimer-mRNA is obtained by purification;

(6) And (3) transfecting the obtained cap-VEE-S-primer-mRNA into BHK-21 cells, and detecting the effective expression of the target protein novel coronavirus S protein through an immunofluorescence experiment.

Wherein, the plasmid VEE-S-Trimer in the step (3) can be linearized by the restriction enzyme Mlu I used;

wherein, in the step (5), the VEE-S-Trimer-mRNA can be subjected to capping modification reaction by using vaccinia virus capping enzyme and related components through a vaccinia virus capping system, and a 7-methylguanosine cap structure is added to the 5' end of the RNA to obtain the cap-VEE-S-Trimer-mRNA after capping.

The invention also provides a replication type mRNA vaccine for expressing the novel coronavirus S protein pre-fusion stable trimeric protein, which comprises the replication type mRNA.

The invention also provides a preparation method of the replicative mRNA vaccine, which comprises the steps of mixing mRNA of the recombinant replicative mRNA synthesis plasmid with cationic lipid, distearoyl phosphatidylcholine (DSPC), cholesterol and polyethylene glycol lipid (DMG-PEG 2000) and obtaining the replicative mRNA vaccine through a microfluidic device or mixed dialysis. The mass molar ratio of the cationic lipid to the distearoyl phosphatidylcholine to the polyethylene glycol lipid to the cholesterol is 50:10:38.5:1.5.

In the present invention, LNP-VEE-S-Trimer-mRNA vaccine products are included as obtained after dialysis by microfluidic devices or mixing. LNP-VEE-S-Trimer-mRNA vaccine can be obtained by capping modified cap-VEE-S-Trimer-mRNA with SM-102, DSPC, DMG-PEG2000 and cholesterol in proportion by microfluidic device or by mixed dialysis.

The invention also provides application of the novel replication type mRNA vaccine of the stable trimer before coronavirus spike protein fusion in preventing pneumonia. The specific antibody for the novel coronavirus S protein exists in animals immunized by LNP-VEE-S-Trimer-mRNA vaccine, and has the effect of preventing pneumonia.

The invention has the beneficial effects that:

the invention prepares a replication type mRNA vaccine for expressing a stable trimeric protein before fusion of a novel coronavirus spike protein, constructs a replication type mRNA vector plasmid containing a novel coronavirus S protein, is used for preparing replication type mRNA, is combined with a liposome encapsulation scheme for preparing the mRNA vaccine, and is applied to immunization of the novel coronavirus. The method has application value in coronavirus research and vaccine creation, and can greatly promote the transformation application of the vaccine.

Drawings

FIG. 1 is a schematic diagram of a recombinant replicative mRNA synthesis plasmid VEE-S-Trimer expressing a pre-fusion stable trimeric protein of a novel coronavirus S protein.

FIG. 2 is a graph showing the results of agarose gel electrophoresis verification of the amplification of the stable trimeric protein gene before fusion of the novel coronavirus S protein; wherein M: DNA Maker,1: spike-primer PCR amplified product.

FIG. 3 is a diagram showing the results of agarose gel electrophoresis verification of the construction of a replicative mRNA synthetic plasmid VEE-S-Trimer by double cleavage of the nsp 1-4 region of the VEE replicase gene and the S gene of the novel coronavirus; wherein, 1: VEE plasmid, 2: VEE plasmid double cleavage product, 3: spike-Trimer gene double cleavage products.

FIG. 4 is a graph showing the result of agarose gel electrophoresis verification of the replicative mRNA synthesis plasmid VEE-S-Trimer restriction enzyme linearization; wherein M: DNA Maker,1: VEE plasmid, 2: the VEE-Spike-Trimer plasmid was digested and linearized.

FIG. 5 is a graph showing the results of agarose gel electrophoresis verification of the preparation of cap-VEE-S-Trimer-mRNA of the cap-modified novel coronavirus S protein; wherein M: DNA Maker,1: VEE plasmid, 2: VEE-Spike-Trimer in vitro transcription product.

FIG. 6 is a graph showing the results of indirect immunofluorescence assay of S protein expressed by replicative cap-VEE-S-Trimer-mRNA of S protein transfected by cells of different novel coronavirus mutants.

FIG. 7 is a graph showing the results of an ELISA assay for specific antibody levels of novel coronavirus S protein in serum after immunization of LNP-VEE-S-Trimer-mRNA vaccine; wherein, 1: VEE-S-Alpha,2: VEE-S-Delta,3: VEE-S-Omicron BA.1.

FIG. 8 is a graph showing the results of determining the effect of neutralizing antibodies against a novel coronavirus in serum after immunization with an LNP-VEE-S-Trimer-mRNA vaccine by virus neutralization; wherein, 1: VEE-S-Alpha,2: VEE-S-Delta,3: VEE-S-Omicron BA.1.

FIG. 9 is a graph showing the results of measuring the level of cytokines in serum after immunization of LNP-VEE-S-Trimer-mRNA vaccine by ELISA; wherein, 1: VEE-S-Alpha,2: VEE-S-Delta,3: VEE-S-Omicron BA.1.

Detailed Description

The technical scheme of the present invention will be further described in detail through the following specific embodiments, but the present invention is not limited to the examples.

The invention aims at the replicative mRNA vaccine developed by the novel coronavirus, mainly adopts (1) preparing replicative mRNA which is modified by capping and expresses stable trimeric protein before fusion of spike protein of the novel coronavirus; (2) Two methods of preparing LNP-VEE-S-Trimer-mRNA vaccine are provided.

EXAMPLE 1 construction of novel replication-competent recombinant mRNA Synthesis plasmid for the pre-fusion stable trimer of coronavirus spike protein

The replication type recombinant mRNA synthesis plasmid VEE-S-Trimer for expressing the novel coronavirus spike protein pre-fusion stable Trimer is designed and constructed. As shown in FIG. 1, the VEE replicon was constructed in the sequence of the T7 promoter sequence, the 5' UTR region, the nsp 1-4 region of the VEE replicase gene, the open reading frame encoding the novel coronavirus fusion pre-trimer S protein (amino acid sequence shown as SEQ ID NO. 1) gene, the 3' UTR region and the 3' terminal Poly (A) tail.

The novel coronavirus S protein is synthesized through the codon optimization sequence of the stable trimer before fusion, as shown in figure 2, and the gene fragment containing the novel coronavirus S protein mutant is obtained through PCR amplification, wherein the target fragment size is 3738bp. Wherein, the fusion protease enzyme cleavage site RRAR-GSAS substitution at positions 682 to 685 of the amino acid sequence of the S protein, the substitution of the amino acid sequences K986P/V987P and F817P/A892P/A899P/A942P, the deletion of positions 1209 to 1273 of the amino acid sequence, and the connection of the carboxyl terminal of the amino acid sequence with the T4 trimer motif.

As shown in FIG. 3, the amplified fragment of the stable trimeric mutant before S protein fusion and the vector of the VEE replicon were subjected to double cleavage reaction using restriction enzymes Not I and Nde I, and the cleavage effect was confirmed by agarose gel electrophoresis, and the VEE gene fragment size was 10800bp. The fragment of the VEE replicase gene nsp 1-4 region and the pre-fusion stable Trimer mutant of the novel coronavirus S protein are connected through T4 ligase reaction, so that the replicative recombinant mRNA for expressing the pre-fusion stable Trimer of the novel coronavirus S protein is obtained to synthesize a plasmid VEE-S-Trimer, and the map of the plasmid VEE-S-Trimer is shown in figure 1.

The plasmid VEE-S-Trimer is stored in the competent strain of the escherichia coli T1, single clones are randomly picked up, inoculated into 200mL of LB liquid medium containing ampicillin resistance, shake-cultured at 37 ℃, and the cloned VEE-S-Trimer plasmid is extracted according to the instruction of a large amount of Omega plasmid DNA kit, and is stored for use after concentration measurement.

Example 2 in vitro transcription preparation of VEE-S-Trimer-mRNA

As shown in FIG. 1, the tail end of the recombinant mRNA synthesis plasmid VEE-S-Trimer of the pre-fusion stable Trimer of the novel coronavirus S protein retains a Mlu I restriction enzyme site, and the vector plasmid is subjected to an enzyme tangential reaction using the restriction enzyme Mlu I, and the procedure is as follows:

(1) The VEE-S-Trimer plasmid constructed according to example 1 was tangentially linearized by Mlu I enzyme;

a) Firstly, 25 mug of VEE-S-Trimer plasmid is taken and digested with MluI for 2 hours at 37 ℃, and a digestion reaction system is prepared as follows:

b) After digestion, 5. Mu.L of 10% SDS solution was added to give a final SDS concentration of 0.5%;

c) Adding 0.5 mu L of 20 mg/mu L of proteinase K to make the final concentration of proteinase 50-100 mu g/mL;

d) Incubation at 37 ℃ for 1h, placing on ice, adding 200 mu L of nuclease-free water and 300 mu L of Phenol/CHCl3/IAA mixed solution, and standing for 5min after vortex;

e) Centrifuging at 12000rpm at room temperature for 10min, collecting supernatant, adding 750 μl of absolute ethanol, and standing at-20deg.C for 30min;

f) Centrifuging at 12000rpm for 15min at 4deg.C, and removing supernatant;

g) Adding 1mL of 75% ethanol, centrifuging at 12000rpm and 4 ℃ for 5min, repeating the steps for 2 times, discarding the supernatant, and airing the centrifuge tube;

h) Adding 20 mu L of nuclease-free water, dissolving DNA, diluting 1 mu L by 10 times, measuring the concentration of linearized DNA, and storing at-20 ℃;

i) Plasmid VEE-S-Trimer enzyme tangentially was verified by agarose gel electrophoresis.

As shown in FIG. 4, the size of the agarose gel electrophoresis band of the VEE plasmid was smaller than that of the VEE-S-Trimer enzyme-digested DNA fragment, demonstrating that the linearization of the VEE-S-Trimer plasmid was completed.

(2) And (3) using the linearized DNA of the step (1) as a template, and carrying out in vitro transcription to obtain VEE-S-Trimer-mRNA.

The mRNA of the stable Trimer before fusion of the novel coronavirus S protein is prepared and expressed by an in vitro transcription reaction by utilizing the T7 promoter of the mRNA synthesis plasmid VEE-S-Trimer upstream of the target gene.

a) Firstly, 1-2 mug of linearization VEE-S-Trimer plasmid is taken, mRNA is synthesized by T7 RNA polymerase at 37 ℃, and an in vitro transcription reaction system is prepared as follows:

b) The in vitro transcription system is incubated for 4h at 37 ℃, then 1 μl DNase is added, and the in vitro transcription system is incubated for 15min at 37 ℃, and the DNA template is digested;

c) VEE-S-Trimer-mRNA (sequence shown as SEQ ID NO. 2) was extracted by Trizol method or affinity chromatography, diluted 10-fold with 1. Mu.L, and the mRNA concentration was determined and stored at-80 ℃.

EXAMPLE 3 preparation of cap-VEE-S-Trimer-mRNA by mRNA capping modification

The 7-methylguanosine cap was added to the 5' end of the mRNA using vaccinia virus capping enzymes and related components. Making mRNA more stable and facilitating transport and translation.

a) The VEE-S-Trimer-mRNA obtained by in vitro transcription is subjected to capping modification reaction by vaccinia virus capping enzyme and Cap 2' -O-methyltransferase at 37 ℃, and a prepared capping modification reaction system is as follows:

b) Incubating the capping modification reaction system at 37 ℃ for 60min;

c) Extracting cap-VEE-S-Trimer-mRNA by Trizol method or affinity chromatography, diluting 1 μL for 10 times, measuring mRNA concentration, and storing at-80deg.C;

d) cap-VEE-S-primer-mRNA of the pre-fusion stable Trimer of the capping modified novel coronavirus S protein was verified by agarose gel electrophoresis.

As shown in FIG. 5, agarose gel electrophoresis showed the band size of mRNA, and the VEE plasmid was used as a control to demonstrate that cap-VEE-S-Trimer-mRNA of the pre-fusion stable Trimer of novel coronavirus S protein was synthesized efficiently.

EXAMPLE 4 immunofluorescence assay detection of novel coronavirus S protein expressed by transfected cap-VEE-S-Trimer-mRNA Pre-fusion stable trimeric protein

Pre-fusion stable Trimer proteins of different epidemic strains of novel coronaviruses were prepared by example 3, including pre-fusion stable Trimer proteins cap-VEE-S-Trimer-mRNA of S protein mutants of Alpha strain, delta strain and Omicron BA.1 strain (VEE-S-Alpha, VEE-S-Delta and VEE-S-Omicron BA.1). Wherein the S protein genes of Alpha strain, delta strain and Omicron BA.1 strain (NC_045512.2, OM858819.1 and OM 858820.1) are obtained by gene synthesis of fragments.

Transfection of cap-VEE-S-Trimer-mRNA in cells enables expression of the novel pre-fusion stable trimeric protein of the coronavirus S protein, and the use of specific antibodies to the novel coronavirus S protein enables detection of efficient translation of cap-VEE-S-Trimer-mRNA, as follows:

a) Adherent cells such as BHK-21 were plated in 48-well plates at 5% CO ₂ Culturing in an incubator at 37 ℃ until the cell density reaches about 70%, and transfecting the obtained cap-VEE-S-Trimer-mRNA;

b) Taking a clean EP tube, adding 300 mu L of Opti-MEM culture medium and 4 mu L of DMRIE-C transfection reagent, uniformly mixing by vortex, incubating for 30min at room temperature, adding 2 mu g of cap-VEE-S-Trimer-mRNA, lightly beating, uniformly mixing, and incubating for 10min;

c) Cells were washed once with Opti-MEM medium, and the incubated mixture was added, and after 24h, specific rabbit polyclonal antibodies to coronavirus S protein were used (sinobiologic, cat: 40590-T62), detecting the expression of the S protein by indirect immunofluorescence;

as shown in FIG. 6, the results show that BHK-21 cells transfected with the Alpha strain, the Delta strain and the Omicron BA.1 strain can obviously observe green fluorescent signals under a fluorescent microscope, and indicate that the cap-VEE-S-Trimer-mRNA can effectively express the stable trimeric protein before S protein fusion of the novel coronavirus Alpha strain, the Delta strain and the Omicron BA.1 strain.

EXAMPLE 5 preparation of lipid nanoparticle mRNA vaccine LNP-VEE-S-Trimer-mRNA

mRNA is a negatively charged biological macromolecule that is difficult to cross negatively charged cell membranes by passive transport. Lipid Nanoparticles (LNP) can be used to deliver RNA, an effective drug delivery means for mRNA vaccines.

The preparation of the lipid nanoparticle mRNA vaccine LNP-VEE-S-Trimer-mRNA is carried out according to the following steps:

a) SM-102, DSPC, DMG-PEG2000 and cholesterol were dissolved in 30. Mu.L absolute ethanol according to a molar ratio of 50:10:38.5:1.5, with a total mass of 200. Mu.g;

b) Under the condition of vortex, rapidly injecting 90 mu L of 20mM sodium acetate buffer solution containing 5 mu g of cap-VEE-S-Trimer-mRNA prepared in the example 3, vigorously stirring for 20S, and then standing for 10 minutes to prepare nano particles;

c) And dialyzing the prepared mixed solution of the sodium ethylacetate containing the nano particles in 10mM PBS solution for 2-4 hours to remove ethanol, and performing ultrafiltration concentration to obtain the final product LNP-VEE-S-Trimer-mRNA.

EXAMPLE 6 mRNA vaccine of stabilized trimeric protein before fusion of novel coronavirus S protein LNP-VEE-S-Trimer-mRNA immunization experiment

The cap-VEE-S-Trimer-mRNA prepared by example 3 was able to efficiently express the pre-fusion stable Trimer protein of the S protein of the novel coronavirus, and LNP-VEE-S-Trimer-mRNA vaccines (VEE-S-Alpha, VEE-S-Delta and VEE-S-Omicron BA.1) of the pre-fusion stable Trimer protein of the S protein of the novel coronavirus Alpha strain, delta strain and Omicron BA.1 strain were prepared in combination with the preparation method of the lipid nanoparticle mRNA vaccine of example 5, and vaccine effects were examined by an immunoassay.

The immune effect evaluation of LNP-VEE-S-Trimer-mRNA vaccine was performed as follows:

a) Female BALB/c strain mice of 6 weeks old were aliquoted into 4 groups (negative control group, VEE-S-Alpha group, VEE-S-Delta group and VEE-S-Omicron BA.1 group) and BALB/c mice were injected with LNP-VEE-S-Trimer-mRNA vaccine at 5 μg/intramuscular route on day 0 and day 30;

b) Collecting blood by orbital route on day 7 after the second immunization, and collecting collected serum sample;

c) Determination of S protein-specific antibody levels in serum after immunization of LNP-VEE-S-Trimer-mRNA vaccine by ELISA: serum samples (diluted 100 times in 5% skim milk powder), HRP-labeled detection antibodies were added in sequence to coated microwells pre-coated with the novel coronavirus S protein, incubated and thoroughly washed. The color was developed with TMB as substrate and converted to a final yellow color by 1M sulfuric acid, and the absorbance (OD value) was measured at a wavelength of 450nm with a microplate reader.

As shown in FIG. 7, the levels of the novel coronavirus S protein-specific antibodies in the samples were determined, and the VEE-S-Alpha, VEE-S-Delta and VEE-S-Omicron BA.1 groups immunized with the LNP-VEE-S-Trimer-mRNA vaccine produced different levels of the novel coronavirus S protein-specific antibodies, respectively, compared to the negative control group, wherein the VEE-S-Delta group antibodies were higher, so that the replication type mRNA vaccine capable of effectively activating humoral immunity to produce specific antibodies by immunizing the stable trimeric protein prior to expression of the novel coronavirus spike protein fusion.

d) Detection of virus neutralizing antibodies after immunization of LNP-VEE-S-Trimer-mRNA vaccine by competitive ELISA assay: in the method, recombinant human ACE2 receptor protein (hACE 2) is coated in a micropore, an HRP-marked RBD antigen protein is used for preparing enzyme-labeled antigen, a serum sample (diluted 100 times in a diluent) and the enzyme-labeled antigen are pre-incubated in a 96-well diluent plate, and then transferred into the hACE2 coating plate, and the enzyme-labeled antigen which is not combined with neutralizing antibodies is combined with the hACE 2. Color development with substrate TMB and conversion to a final yellow color by 1M sulfuric acid, absorbance (OD value) was measured with a microplate reader at a wavelength of 450nm, the absorbance being inversely proportional to the effective activity of neutralizing antibodies in the sample.

As shown in FIG. 8, the levels of the neutralizing antibodies against the novel coronavirus in the samples were determined, and the VEE-S-Alpha, VEE-S-Delta and VEE-S-Omicron BA.1 groups immunized with the LNP-VEE-S-Trimer-mRNA vaccine produced different levels of the neutralizing antibodies against the novel coronavirus, respectively, compared with the negative control group, and thus the neutralizing antibodies in the specific antibodies were produced by the organisms effectively activated by the replication type mRNA vaccine which expressed the stable trimeric proteins prior to fusion of the novel coronavirus spike protein.

e) Determination of cytokine levels in serum following immunization of LNP-VEE-S-Trimer-mRNA vaccine by ELISA: serum samples (diluted 100 times in 5% skim milk powder), HRP-labeled detection antibodies were added in sequence to coated microwells pre-coated with IL-4, IL-6, IFN-gamma, IL-10, incubated and thoroughly washed. The color was developed with TMB as substrate and converted to a final yellow color by 1M sulfuric acid, and the absorbance (OD value) was measured at a wavelength of 450nm with a microplate reader.

As shown in FIG. 9, the level of the cytokines in the sample was judged, and the levels of IL-4, IL-6, IFN-gamma and IL-10 in serum were all increased in the VEE-S-Alpha group, the VEE-S-Delta group and the VEE-S-Omicron BA.17 group immunized with the LNP-VEE-S-Trimer-mRNA vaccine compared with the negative control group, so that the replication type mRNA vaccine capable of expressing the stable trimeric protein before fusion of novel coronavirus spike protein exerted a certain immunological adjuvant effect and activated the natural immune level of the organism.

Claims

1. A recombinant replicative mRNA synthesis plasmid comprising a plasmid backbone, replicase genes, and genes encoding novel coronavirus S protein mutants;

2. The recombinant replicative mRNA synthesis plasmid of claim 1, wherein said novel coronavirus is Alpha strain, delta strain, and Omicron ba.1 strain.

3. The recombinant replicative mRNA synthesis plasmid of claim 1, wherein said mutation that eliminates or disrupts the Furin protease cleavage site is a R682G/R683S/R685S substitution;

4. The recombinant replication type mRNA synthesis plasmid of claim 3, wherein said novel coronavirus S protein mutant further comprises deletion of amino acids 1209 to 1273 of the novel coronavirus S protein amino acid sequence and addition of a motif at the C-terminal end of the novel coronavirus S protein; the motif is a T4 or GCN4 motif.

5. The recombinant replicative mRNA synthesis plasmid of claim 1, wherein said plasmid backbone comprises a T7 promoter sequence, a 5' utr region, a 3' utr region, and a 3' terminal Poly a tail; the 3' end of the Poly A tail contains a restriction enzyme site for plasmid linearization preparation; the gene sequences encoding the novel coronavirus S protein mutants are codon optimized.

6. The recombinant replicative mRNA synthesis plasmid of claim 1, wherein said replicase gene is derived from venezuelan equine encephalitis virus; the replicase gene is a replicase 1-4 coding region.

7. Use of a recombinant replicative mRNA synthetic plasmid according to any of claims 1 to 6 for the preparation of a vaccine for the prevention of pneumonia caused by novel coronavirus infection.

8. A replicative mRNA comprising a 5' utr region, a 3' utr region, and a 3' terminal Poly a tail, replicase 1-4 coding region sequences, mRNA encoding a novel coronavirus S protein mutant, and a T4 motif.

9. The replicative mRNA of claim 8, further comprising a 5 'cap structure, said 5' cap structure being a 7-methylguanosine cap structure.

10. A replicative mRNA vaccine comprising the replicative mRNA of claim 8 or 9.