Performance-extended new coronavirus self-replicating nucleic acid vaccine

Abstract

The invention provides an efficacy-prolonged new coronavirus self-replicating nucleic acid vaccine, a product capable of being prepared from the vaccine and application of the vaccine. According to the saRNA vaccine provided by the invention, the innate immunomodulator is introduced to improve the immunogenicity of saRNA, so that enough immune response is induced under single-needle inoculation with lower dose, and immune protection on various new coronaviruses and mutants thereof is provided.

Description

Technical Field

[0001] The present invention relates to a novel coronavirus self-replicating nucleic acid vaccine with extended efficacy, and relates to the technical field of vaccine compositions. Background Art

[0002] Novel coronavirus infection is an acute infectious disease caused by the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The original strain and variants of the virus, such as Lambda, Delta, Beta and Omicron, are highly contagious and spread rapidly around the world. The clinical symptoms of novel coronavirus infection are mainly fever, dry cough, fatigue, and may also include nasal congestion, runny nose, sore throat, decreased or loss of smell and taste. Some patients may not have any clinical symptoms during the course of the disease, and are called asymptomatic infections. Severe cases may develop acute respiratory distress syndrome or septic shock, or even death. The disease is contagious, and the source of infection is mainly patients infected with the novel coronavirus, which is contagious during the incubation period. All people are likely to be infected with the novel coronavirus, but the mortality rate is higher in the elderly and those with chronic underlying diseases.

[0003] The main method of treating novel coronavirus infection is to alleviate the patient's clinical symptoms and maintain the stability of his body functions. The prognosis varies from individual to individual, and most patients can recover after treatment. An effective way to prevent novel coronavirus infection is vaccination. There are many types of new crown vaccines currently, including inactivated virus vaccines, attenuated vaccines, adenovirus vector vaccines, recombinant protein vaccines and mRNA vaccines. These vaccines usually require multiple vaccinations to fully stimulate and maintain immune responses. Generally, two doses of vaccine are required to induce an effective immune response, which makes the vaccine response cycle longer. Two doses of vaccination not only increase the demand for vaccine production, but also bring higher time and financial costs.

[0004] Based on the above background, there is an urgent need for a new coronavirus vaccine that can fully induce and maintain an immune response with a single injection, thereby reducing the vaccine response cycle, reducing production time, and reducing production costs, and providing higher efficiency and economy in the manufacture and distribution of vaccines. The present invention aims to provide a new type of extended-effective new coronavirus nucleic acid vaccine, which achieves effective immune protection against the new coronavirus through innovative design. The development of this vaccine not only demonstrates the progress of modern molecular biology and immunology, but also provides new ideas and methods for the design and development of future vaccines. Summary of the invention

[0005] In order to make up for the shortcomings of the prior art, the object of the present invention is to provide a self-replicating nucleic acid vaccine (saRNA vaccine) of the new coronavirus with prolonged efficacy.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a vaccine for the new coronavirus comprising a self-replicating nucleic acid, wherein the vaccine comprises a combination of a self-replicating RNA system and a circular RNA encoding an innate immune regulator.

[0008] Furthermore, the strains of the new coronavirus include the original strain, Lambda strain, Delta strain, Beta strain and Omicron strain.

[0009] The self-replicating RNA system includes the following components:

[0010] 1) Self-replicating RNA encoding conserved antigenic epitopes;

[0011] 2) negative-strand RNA generated by replication of the self-replicating RNA encoding the conserved antigenic epitope;

[0012] 3) double-stranded dsRNA formed by pairing component 1) and component 2);

[0013] 4) an RNA polymerase complex formed by polymerization of one or more of the non-structural proteins nsP1 to nsP4 in the self-replicating RNA encoding the conserved antigenic epitope; and

[0014] 5) Subgenomic RNA produced by transcription of the self-replicating RNA encoding the conserved antigenic epitope.

[0015] The vaccine may further comprise a carrier.

[0016] Furthermore, the carrier includes lipid nanoparticles, cationic nanoemulsions, polymer nanoparticles, and viral replicon particles.

[0017] Furthermore, the carrier is a lipid nanoparticle.

[0018] Furthermore, the lipid nanoparticles include SM-102, DSPC, PEG-DMG and cholesterol.

[0019] The self-replicating RNA encoding the conserved antigenic epitope comprises a sequence encoding the conserved antigenic epitope and a carrier sequence.

[0020] Furthermore, the vector sequence is obtained based on alpha virus gene modification, and comprises a 5' untranslated region derived from alpha virus, non-structural protein sequences nsP1-nsP4, a subgenomic promoter sequence SGP, a 3' untranslated region and a poly(A) tail.

[0021] Further, the alphavirus is selected from at least one of Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Everglades virus, Semliki Forest virus, Chikungunya virus, Ross River virus, Western equine encephalitis virus, Highland J virus, salmon alphavirus and Buggy Creek virus.

[0022] Furthermore, the alpha virus is Venezuelan equine encephalitis virus.

[0023] Furthermore, the conserved antigenic epitopes include Spike protein, Spike protein S1 subunit, receptor binding domain RBD, and receptor binding domain RBD extension.

[0024] Furthermore, the conserved antigen epitope is the receptor binding domain RBD.

[0025] Furthermore, the sequence encoding the conserved antigenic epitope is shown in SEQ ID NO:1.

[0026] Furthermore, the sequence of the self-replicating RNA encoding the conserved antigenic epitope is shown in SEQ ID NO:3.

[0027] Furthermore, the sequence of the self-replicating RNA encoding the conserved antigenic epitope has more than 90% identity with the sequence shown in SEQ ID NO:3.

[0028] The circular RNA includes sequences encoding innate immune regulators and the internal ribosome entry site sequence of Coxsackievirus B3.

[0029] Furthermore, the innate immune regulators include vaccinia virus B18R protein, herpes simplex virus type 2 US1 protein, herpes simplex virus type 1 US1 protein, herpes simplex virus type 1 US11 protein, bovine viral diarrhea virus Npro protein, parainfluenza virus type 5 V protein, Middle East respiratory syndrome coronavirus M protein, Middle East respiratory syndrome coronavirus ORF4a protein, Langat virus NS5 protein and influenza virus NS1 protein.

[0030] Furthermore, the innate immune regulator is vaccinia virus B18R protein.

[0031] Furthermore, the sequence encoding the innate immune regulator is shown as SEQ ID NO:2.

[0032] Furthermore, the sequence of the circular RNA encoding the innate immune regulator is shown in SEQ ID NO:4.

[0033] Furthermore, the sequence of the circular RNA encoding the innate immune regulator has more than 90% identity with the sequence shown in SEQ ID NO:4.

[0034] The ratio of the combination of the self-replicating RNA encoding the conserved antigen epitope and the circular RNA encoding the innate immune regulator is 10:1 to 1:1.

[0035] Furthermore, the ratio of the combination of the self-replicating RNA encoding the conserved antigen epitope and the circular RNA encoding the innate immune regulator is 1:1.

[0036] The second aspect of the present invention provides a product for preventing or treating novel coronavirus infection, wherein the product comprises the vaccine described in the first aspect of the present invention.

[0037] Furthermore, the product includes a test kit, a chip, and a nucleic acid membrane strip.

[0038] The third aspect of the present invention provides use of the vaccine described in the first aspect of the present invention in the preparation of the product described in the second aspect of the present invention.

[0039] The fourth aspect of the present invention provides a pharmaceutical composition for preventing novel coronavirus infection, characterized in that the pharmaceutical composition includes the vaccine described in the first aspect of the present invention.

[0040] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable adjuvant, a pharmaceutically acceptable carrier, a diluent and / or an excipient.

[0041] The fifth aspect of the present invention provides a method for preparing the vaccine described in the first aspect of the present invention, the method comprising the following steps: preparing the self-replicating RNA system described in the first aspect of the present invention and the circular RNA encoding the innate immune regulator described in the first aspect of the present invention, and combining them in a ratio of 10:1 to 1:1.

[0042] Furthermore, the method includes the following steps: preparing saRNA expressing the receptor binding domain RBD and cRNA expressing vaccinia virus B18R by in vitro transcription, combining them in a ratio of 10:1 to 1:1, and encapsulating them in lipid nanoparticles.

[0043] Furthermore, the encapsulation form includes premixing followed by co-encapsulation or single encapsulation followed by mixing.

[0044] Advantages and beneficial effects of the present invention:

[0045] The present invention provides a novel coronavirus vaccine (saRNA vaccine) containing self-replicating nucleic acid with extended efficacy, which improves the immunogenicity of saRNA itself by introducing innate immune regulators, achieves full induction of immune response under a single injection at a lower dose, and maintains for a longer time, and can provide immune protection against a variety of novel coronavirus original strains and variants. Compared with traditional vaccines, the saRNA vaccine provided by the present invention significantly shortens the immune response cycle, reduces production time and reduces production costs, thereby providing higher efficiency and economy in the manufacture and distribution of vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 A schematic diagram of the structure of the self-replicating nucleic acid vaccine provided by the present invention;

[0047] Figure 2 This is a graph showing the integrity of saRNA detected by capillary gel electrophoresis;

[0048] Figure 3 This is a result diagram for continuous detection of cell expression of luciferase;

[0049] Figure 4 The imaging results of nude mice injected with LNP-saRNA (left hind limb) and LNP-mRNA (right hind limb) respectively;

[0050] Figure 5 This is a comparison chart of the quantitative data of LNP-saRNA and LNP-mRNA mouse imaging results;

[0051] Figure 6 This is a comparison chart of the cell expression RBD results of LNP-saRNA and LNP-mRNA;

[0052] Figure 7 It is a Western blot diagram of LNP-cRNA and LNP-mRNA expressing B18R, wherein band 1 represents LNP-cRNA and band 2 represents LNP-mRNA;

[0053] Figure 8 The results of antibody titer after vaccination with different doses of LNP-saRNA and different ratios of LNP-cRNA vaccine are shown;

[0054] Fig. 9 The graph shows the antibody titer results of different vaccination groups. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvement by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0056] In the present invention, the novel coronavirus refers to a new strain of coronavirus, which was named SARS-CoV-2 by the International Committee on Taxonomy of Viruses on February 11, 2020. Coronavirus is a large family of viruses known to cause more serious diseases such as colds, Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The novel coronavirus is a new strain of coronavirus that has never been found in humans before. Common signs of novel coronavirus infection include respiratory symptoms, fever, cough, shortness of breath and difficulty breathing. In more severe cases, infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. The new genome of the novel coronavirus is generated at a rate that exceeds the mutation rate of other coronaviruses, so it has a large number of variants.

[0057] In the present invention, lipid nanoparticles (Lipid Nanoparticles, LNP) is one of the important technologies in the lipid carrier drug delivery system, and has become an important progress based on oligonucleotide therapeutic drugs. Oligonucleotides encapsulated in lipid nanoparticles are protected during delivery, are not degraded by enzymes, and are effectively delivered to cells, where the contents in the carrier particles are released and translated into therapeutic proteins. In the preparation of nucleic acid vaccines, the application of LNP not only improves the stability and delivery efficiency of RNA, but also helps to activate the host's immune system. LNP can be recognized and taken in by the host's immune cells (such as dendritic cells), and then presented to T cells to stimulate immune response. In addition, LNP itself can also be used as an adjuvant to enhance the immunostimulatory effect of the vaccine.

[0058] In some embodiments, lipid nanoparticles are spherical vesicles composed of one (monolayer) or multiple (multilayer) phospholipid bilayers, usually composed of four components: cationic lipids, helper lipids, cholesterol, and pegylated lipids (PEG-lipids). Preferably, LNPs prepared from four lipids, SM-102, DSPC, PEG-DMG, and cholesterol, are used as nucleic acid vaccine vectors provided by the present invention.

[0059] In the present invention, self-amplifying RNA (saRNA) refers to a synthetic RNA that has the ability to replicate itself in host cells, resulting in increased expression of target proteins, a property that makes it a promising tool for vaccine development and gene therapy. Due to its replication properties, saRNA only requires about 1 / 6 of the linear mRNA dose of the vaccine to achieve the same protein expression. The advantage of the dose can significantly reduce its production cost and the immunogenicity brought by the dose, making it an ideal vaccine delivery platform.

[0060] In some embodiments, both saRNA and mRNA are synthesized by in vitro transcription (IVT). In terms of sequence composition, saRNA contains the basic elements of conventional mRNA, such as 5' untranslated region (5'UTR), target protein coding region (CDS), 3' untranslated region (3'UTR) and poly (A) tail. Based on the transformation of the alpha virus genome, saRNA, in addition to these components of conventional mRNA, also contains a non-structural protein (nsP) gene sequence derived from alpha virus downstream of 5'UTR, and there is a subgenomic promoter sequence (SGP) upstream of the target protein coding sequence.

[0061] In the present invention, in vitro transcription (IVT) refers to the use of DNA as a template in an in vitro cell-free system to mimic the in vivo transcription process to generate RNA, thereby controlling the transcribed gene, the transcription process, and the use of the RNA after transcription. Usually, the exogenous target gene is connected to a plasmid, the plasmid is extracted, and the linearized template is obtained by enzyme digestion, and RNA is synthesized using this linearized plasmid template.

[0062] In the present invention, alphaviruses refer to a class of mosquito-borne arboviruses that cause musculoskeletal and neurological diseases in humans. Debilitating acute illnesses are often reported in the context of concentrated outbreaks in specific areas, which can persist as chronic manifestations for many years after infection, and these viruses can be distributed along specific geographic locations.

[0063] In some embodiments, the alphavirus may be selected from Venezuelan Equine Encephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV), Everglades Virus (EVEV), Semliki Forest Virus (SFV), Chikungunya Virus (CHIKV), Ross River Virus (RRV), Western Equine Encephalitis Virus (WEEV), Highlands J Virus (HJV), Salmonid Alphavirus (SAV), and Beechey Ground Squirrel Virus (BCRV), or any combination thereof. Preferably, the alphavirus is Venezuelan Equine Encephalitis Virus.

[0064] In the present invention, nsP sequences (nonstructural proteins) refer to nonstructural proteins of viruses, i.e. proteins encoded by the viral genome, which have certain functions in the process of viral replication or gene expression regulation, but do not ultimately bind to form mature viruses and are not part of the viral structure. Nonstructural proteins of viruses play different roles in various stages of viral replication, such as viral RNA replication, transcription, translation, etc., which require the participation of nonstructural proteins.

[0065] In some embodiments, when saRNA enters the host cell, the nsP sequence is translated into four non-structural proteins (nsP1 to nsP4), each of which has unique and diverse functions. For example, nsP1 is involved in RNA capping reaction, nsP2 has RNA helicase and protease activity, nsP3 plays a role in mediating protein interactions, and nsP4 is an RNA-dependent RNA polymerase (RdRp). These non-structural proteins assemble into an RNA-dependent RNA polymerase complex (RdRp complex), which first synthesizes complementary negative-strand RNA using the saRNA positive strand as a template, and then replicates using the negative-strand RNA as a template to generate multiple positive-strand RNAs (usually 10 to 100 times). Among these generated positive-strand RNAs, the first is a copy of the full-length saRNA, and the second is a subgenomic RNA encoding the target protein. Subgenomic RNA accumulates in large quantities during the replication process, up to 10 6The saRNA can achieve high-level and long-lasting expression of the target protein at a low dose. This high-efficiency expression enables saRNA to achieve an immune response effect comparable to that of high-dose mRNA at a low dose in vaccine applications.

[0066] During the replication of saRNA, the double-stranded RNA (dsRNA) structure temporarily formed by the positive and negative strands of RNA can be recognized by pattern recognition receptors (PRRs) in host cells, such as TLR3, RIG-I, and MDA5, activating downstream signaling pathways, promoting the production of type I interferons (such as IFN-α and IFN-β), and then activating innate immune responses. The immunogenicity of saRNA itself can act as an immune adjuvant, and proper innate immune activation helps promote the development of acquired immunity. If the immune response is overactivated, it may cause cell death or immunopathology through pathways such as PKR, thereby affecting the effectiveness of saRNA as a vaccine.

[0067] In order to balance the immune response and avoid pathological effects caused by over-activation, different strategies can be used to reduce the immunogenicity of saRNA. For example, use immunomodulators or optimize saRNA vector sequences. In a 2021 study, Anna K. Blakney et al. constructed a saRNA vaccine that cis-encodes a natural immune response inhibitory protein, and verified in animal models and human skin explants that the use of immunomodulators can reduce the immunogenicity of saRNA and significantly enhance protein expression levels. In a study published in 2023, Mark W. Grinstaff et al. pointed out that the use of three modified nucleosides, 5OHmC, 5mC, and 5mU, can significantly improve transfection efficiency by 14 times, 10 times, and 8 times, respectively. In in vitro and in vivo experiments, these modified nucleotides enhanced the expression and efficacy of saRNA while reducing early interferon responses.

[0068] The saRNA-encoded novel coronavirus antigen of the present invention contains the complete RBD sequence of the Spike protein, which is the key part of the virus invading host cells and is highly conserved in multiple virus mutants. By guiding the host to produce a specific immune response against RBD, the virus infection can be effectively blocked, and as a universal vaccine, cross-immunity effects can be produced on different mutants. The vaccine provided by the present invention can induce strong cellular and humoral immune responses, providing comprehensive protection for the host. The novel coronavirus strain can be selected from one of the original novel coronavirus strain, lambda strain, delta strain, beta strain and Omicron strain. The nucleic acid sequence containing the complete RBD can be selected from any one of the full-length Spike, S1, RBD, RBD upstream extension, RBD downstream extension or RBD upstream and downstream extension. During the replication process of saRNA in the host cell, double-stranded RNA structures are formed, which can be recognized by pattern recognition receptors (PRRs) in the host cell, thereby activating downstream signaling pathways, promoting the production of type I interferons (such as IFN-α and IFN-β), and stimulating innate immune responses. However, an overly strong immune response can accelerate the in vivo clearance of saRNA. Therefore, by rationally regulating this immune response process, it can be used as an immune adjuvant to enhance the effectiveness of the vaccine.

[0069] In some embodiments, the saRNA active ingredient in the vaccine provided by the present invention can be obtained by the self-replicating RNA system provided by the present invention, including 1) a self-replicating RNA encoding a conserved antigenic epitope; 2) a negative-strand RNA replicated by the self-replicating RNA encoding the conserved antigenic epitope; 3) a double-stranded dsRNA formed by pairing component 1) and component 2); 4) an RNA polymerase complex formed by polymerization of one or more of the non-structural proteins nsP1 to nsP4 in the self-replicating RNA encoding the conserved antigenic epitope; and 5) one or more of the subgenomic RNAs transcribed by the self-replicating RNA encoding the conserved antigenic epitope.

[0070] Circular RNA (cRNA) is a covalently closed ring structure without a 5' cap structure and a 3' poly (A) tail, and is formed by reverse splicing of precursor RNA. Compared with linear mRNA, due to the lack of exposed ends, it can be protected from degradation by RNA exonucleases, so it is more stable in cells and has a longer half-life. In order to further improve the long-term expression ability of cRNA, Howard Chang's research group at Stanford University School of Medicine has developed a systematic approach that significantly improves the protein yield of cRNA through optimized design, enabling it to achieve more sustained translation in vivo. These optimizations include vector topology, 5'UTR, 3'UTR, internal ribosome entry site (IRES), and synthetic aptamer-initiated translation mechanism. With these improvements, the expression intensity of engineered cRNA in vivo is similar to that of modified mRNA, but lasts longer.

[0071] In the present invention, innate immunity is the first line of defense for the host to resist viral infection. The reason why innate immunity can recognize viruses in a broad spectrum and quickly exert its effects is mainly based on the innate immune system recognizing the molecular pattern (molecular pattern) of viruses with certain structural characteristics through pattern recognition receptors (PRRs), triggering signal pathways, and inducing a wide range of antiviral responses; on the other hand, in order to escape the host's immune clearance, the virus has evolved the ability to escape the host's innate immune response. Therefore, the protein in the virus that has the ability to escape the innate immune response is called an innate immune regulator or an innate immune inhibitory protein, which can play an important role in enhancing the immune efficacy of the vaccine in vaccine research.

[0072] In some embodiments, the innate immune suppressor protein is selected from B18R protein of vaccinia virus (Vaccinia Virus), US1 protein of herpes simplex virus type 2 (HSV-2), US1 protein and US11 protein of herpes simplex virus type 1 (HSV-1), Npro protein of bovine viral diarrhea virus (BVDV), V protein of parainfluenza virus type 5 (PIV-5), M protein and ORF4a protein of Middle East respiratory syndrome coronavirus (MERS CoV), NS5 protein of Langat virus and NS1 protein of influenza virus. One of them. The immunosuppressive protein expressed by cRNA can inhibit or antagonize signal molecules in the innate immune signaling pathway, block the body from producing an innate immune response, thereby reducing the body's inherent clearance efficiency of saRNA and ensuring the effective expression of saRNA. By mixing the saRNA expressing the antigen and the cRNA expressing the immunosuppressive protein in an appropriate proportion, the immunosuppressive protein expressed by cRNA can achieve trans-regulation of saRNA antigen expression after translation, thereby achieving a better immune effect.

[0073] The cRNA in the present invention adopts the Group I Intron self-splicing strategy to form a loop, and has an internal ribosome entry site (Internal Ribosome Entry Site, IRES) of Coxsackievirus B3 (CVB3), so as to recruit ribosomes and initiate the translation of downstream innate immune inhibitory proteins. Coxsackievirus is an enterovirus (enterovirus), which is divided into two categories, A and B. It is a common virus that infects the human body through the respiratory tract and digestive tract. After infection, people will have cold symptoms such as fever, sneezing, and coughing. Studies have shown that the IRES derived from CVB3 can maximize the protein translation efficiency of cRNA.

[0074] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Simple improvements to the present invention according to the essence of the present invention all belong to the scope of protection claimed in the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and the materials and reagents used in the following examples, unless otherwise specified, are all obtained from commercial sources.

[0075] Example 1 Preparation and integrity detection of saRNA

[0076] 1. Experimental Materials

[0077] In vitro transcription IVT kit (Suzhou Nearshore Protein Technology Co., Ltd.), NTP, plasmid template, endonuclease, chromatography instrument.

[0078] 2. Experimental Methods

[0079] 1. Linearize the plasmid template using endonuclease;

[0080] 2. Purify the linearized plasmid with phenol-chloroform;

[0081] 3. Experimental preparation of saRNA by in vitro transcription using linearized template;

[0082] 4. Purify saRNA using chromatography;

[0083] 5. Use capillary gel electrophoresis to detect the integrity of saRNA.

[0084] 3. Experimental Results

[0085] The results are as follows Figure 2 As shown, it can be seen that the integrity of saRNA after purification by chromatography reached 93.6%.

[0086] Example 2 Detection of saRNA and mRNA expression ability

[0087] 1. Experimental Materials

[0088] saRNA and mRNA expressing luciferase or RBD, SM-102, DSPC, PEG-DMG, cholesterol, nanoparticle preparation instrument, Huh7 cells, flow cytometer, BALB / c nude mice (Viton River), small animal imaging system, Pan02 cells, RBD-specific ELISA detection kit.

[0089] 2. Experimental Methods

[0090] 1. Use a nanoparticle preparation instrument to prepare LNPs with four lipids: SM-102, DSPC, PEG-DMG, and cholesterol, and encapsulate saRNA and mRNA expressing luciferase respectively;

[0091] 2. After transfecting Huh7 cells, continue to observe the expression time of the two;

[0092] 3. Inject LNP-saRNA (left hind limb) and LNP-mRNA (right hind limb) on both sides of the hind limbs of nude mice, and use the small animal imaging system to regularly take imaging pictures of the mice;

[0093] 4. Use a nanoparticle preparation instrument to prepare LNPs with four lipids: SM-102, DSPC, PEG-DMG, and cholesterol, to encapsulate saRNA and mRNA expressing RBD, respectively;

[0094] 5. After transfecting Pan02 cells, use an ELISA kit to detect the RBD content in the cell supernatant.

[0095] 3. Experimental Results

[0096] 1. In vitro expression ability: the results are as follows Figure 3 As shown, in Huh7 cells, the expression of LNP-mRNA gradually weakened over time, and was almost not expressed on Day 11. However, LNP-saRNA was able to express stably and evenly, and continued to express for more than one month.

[0097] 2. In vivo expression ability: The results are as follows Figure 4 , Figure 5 As shown, it can be seen that the LNP-saRNA expression in the left hind limb of the mouse lasts longer than the LNP-mRNA expression in the right hind limb, with continuous expression for more than one month, and the expression level increases significantly over time.

[0098] 3. Target gene RBD expression ability: The results are as follows Figure 6 As shown, it can be seen that the expression level of RBD in the supernatant of cells transfected with LNP-saRNA is more than 10 times that in the supernatant of cells transfected with LNP-mRNA.

[0099] The above results show that the LNP-saRNA provided by the present invention has stable and sustained expression ability in vivo and in vitro, and its expression ability for target genes far exceeds that of LNP-mRNA.

[0100] Example 3 Detection of cRNA and mRNA expression ability

[0101] 1. Experimental Materials

[0102] cRNA and mRNA expressing B18R, SM-102, DSPC, PEG-DMG, cholesterol, nanoparticle preparation instrument, Huh7 cells, Western blot (WB) experimental reagents, chemiluminescence imager.

[0103] 2. Experimental Methods

[0104] 1. Use a nanoparticle preparation instrument to prepare LNPs with four lipids: SM-102, DSPC, PEG-DMG, and cholesterol, and encapsulate cRNA and mRNA expressing B18R respectively;

[0105] 2. After transfecting Huh7 cells, collect the cell supernatant and perform WB to detect the expression of B18R.

[0106] 3. Experimental Results

[0107] The results are as follows Figure 7 As shown, band 1 represents LNP-cRNA, and band 2 represents LNP-mRNA. It can be seen that the amount of B18R protein expressed by the LNP-cRNA designed by the present invention is significantly greater than the amount of protein expressed by the LNP-mRNA. This shows that the circular RNA designed by the present invention is significantly better than mRNA in expressing the target protein B18R.

[0108] Example 4 Detection of immune efficacy of saRNA / cRNA vaccine and mRNA vaccine

[0109] 1. Experimental Materials

[0110] cRNA expressing B18R, saRNA and mRNA expressing RBD, SM-102, DSPC, PEG-DMG, cholesterol, nanoparticle preparation instrument, Balb / c mice, and ELISA kit for detection of novel coronavirus-specific IgG antibodies.

[0111] 2. Experimental Methods

[0112] 1. Use a nanoparticle preparation instrument to prepare LNPs with four lipids: SM-102, DSPC, PEG-DMG, and cholesterol to encapsulate cRNA expressing B18R, saRNA expressing RBD, and mRNA;

[0113] 2. Inject LNP-saRNA and LNP-cRNA into mice within a certain range (10:1-1:1), and use the LNP-mRNA injection group as a control;

[0114] 3. 14 days after vaccination, use ELISA kit to detect the IgG titer of mice injected by both methods.

[0115] 3. Experimental Results

[0116] 1. Antibody titer results after vaccination with different doses of LNP-saRNA and different ratios of LNP-cRNA Figure 8 As shown, it can be seen that mice vaccinated with LNP-saRNA / cRNA have higher IgG titers, and the effect is better when the ratio of saRNA to cRNA is 1:1 than 10:1.

[0117] 2. Antibody titer results of different vaccination groups Fig. 9 As shown, it can be seen that at a ratio of 1:1, 1 μg saRNA is better than 0.5 μg saRNA, while 5 μg mRNA still does not achieve the immune efficacy that saRNA can achieve.

[0118] The above results show that the LNP-saRNA / cRNA provided by the present invention is a novel coronavirus self-replicating nucleic acid vaccine with better immune efficacy and prolonged immune duration. The sequence table of the present invention is shown in Table 1.

[0119] Table 1. LNP-saRNA / cRNA vaccine sequence list

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A vaccine comprising a self-replicating nucleic acid for a novel coronavirus, characterized in that: The vaccine comprises a combination of a self-replicating RNA system and a circular RNA encoding an innate immune modulator; Preferably, the strains of the new coronavirus include the original strain, Lambda strain, Delta strain, Beta strain and Omicron strain.

2. The vaccine according to claim 1, characterized in that The self-replicating RNA system includes the following components: 1) Self-replicating RNA encoding conserved antigenic epitopes; 2) negative-strand RNA generated by replication of the self-replicating RNA encoding the conserved antigenic epitope; 3) double-stranded dsRNA formed by pairing component 1) and component 2); 4) an RNA polymerase complex formed by polymerization of one or more of the non-structural proteins nsP1 to nsP4 in the self-replicating RNA encoding the conserved antigenic epitope; and 5) Subgenomic RNA produced by transcription of the self-replicating RNA encoding the conserved antigenic epitope.

3. The vaccine according to claim 1, characterized in that The vaccine may further include a carrier; Preferably, the carrier comprises lipid nanoparticles, cationic nanoemulsions, polymer nanoparticles, viral replicon particles; Preferably, the carrier is a lipid nanoparticle; Preferably, the lipid nanoparticles include SM-102, DSPC, PEG-DMG and cholesterol.

4. The vaccine according to claim 2, characterized in that The self-replicating RNA encoding the conserved antigenic epitope comprises a sequence encoding the conserved antigenic epitope and a vector sequence; Preferably, the vector sequence is obtained based on alphavirus gene modification, and comprises a 5' untranslated region derived from alphavirus, non-structural protein sequences nsP1 to nsP4, a subgenomic promoter sequence SGP, a 3' untranslated region and a poly(A) tail; Preferably, the alphavirus is selected from at least one of Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Everglades virus, Semliki Forest virus, Chikungunya virus, Ross River virus, Western equine encephalitis virus, Highland J virus, salmon alphavirus, and Bugey Creek virus; Preferably, the alphavirus is Venezuelan equine encephalitis virus; Preferably, the conserved antigenic epitopes include Spike protein, Spike protein S1 subunit, receptor binding domain RBD, and receptor binding domain RBD extension; Preferably, the conserved antigenic epitope is the receptor binding domain RBD; Preferably, the sequence encoding the conserved antigenic epitope is as shown in SEQ ID NO: 1; Preferably, the sequence of the self-replicating RNA encoding the conserved antigenic epitope is as shown in SEQ ID NO: 3; Preferably, the sequence of the self-replicating RNA encoding the conserved antigenic epitope has more than 90% identity with the sequence shown in SEQ ID NO:

3.

5. The vaccine according to claim 1, characterized in that The circular RNA includes a sequence encoding an innate immune regulator, an internal ribosome entry site sequence of Coxsackievirus B3; Preferably, the innate immune modulators include vaccinia virus B18R protein, herpes simplex virus type 2 US1 protein, herpes simplex virus type 1 US1 protein, herpes simplex virus type 1 US11 protein, bovine viral diarrhea virus Npro protein, parainfluenza virus type 5 V protein, Middle East respiratory syndrome coronavirus M protein, Middle East respiratory syndrome coronavirus ORF4a protein, Langat virus NS5 protein and influenza virus NS1 protein; Preferably, the innate immune regulator is vaccinia virus B18R protein; Preferably, the sequence encoding the innate immune regulator is as shown in SEQ ID NO: 2; Preferably, the sequence of the circular RNA encoding the innate immune regulator is as shown in SEQ ID NO: 4; Preferably, the sequence of the circular RNA encoding the innate immune regulator has more than 90% identity with the sequence shown in SEQ ID NO:

4.

6. The vaccine according to claim 1, characterized in that The ratio of the combination of the self-replicating RNA system and the circular RNA encoding the innate immune regulator includes 10:1 to 1:1; Preferably, the ratio of the combination of the self-replicating RNA system and the circular RNA encoding the innate immune regulator is 1:

1.

7. A product for preventing or treating novel coronavirus infection, characterized in that: The product comprises the vaccine according to any one of claims 1 to 6; Preferably, the product includes a kit, a chip, and a nucleic acid membrane strip.

8. Use of the vaccine according to any one of claims 1 to 6 in the preparation of the product according to claim 7.

9. A pharmaceutical composition for preventing novel coronavirus infection, characterized in that: The pharmaceutical composition comprises the vaccine according to any one of claims 1 to 6; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant, a pharmaceutically acceptable carrier, a diluent and / or an excipient.

10. A method for preparing a vaccine according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: preparing the self-replicating RNA system of claim 1 and the circular RNA encoding the innate immune modulator of claim 1, and combining them in a ratio of 10:1 to 1:1; Preferably, the method comprises the following steps: preparing saRNA expressing the receptor binding domain RBD and cRNA expressing vaccinia virus B18R by in vitro transcription, combining them in a ratio of 10:1 to 1:1, and encapsulating them in lipid nanoparticles; Preferably, the lipid nanoparticle encapsulation form includes premixing followed by co-encapsulation or single encapsulation followed by mixing.

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