Foot-and-mouth disease vaccine and preparation method thereof

Abstract

The invention discloses a foot-and-mouth disease vaccine and a preparation method thereof. The foot-and-mouth disease virus vector comprises a circular RNA molecule for coding foot-and-mouth disease virus VP1 truncated and mutant protein and a pharmaceutically acceptable vector, wherein the amino acid sequence of the foot and mouth disease virus VP1 truncated mutant protein is shown as SEQ ID No.13 or a degenerate sequence thereof, and the nucleotide sequence is shown as SEQ ID No.14 or a degenerate sequence thereof; the nucleotide sequence of the circular RNA molecule is as shown in SEQ ID No. 17. At present, no RNA vaccine for resisting foot-and-mouth disease virus infection exists, and the circular RNA molecule for coding foot-and-mouth disease virus VP1 truncation and mutant protein and the lipid nanoparticles are used for preparing the vaccine, so that the vaccine has the advantages of safety, high efficiency, low cost and the like. A high-level virus blocking antibody can be induced and CD8 + T cell immune response can be successfully activated by injecting a low-dose circular RNA vaccine (80mu g / ml), the vaccine is good in safety and stability, and a basis is provided for large-scale vaccine preparation and application.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a foot-and-mouth disease vaccine and a preparation method thereof. Background Art

[0002] Foot-and-mouth disease virus (FMDV) belongs to the Picornaviridae family and the genus FMDV. FMDV virus particles consist of an icosahedral shell, containing four structural proteins (VP1, VP2, VP3 and VP4) and eight non-structural proteins, of which four structural proteins can form virus-like particles (VLPs) alone. There are seven serotypes of FMDV, namely O, A, Asia1, SAT1, SAT2, SAT3 and C, and there is no cross-protection reaction between the types.

[0003] Foot-and-mouth disease vaccine is the main measure to prevent foot-and-mouth disease virus infection in the process of animal husbandry production. It is a vaccine that is compulsory for vaccination during animal husbandry. The existing vaccine technology route is mainly inactivated vaccine, which is made by amplification, inactivation, emulsification and other processes of the strain, and plays an important role in the prevention of foot-and-mouth disease. However, the traditional inactivated vaccine has high requirements on the production environment and production personnel during the production and preparation process. There is a risk of leakage of virus strains and incomplete inactivation during the production and preparation process, so the production cost is high. Another vaccine technology route is foot-and-mouth disease recombinant subunit vaccine, which is a VLP particle formed by enzyme shearing of the P1 protein encoded by the genome open reading frame (OpenReading Frame, ORF), but because the viral gene sequence is highly toxic to cells during in vitro cell fermentation, the VLP yield is low, and there are purification process difficulties. At present, related products have not yet entered clinical application in mass production. There is still a breakthrough in the development of new vaccines for foot-and-mouth disease virus.

[0004] Messenger Ribonucleic Acid (mRNA) is transcribed from DNA and provides the genetic information required for the next step of protein translation. When mRNA encoding antigenic proteins is injected into an organism, it can synthesize antigenic proteins in vivo, thereby inducing strong cellular and humoral immune responses, and exhibiting the characteristics of autoimmune adjuvants, making it an excellent vaccine method. In addition, mRNA has many other advantages as a vaccine or the production of therapeutic proteins. For example, compared with DNA vectors, mRNA is expressed transiently in cells, there is no risk of integration into the genome, and it is not dependent on the cell cycle, so it has higher safety; compared with viral vectors, mRNA does not have the immune resistance brought by the vector itself, so protein expression is easier to achieve; compared with recombinant proteins, viruses, etc., the production process of mRNA is a cell-free system, involving only enzyme-catalyzed reactions in vitro, so the production process is simpler, controllable and low-cost. At present, mRNA has shown broad application potential as a vaccine, the production of therapeutic proteins, and as a means of gene therapy.

[0005] mRNA vaccines are vaccines that directly introduce mRNA containing antigenic proteins into the body, so that it is directly translated in the body to complete the expression of the target protein, thereby causing a specific immune response in the body. They have played an important role in the prevention of major respiratory infectious diseases in humans. At present, the mRNA used in clinical or preclinical applications is mainly linear mRNA. The structure of linear mRNA includes 5' cap structure (5'Cap), 3' polyadenosine tail (PolyA tail), 5' untranslational sequence (5'untranslational region, 5'UTR), 3' untranslational sequence (3'untranslational region, 3'UTR) and open reading frame (Open reading frame, ORF). The 5' cap structure is a basic feature of eukaryotic mRNA, which is obtained by adding N7-methylguanosine to the 5' end of mRNA. The 5' cap structure and 3' polyadenosine tail are necessary components of linear RNA, which can effectively prevent mRNA degradation and promote the translation of mRNA into protein. The 5' cap structure promotes mRNA translation by binding to the translation initiation complex eif4E, and can effectively prevent mRNA degradation and reduce the immunogenicity of mRNA. The main function of the 3' polyadenosine tail is to bind to PolyA binding protein (PABP), which interacts with eIF4G and eIF4E, mediates the formation of mRNA into a ring, promotes the translation process, and prevents mRNA degradation.

[0006] Circular RNA (circRNAs) is a common type of RNA in eukaryotes. Currently, the most efficient technology for constructing circular RNA in vitro is the self-cleavage circular system design technology (patent CN 114574483 A). The design principle of this technology is to connect the exon E1 and E2 sequences end to end through molecular cloning to form a continuous circular plasmid. The intron is cut and broken by restriction endonuclease to obtain a linear plasmid. Then, in vitro transcription is performed through the T7 promoter upstream of the inverted 3' intron to obtain a linear RNA containing a 3' intron-E2-E1-5' intron structure. Similar to the natural class I intron system, the specific conserved sequence of the splicing site of exon E1 is broken by the nucleophilic attack of the free guanosine 3' hydroxyl, and exon E1 produces a naked 3' hydroxyl, while guanosine binds to the broken 5' intron. Afterwards, the exposed 3'hydroxyl group of exon E1 attacks the conserved sequence between the 3'intron and exon E2, excises the 3'intron, and exons E2 and E1 undergo a circularization reaction to obtain a circular E1-E2 RNA. Compared with linear mRNA, circular mRNA molecules often have a longer half-life and better stability, and have important application prospects in protein expression and clinical treatment. In addition, the preparation process of circular RNA designed based on the self-cleaving circularization system is simpler and has lower process costs. After transcription, only circularization is performed, and there is no need to use expensive capping enzymes and tailing enzymes to modify additional 5' cap structures and 3' polyadenosine tail structures, nor is there a need for additional circularization processes using T4 RNA ligase as in traditional circular RNA. In addition, it can avoid the introduction of additional exon sequences in circular RNA, thereby improving the sequence accuracy of circular RNA molecules.

[0007] As of now, no new vaccines for foot-and-mouth disease virus, including RNA vaccines, have entered clinical use worldwide, and breakthroughs in related fields are yet to be made. Summary of the invention

[0008] In view of the many problems existing in the existing inactivated foot-and-mouth disease vaccines and recombinant subunit vaccines in clinical application: for example, the inactivated foot-and-mouth disease vaccines have the risk of virus strain leakage, incomplete inactivation, high production cost, low recombinant subprotein production efficiency, and difficult purification process, while the linear RNA vaccine for foot-and-mouth disease under development cannot efficiently activate CD8+T cell immune response and has insufficient immune protection efficacy (Development of O-type foot-and-mouth disease mRNA vaccine [D], 2022), etc., the present invention provides a foot-and-mouth disease vaccine and a preparation method thereof. The circular RNA molecule contains an optimized coding element of the foot-and-mouth disease virus VP1 protein and an optimized circular element, which can be produced on a large scale. The circular RNA molecule can induce protein expression, produce high levels of virus blocking antibodies, and successfully activate CD8+T cell immune response when entering the animal body, providing a basis for the preparation and application of circular mRNA vaccines for the prevention and / or treatment of foot-and-mouth disease.

[0009] The objective of the present invention is achieved through the following technical solutions:

[0010] The invention provides a foot-and-mouth disease vaccine, which comprises a circular RNA molecule encoding a truncated and mutant protein of VP1 of a foot-and-mouth disease virus and a pharmaceutically acceptable carrier.

[0011] As an embodiment of the present invention, the amino acid sequence of the truncated and mutant protein of foot-and-mouth disease virus VP1 is shown in SEQ ID No. 13 or its degenerate sequence.

[0012] As an embodiment of the present invention, the optimized nucleotide sequence of the truncated and mutant protein of foot-and-mouth disease virus VP1 is shown as SEQ ID No. 14 or its degenerate sequence.

[0013] As one embodiment of the present invention, the nucleotide sequence of the circular RNA molecule (PPV2-VP1-5147) is as SEQ ID No.17.

[0014] As an embodiment of the present invention, the content of circular RNA molecule (PPV2-VP1-5147) in the foot-and-mouth disease vaccine is 30-200 μg / ml, including 30-60 μg / ml, 60-90 μg / ml, 90-120 μg / ml, 120-150 μg / ml, 150-180 μg / ml, 180-200 μg / ml, preferably 60-100 μg / ml.

[0015] As one embodiment of the present invention, the pharmaceutically acceptable carrier comprises lipid nanoparticles.

[0016] As an embodiment of the present invention, the ratio of lipid nanoparticles to circular RNA molecules in the foot-and-mouth disease vaccine is 1:1-1:3 (V / V).

[0017] As an embodiment of the present invention, the raw material composition of the lipid nanoparticles includes: 45%-55% cationic lipid, 35%-44% cholesterol, 3%-10% neutral lipid and 0.8%-1.8% PEG-modified lipid.

[0018] As one embodiment of the present invention, the cationic lipid is CMAX4 (4-[(3-{[3-({3-[bis(3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxypropyl)amino]propyl}(methyl)amino)propyl](3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxypropyl)amino}propoxy)oxy]butyl 2-octanoate) or SM-102 (1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecanyloxy)hexyl]amino]-octanoate.

[0019] As an embodiment of the present invention, the cholesterol is 5-cholesten-3β-ol.

[0020] As an embodiment of the present invention, the neutral lipid is DSPC (distearoylphosphatidylcholine) or DOPE (dioleoylphosphatidylethanolamine).

[0021] As an embodiment of the present invention, the PEG-modified lipid is PEG-DMG (polyethylene glycol-dimyristyl glyceride), PEG-DSG (bis-distearoyl-rac-glycerol-polyethylene glycol) or PEG-DPG (polyethylene glycol diphosphoglycerate).

[0022] As an embodiment of the present invention, the preparation method of the lipid nanoparticles comprises: dissolving 45%-55% cationic lipids, 35%-44% cholesterol, 3%-10% neutral lipids, and 0.8%-1.8% PEG-modified lipids in ethanol.

[0023] As an embodiment of the present invention, the cationic lipid is CMAX4; the cholesterol is 5-cholesten-3β-ol; the neutral lipid is DSPC; the PEG-modified lipid is PEG-DMG; the molar ratio of CMAX4:5-cholesten-3β-ol:DSPC:PEG-DMG is 45.9:42.6:9.2:1.5.

[0024] The present invention also provides a method for preparing a foot-and-mouth disease vaccine, the method comprising the following steps:

[0025] S1, synthesizing and inserting the truncated VP1 gene plus mutant protein and looping element of the foot-and-mouth disease virus into a cloning vector to obtain a recombinant vector containing the truncated VP1 gene plus mutant protein and looping element of the foot-and-mouth disease virus;

[0026] S2, transforming or transducing a host with the recombinant vector containing the truncated VP1 plus mutant protein gene and the looping element in the foot-and-mouth disease virus obtained in step S1 to obtain a recombinant containing the recombinant vector;

[0027] S3, culturing the recombinant obtained in step S2 to prepare a plasmid containing the truncated VP1 plus mutant protein gene and the circular element in the foot-and-mouth disease virus;

[0028] S4, using the plasmid containing the truncated and mutant protein gene of VP1 in foot-and-mouth disease virus and the circular element obtained in step S3, to prepare a circular mRNA encoding the truncated and mutant protein of VP1 in foot-and-mouth disease virus;

[0029] S5. Add the circular mRNA encoding the truncated and mutant protein of foot-and-mouth disease virus VP1 obtained in step S4 to a pharmaceutically acceptable carrier to obtain the foot-and-mouth disease vaccine.

[0030] The present invention also provides an application of a foot-and-mouth disease vaccine in preparing a medicine for preventing and / or treating foot-and-mouth disease virus infection.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) There is currently no FMD virus RNA vaccine. The present invention uses a circular RNA molecule encoding a truncated VP1 plus a mutant protein optimized nucleotide sequence as an antigen to prepare an anti-FMD virus circular RNA vaccine, which has many advantages such as simple process, safety, and high efficiency.

[0033] (2) Compared with the disclosed elements, the total yield of circular RNA molecules containing the optimized elements is increased, which meets the requirements of large-scale production process.

[0034] (3) Injection of a low dose of circular RNA vaccine against foot-and-mouth disease virus (RNA content of 80 μg / ml) can produce high levels of virus-blocking antibodies and cellular immune responses, and the vaccine is highly effective. Compared with linear RNA vaccines and commercially available inactivated vaccines that cannot efficiently activate CD8+ T cell immune responses, circular RNA vaccines against foot-and-mouth disease virus successfully activate CD8+ T cell immune responses.

[0035] (4) In the present invention, the circular RNA vaccine against foot-and-mouth disease infection is safe for use in piglets.

[0036] (5) The circular RNA vaccine against foot-and-mouth disease virus prepared by the optimized process of the present invention is stable when stored at -20°C for 6 months, providing a basis for large-scale vaccine preparation and clinical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0038] Figure 1 This is the proportion of CD4+CD8+T lymphocytes in PBMC cells of pigs in each group after vaccine immunization (14 days after the second immunization). DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are implemented on the premise of the technical solution of the present invention, provide detailed implementation methods and specific operation processes, and will help those skilled in the art to further understand the present invention. It should be pointed out that the protection scope of the present invention is not limited to the following embodiments, and several adjustments and improvements made under the premise of the concept of the present invention all belong to the protection scope of the present invention.

[0040] In the present invention, the term "a" or "an" or "the" may mean "one", and may also mean "one or more", "at least one" and "one or more than one".

[0041] In the present invention, the terms "comprising", "having", "including" or "containing" may mean inclusive or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising", "having", "including" or "containing" may also mean closed, excluding additional, uncited elements or method steps.

[0042] In the present invention, the word "may" includes both performing a certain process and not performing a certain process.

[0043] In the present invention, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances where the event occurs and instances where it does not occur.

[0044] In the present invention, the term "about" may mean that a value includes the standard deviation of the error of the device or method used to determine the value. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are modified by "about".

[0045] In the present invention, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and are amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0046] In the present invention, the term "circular RNA" refers to an RNA molecule in a closed circular shape. In the present invention, the circular RNA used has protein translation activity and can also be called "circular mRNA".

[0047] In the present invention, the term "linear RNA" refers to an RNA precursor that can be circularized to form a circular RNA, which is generally transcribed from a linear DNA molecule and can also be called "linear mRNA".

[0048] In the present invention, the term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide may be included in a suitable vector, and the vector may be used to transform into a suitable host cell. The polynucleotide is then expressed in a recombinant host cell to produce, for example, a "recombinant polypeptide", "recombinant protein", "fusion protein", etc. In the present invention, the recombinant nucleic acid molecule comprises a coding element encoding a target polypeptide, and a translation initiation element connected upstream of the coding element.

[0049] In the present invention, the term "translation initiation element" refers to any sequence that can recruit ribosomes and initiate the translation process of RNA molecules. Exemplary translation initiation elements include IRES sequences, m 6 A modification sequence, or the initiation sequence of rolling circle translation, etc.

[0050] In the present invention, the term "IRES" (Internal ribosome entry site, IRES) is also called internal ribosome entry site, "internal ribosome entry site" (IRES) belongs to the translation control sequence, which is usually located at the 5' end of the gene of interest and enables the translation of RNA in a cap-independent manner. The transcribed IRES can directly bind to the ribosomal subunit so that the mRNA start codon is properly oriented in the ribosome for translation. The IRES sequence is usually located in the 5'UTR of the mRNA (just upstream of the start codon). IRES functionally replaces the need for various protein factors that interact with the eukaryotic translation machinery.

[0051] In the present invention, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0052] In the present invention, the term "wild type" is synonymous with "naturally occurring", and refers to an object that can be found in nature and has not been modified by humans.

[0053] In the present invention, the terms "variant" and "mutant" are used interchangeably and refer to nucleotides or amino acids that contain changes (i.e., substitutions, insertions and / or deletions) at one or more (e.g., several) positions relative to a "wild-type" polynucleotide or polypeptide. Substitution refers to replacing a nucleotide / amino acid occupying a position with a different nucleotide / amino acid. Deletion refers to the removal of a nucleotide / amino acid occupying a position. Insertion refers to the addition of a nucleotide / amino acid adjacent to and immediately following the nucleotide / amino acid occupying the position.

[0054] In the present invention, the terms "sequence identity" and "percentage of identity" refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. The polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). The polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). The sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the identical nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0055] In the present invention, the term "individual", "patient" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0056] In the present invention, "treatment" means: after suffering from a disease, the subject is exposed to (e.g., administered) the RNA molecule described in the present invention, thereby alleviating the symptoms of the disease compared to when the subject is not exposed, and does not necessarily mean that the symptoms of the disease must be completely suppressed. Suffering from a disease means: the body has symptoms of the disease.

[0057] In the present invention, "prevention" means that before contracting a disease, by exposing a subject to the RNA molecule described in the present invention, the probability of contracting a disease and / or the symptoms after contracting a disease are reduced compared to when the subject is not exposed, and it does not mean that the disease must be completely suppressed.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include inevitable systematic errors.

[0059] <circular RNA>

[0060] The present invention provides a circular RNA, which comprises a coding element, wherein the coding element encodes a truncated and mutant protein of VP1 of a foot-and-mouth disease virus; the amino acid sequence of the truncated and mutant protein of VP1 of a foot-and-mouth disease virus is shown as SEQ ID No. 13 or a degenerate sequence thereof; the optimized nucleotide sequence of the truncated and mutant protein of VP1 of a foot-and-mouth disease virus is shown as SEQ ID No. 14 or a degenerate sequence thereof; the nucleotide sequence of the circular RNA molecule (numbered PPV2-VP1-5147) is shown as SEQ ID No. 17.

[0061] In some embodiments, the circular RNA described in the present invention is obtained by transcription and circularization of a vector DNA comprising the following elements (in the direction from 5' to 3'): intron fragment II (numbered B-II, nucleotide sequence such as SEQ ID NO.8), translation initiation element truncated fragment II (numbered PPV-II, nucleotide sequence such as SEQ ID NO.10), protein coding element (numbered VP1-5147, nucleotide sequence such as SEQ ID NO.14), insertion element (numbered B-polyAC, nucleotide sequence such as SEQ ID NO.12), translation initiation element truncated fragment I (numbered PPV-I, nucleotide sequence such as SEQ ID NO.11), intron fragment I (numbered BI, nucleotide sequence such as SEQ ID NO.9).

[0062] In other embodiments, the circular RNA of the present invention is prepared by a clean PIE system, under which conditions, the circular RNA comprises elements arranged in the order shown in (c) or (d):

[0063] (c) translation initiation elements and coding elements;

[0064] (d) Translation initiation elements, coding elements and insertion elements.

[0065] In some preferred embodiments, the circular RNA of the present invention is prepared by a clean PIE system, and the circular RNA comprises the elements arranged in the order shown in (d) above. The clean PIE system for preparing circular RNA can refer to the contents shown in patents CN202210200112.6 and CN202210200186.X, and the entire contents of the above patents are incorporated into this application by reference.

[0066] In some embodiments, the translation initiation element described herein can be any type of element that can initiate translation of a coding element in a circular RNA.

[0067] In some specific embodiments, the translation initiation element of the present invention comprises one or more of the following sequences with translation initiation activity: IRES sequence, 5'UTR sequence, Kozak sequence, m 6 A modified (N(6) methyladenosine modification) sequence, complementary sequence of ribosomal 18S rRNA.

[0068] In some preferred embodiments, the translation initiation element of the present invention comprises an IRES sequence. Exemplary, the IRES sequence includes but is not limited to an IRES sequence derived from Echovirus, Human poliovirus, Human Enterovirus, Coxsackievirus, Human rhinovirus, Canine picornavirus, Turdivirus 3, Hepatovirus, Passerivirus, Picornaviridae, Tremovirus A, Feline kobuvirus, Murine kobuvirus, Kobuvirus sewage Kathmandu, Ferret kobuvirus, Marmot kobuvirus, Humanparechovirus, Chicken picornavirus, Falcon picornavirus, Feline picornavirus, French Guiana picornavirus, etc.

[0069] In some embodiments, the insertion element described in the present invention can be used to regulate the transcription of recombinant nucleic acid molecules, to regulate the translation of circular RNA, to achieve specific expression of circular RNA in different tissues, or to purify circular RNA, etc.

[0070] In some specific embodiments, the insertion element of the present invention comprises one or more of the following sequences: untranslated region sequence, polyN sequence, aptamer sequence, ribosomal switch sequence, and sequence binding to transcriptional regulatory factors; wherein, in the polyN sequence, N is selected from at least one of A, T, G, and C. In some preferred embodiments, the insertion element of the present invention comprises a polyAC sequence; preferably, the polyAC sequence comprises the sequence shown in SEQ ID NO.12.

[0071] In some more preferred embodiments, the circular RNA described in the present invention has a sequence as shown in any one of SEQ ID NOs. 15 to 17.

[0072] <Recombinant Nucleic Acid Molecule>

[0073] The present invention provides a recombinant nucleic acid molecule, which forms the above-mentioned linear RNA after transcription.

[0074] In some embodiments, the recombinant nucleic acid molecule of the present invention may contain a regulatory sequence in addition to the elements contained in the linear RNA described above. Exemplarily, the regulatory sequence is a T7 promoter.

[0075] <Nucleic acid lipid nanoparticle composition>

[0076] The present invention provides a nucleic acid lipid nanoparticle composition, which comprises lipid nanoparticles and the above-mentioned circular RNA.

[0077] By encapsulating the above circular RNA into lipid nanoparticles, the body's uptake efficiency of circular RNA can be improved, while the storage stability of circular RNA is improved, providing assistance for the development and application of foot-and-mouth disease vaccines.

[0078] <Medical Use>

[0079] The circular RNA, linear RNA, recombinant nucleic acid molecule and / or nucleic acid lipid nanoparticle composition provided by the present invention can be used to prevent and / or treat foot-and-mouth disease.

[0080] The circular RNA, linear RNA, recombinant nucleic acid molecule and / or nucleic acid lipid nanoparticle composition provided by the present invention can be used to prepare drugs for preventing and / or treating foot-and-mouth disease.

[0081] The present invention provides a method for preventing and / or treating foot-and-mouth disease, comprising administering an effective amount of the above-mentioned circular RNA, linear RNA, recombinant nucleic acid molecule and / or nucleic acid lipid nanoparticle composition to a subject; preferably, the administration is performed by injection.

[0082] The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the present invention. Without departing from the spirit and scope of the present invention, those skilled in the art may make any modifications or changes to the technical solution of the present invention, and such modifications and changes are also included in the scope of the present invention.

[0083] In the examples, if the specific conditions are not specified, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can be implemented equally without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0084] Example 1: Gene design and synthesis

[0085] Plasmid vector DNA encoding different RNA molecules was designed for foot-and-mouth disease virus VP1 (see Table 1). The vector DNA contained the following elements arranged in the following order from 5' to 3': intron fragment II, translation initiation element truncated fragment-II, protein coding element, insertion element, translation initiation element truncated fragment-I, intron fragment I.

[0086] Table 1 Vector DNA design and construction

[0087]

[0088] Among them, intron fragment II (numbered A-II), translation initiation element truncated fragment II (numbered EVE-II), insertion element (A-polyAC), translation initiation element truncated fragment I (numbered EVE-I), intron fragment I (numbered AI) are designed with reference to patent CN 114574483 A, intron fragment II (numbered B-II), intron fragment I (numbered BI), translation initiation element truncated fragment II (numbered PPV-II), translation initiation element truncated fragment I (numbered PPV-I), and insertion element (B-polyAC) are obtained through design optimization screening, and the specific information is as follows:

[0089] The nucleotide sequence of intron fragment II (numbered A-II) is shown in SEQ ID NO.1;

[0090] The nucleotide sequence of the translation initiation element truncation fragment II (numbered as EVE-II) is shown in SEQ ID NO.2;

[0091] The protein coding element comprises: a nucleotide sequence (as shown in SEQ ID NO.4) encoding the VP1 wild-type protein of foot-and-mouth disease virus (numbered VP1-1501, the amino acid sequence is shown in SEQ ID NO.3), and a stop codon (as shown in (TAA)x(TAG)y(TGA)z, wherein x+y+z≥1, x, y, z are independently integers greater than or equal to 0, and the stop codon is preferably TGA);

[0092] The nucleotide sequence of the insertion element (A-polyAC) is shown in SEQ ID NO.5;

[0093] The nucleotide sequence of the translation initiation element truncation fragment I (numbered as EVE-I) is shown in SEQ ID NO.6;

[0094] The nucleotide sequence of intron fragment I (numbered as AI) is shown in SEQ ID NO.7.

[0095] The nucleotide sequence of intron fragment II (numbered B-II) is shown in SEQ ID NO.8;

[0096] The nucleotide sequence of intron fragment I (numbered BI) is shown in SEQ ID NO.9;

[0097] The nucleotide sequence of the translation initiation element truncated fragment II (numbered as PPV-II) is shown in SEQ ID NO.10;

[0098] The nucleotide sequence of the translation initiation element truncation fragment I (numbered as PPV-I) is shown in SEQ ID NO.11;

[0099] The nucleotide sequence of the insertion element (B-polyAC) is shown in SEQ ID NO.12;

[0100] The protein coding element includes: an optimized nucleotide sequence (as shown in SEQ ID NO.14) encoding a truncated plus mutant protein of VP1 of foot-and-mouth disease virus (29 amino acids truncated at the N-terminus and containing mutations L61P and K202Q, numbered VP1-5147, and the amino acid sequence is shown in SEQ ID NO.13), and a stop codon (as shown in (TAA)x(TAG)y(TGA)z, wherein x+y+z≥1, x, y, z are independently integers greater than or equal to 0, and the preferred stop codon is TGA); the above gene was commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis and cloning. The obtained gene fragment was connected to the BsaI restriction enzyme site of the pUC57 vector and verified to be correct by gene sequencing.

[0101] In the subsequent steps, the above vectors are transcribed to obtain linear RNA molecules with corresponding structures. The mechanism of forming circular RNA from linear RNA molecules is as follows: ribozyme recognizes the connection position between translation initiation element truncated fragment I and intron fragment I to first generate a break, releasing intron fragment I; then ribozyme recognizes the connection position between translation initiation element truncated fragment II and intron fragment II to generate a break, releasing intron fragment II. The 3' end of translation initiation element truncated fragment I and the 5' end of translation initiation element truncated fragment II are connected to form a circular molecule.

[0102] Example 2: Construction of recombinant bacteria

[0103] 2.1 Construction of original seeds. The above-mentioned vector DNA molecules pEVE-VP1-1501, pPPV-VP1-5147, and pPPV2-VP1-5147 were transformed into E. coli DH5α competent cells, respectively, and spread on LB medium plates containing corresponding antibiotics (100 μg / ml ampicillin), and cultured at 37°C. When the colonies on the plates were clearly visible, single colonies containing high-copy plasmids were picked and placed in 3 ml liquid LB medium containing corresponding antibiotics (100 μg / ml ampicillin), cultured at 37°C, and 1 ml of bacterial liquid was added to a final concentration of 8% glycerol, and stored at -80°C to obtain recombinant engineered bacteria (numbered b-pEVE-VP1-1501, b-pPPV-VP1-5147, and b-pPPV2-VP1-5147, respectively) as original seed banks.

[0104] 2.2 Identification of original seeds.

[0105] (1) Morphological and biochemical characteristics: This engineered bacterium is a Gram-negative short rod. It can ferment and decompose glucose to produce acid and gas. The indole test and methyl red test are both positive; the Voges-Proskauer test (VP test) and citrate test are both negative.

[0106] (2) Culture characteristics: It grows on LB solid medium to form round, shiny, smooth, milky white colonies with neat edges.

[0107] (3) Pure test: The test is carried out according to the pure test method in the appendix of the "Pharmacopoeia of the People's Republic of China", and the results are pure.

[0108] Example 3: Fermentation of recombinant bacteria

[0109] The b-pEVE-VP1-1501, b-pPPV-VP1-5147, and b-pPPV2-VP1-5147 strains were inoculated into 500 mL of LB medium containing the corresponding antibiotics (100 μg / ml ampicillin), and cultured under shaking at 28-32°C until the OD600 value was about 2.0-4.0. The seed liquid was inoculated into a 5L fermentation tank at an inoculation amount of 0.5-3% for fermentation culture. When the OD600 value of the bacteria reached about 80-100, the bacteria were collected by centrifugation with a wet weight of about 1000 g.

[0110] Example 4 Plasmid DNA preparation and testing

[0111] 4.1 Bacterial lysis

[0112] (1) Take 200 g of the E. coli fermented cells obtained in Example 3, add 1.6 L of solution I stored at 4° C. in a mass volume ratio of 1:8 between the cells and solution I (50 mM Tris-Hl, 10 mM EDTA, pH 8.0), and stir until dissolved.

[0113] (2) After dissolution, add 1.6 L of solution II (0.2 M NaOH, 1.0% SDS), stir slowly, and then let stand to react for 3-10 minutes.

[0114] (3) After the reaction is completed, add 1.6 L of solution III (3 M KAC, pH 5.5, pre-cooled at 4°C), shake well, and let it stand for reaction for 30 min.

[0115] (4) After the reaction is completed, add 1.6 L of 2M CaCl2 solution, shake gently, and let it stand for 1 h. The cleavage solution is divided into two layers, the upper layer is solid and the lower layer is liquid. The liquid is discharged with a peristaltic pump and filtered through a filter element with a pore size of 1.0 μm to remove impurities.

[0116] 4.2 Ultrafiltration concentration

[0117] The lysate was filtered through a Quickstand ultrafiltration system using a 300KD hollow fiber ultrafiltration membrane (GE Healthcare Life Science, USA, membrane area 850 cm 2 , membrane fiber tube diameter 1mm) filtration, membrane negative pressure (TMP) is 1psi, operation time is 1.5h, and concentration multiple is 5-10 times volume.

[0118] 4.3 Ion exchange chromatography

[0119] Plasmid was purified by AKTA purifier TM 100 purification system, Capto TMDEAE (GE Healthcare Life Science, USA) was used to purify the plasmid. Before purification, 5 column volumes were equilibrated with a cleaning solution (0.55 M NaCl, 50 Mm Tris-HCL, 10 mm EDTA, pH 8.0). After loading the sample, the column was cleaned with a cleaning solution (0.55 M NaCl, 50 Mm Tris-HCL, 10 mm EDTA, pH 8.0); then the column was gradient eluted with an eluent (0.65 M NaCl, 50 Mm Tris-HCl, 10 Mm EDTA, pH 8.0) solution, and the eluted sample was collected; then the column was cleaned and regenerated with CIP (2 M NaCl, 0.5 M NaOH), and then equilibrated with a balancing solution.

[0120] 4.4 Gel filtration chromatography

[0121] First, Capto TM Core 700 (GE Healthcare Life Science, USA) column was equilibrated with PBS (NaCl 8.5 g / L, Na2HPO4·12H2O 2.9 g / L, KH2PO4 0.25 g / L, pH 7.4) for 3 column volumes, and the sample collected by ion exchange column purification was loaded onto Capto TM core 700, after the sample loading is completed, use PBS to elute and collect the eluted sample.

[0122] 4.5 Ultrafiltration and Diafiltration

[0123] The cells were separated by a Quickstand ultrafiltration system using a 100 KD hollow fiber ultrafiltration membrane (GE Healthcare LifeScience, USA, membrane area 500 cm 2 , membrane fiber tube diameter 1mm) concentration and diafiltration, the membrane negative pressure (TMP) is 1psi, the concentration multiple is 5-10 times the volume, and the diafiltration multiple is 4-12 times.

[0124] 4.6 Plasmid DNA yield and yield / impurity removal effect

[0125] For plasmid DNA (pPPV2-VP1-5147), according to the "Technical Guidelines for Preclinical Research of DNA Vaccines for Prevention", the plasmid DNA concentration was determined by spectrophotometry, the bacterial endotoxin residue was detected by the horseshoe crab reagent gel method, the RNA residue was detected by agarose gel electrophoresis, and the protein residue was tested by BCA method to confirm the process.

[0126] By optimizing the culture medium and culture conditions, the Escherichia coli DH5α strain was expanded to obtain bacterial cells. The lysis method was used to remove large molecular RNA and protein impurities by adding calcium chloride to obtain a crude plasmid DNA extract sample. Ultrafiltration, chromatography, etc. can be used to further remove impurities.

[0127] The yield of plasmid DNA produced at each stage of this process and the preliminary quality inspection results are shown in Table 2. Using the optimized process, for the pPPV2-VP1-5147 plasmid, the lysis, ultrafiltration concentration, ion exchange chromatography, gel filtration chromatography, and ultrafiltration diafiltration yields reached 79%, 91%, 87%, 93%, and 85%, respectively, and the total yield reached 49.7%. The production process met the requirements.

[0128] Table 2 Plasmid DNA yield (pPPV2-VP1-5147)

[0129]

[0130] 4.7 Plasmid testing

[0131] The plasmids (pEVE-VP1-1501, pPPV-VP1-5147, pPPV2-VP1-5147) were tested according to Analytical Procedures for mRNA Vaccine Quality 2rd USP, and all indicators met the requirements.

[0132] (1) Identification: The plasmid was sequenced and analyzed, and the sequence was consistent with the target sequence.

[0133] (2) Concentration analysis: The concentration of the plasmid was measured by UV spectrophotometer. The results are shown in Table 3.

[0134] (3) Purity analysis: A260 / A280, supercoil content, and impurity residues were analyzed according to the methods specified by regulations. A260 / A280 was between 1.8 and 2.0, supercoil content was not less than 90%, host RNA residue was less than 100 pg / ml, host DNA was less than 1 ng / ml, host protein residue was less than 100 pg / ml, and antibiotic residual activity was negative.

[0135] (4) Safety: Endotoxin test was performed according to the method specified by the regulations, and the endotoxin content was less than 10EU / mg.

[0136] (5) Others: Analyze appearance and pH according to the methods specified by regulations. The appearance inspection should be free of foreign matter and the pH value should be between 6.5 and 7.5.

[0137] Table 3 DNA plasmid yield and purity

[0138]

[0139] Example 5: Preparation and testing of circular RNA

[0140] 5.1 CircRNA Preparation

[0141] (1) Plasmid digestion and purification: The plasmid was digested by restriction endonuclease Bsa I (nearshore protein) / Bsa I (Takara), and the digestion system was as follows: 10× buffer: 2 ml, plasmid: 100 mg, Bsa I: 3 ml, water was added to 20 ml, and digestion was carried out at 37°C overnight. After digestion, Capto Q ImpRes (Source 30Q) (Cytiva) column was used for chromatography purification to obtain linearized plasmid.

[0142] (2) In vitro transcription: The transcription system is configured as shown in Table 1 below:

[0143] Table 4

[0144]

[0145]

[0146] Reaction conditions: Constant temperature shaking reaction at 20-40°C / 220rpm for 1-4h, then add 100ml of deoxyribonuclease I (DNaseI) (1000U / ml) to the transcription system, and constant temperature shaking reaction at 37°C / 220rpm for 15min.

[0147] It is difficult to obtain stable and complete RNA molecules using conventional in vitro transcription reaction systems for long linear RNA. In this study, the following conditions were optimized one by one for plasmid pPPV2-VP1-5147 to obtain the optimal transcription conditions:

[0148] 1) Optimization of linearized plasmid template concentration: The final concentrations of the plasmid template in the system were set to 25 mg, 50 mg, 75 mg, and 100 mg respectively.

[0149] 2) Optimization of nucleotide concentration: Set the GTP / ATP / CTP / N concentration in the system in sequence. 1 The final concentrations of Ψ were 8mM / 8mM / 8mM / 8mM, 32mM / 8mM / 8mM / 8mM, 16mM / 32mM / 8mM / 8mM, and 16mM / 16mM / 8mM / 8mM respectively.

[0150] 3) Optimization of T7 RNA polymerase (RNA Polymerase) concentration: The concentrations of T7 RNA polymerase (RNA Polymerase) in the system were set to 750KU, 900KU, 1000kU, and 1250kU respectively.

[0151] 4) Optimization of enzymatic reaction temperature: The enzymatic reaction temperatures were set to 20°C, 30°C, 37°C, and 40°C, respectively.

[0152] 5) Optimization of enzymatic reaction duration: The enzymatic reaction duration was set to 1 h, 2 h, 3 h, and 4 h respectively.

[0153] The RNA content was determined by UV spectrophotometry, and the integrity of the RNA was analyzed by capillary electrophoresis.

[0154] The above conditions have a great influence on the results of RNA in vitro transcription. Without optimization of the conditions, the RNA content obtained by in vitro transcription of plasmid pPPV2-VP1-5147 is only 0.6 g / 1000 ml reaction, which contains a lot of incomplete RNA.

[0155] The optimal in vitro transcription conditions were obtained through experimental screening: the linearized plasmid template concentration was 50 mg, GTP / ATP / CTP / N 1 The final concentration of Ψ is 16mM / 16mM / 8mM / 8mM, the final concentration of T7 RNA polymerase is 900KU, the enzymatic reaction temperature is 37°C, and the enzymatic reaction time is 3h. Under these conditions, the RNA content obtained by in vitro transcription of plasmid pPPV2-VP1-5147 can be increased to 3.4g / 1000ml reaction, and the production process meets the requirements.

[0156] (3) Precipitation of mRNA of the transcription product: Add LiCl solution to the above transcription product and place it at -20°C for overnight precipitation; centrifuge the overnight treatment solution to obtain mRNA precipitate, dry it at room temperature, and resuspend the precipitate in enzyme-free water to obtain a linear mRNA aqueous solution.

[0157] (4) mRNA cyclization and concentration: The cyclization system and conditions are shown in Table 5 below:

[0158] Table 5

[0159]

[0160]

[0161] The above solutions are mixed thoroughly and heated at 48-60°C for 8-20 minutes. The circularized mRNA product is concentrated by ultrafiltration tube centrifugation, and the filtrate is discarded; and the ultrafiltration liquid is transferred to a new centrifuge tube.

[0162] It is difficult to obtain stable and complete circular RNA molecules using conventional reaction systems. In this study, the following conditions were optimized one by one for plasmid pPPV2-VP1-5147 to obtain the optimal circularization conditions:

[0163] 1) Optimization of linear mRNA concentration: The final concentrations of linear mRNA in the system were set to 3 g, 3.5 g, and 4 g, respectively.

[0164] 2) Optimization of magnesium ion concentration: set the final magnesium ion concentration in the system to 5mM, 8mM, 10mM, 12mM, and 15mM, respectively.

[0165] 3) Optimization of cyclization reaction temperature: the reaction temperatures were set to 48°C, 52°C, 55°C, 58°C, and 62°C, respectively.

[0166] 4) Optimization of cyclization reaction time: set the reaction time to 8 min, 10 min, 12 min, 15 min, and 20 min respectively.

[0167] The above conditions have a great influence on the results of RNA cyclization, and complete circular RNA cannot be prepared without optimization. The optimal cyclization conditions were obtained through experimental screening: the linear mRNA concentration was 3.5g, the magnesium ion concentration was 8mM, the cyclization reaction temperature was 52℃, and the cyclization reaction time was 12min. Under these conditions, the content of circular RNA obtained by in vitro transcription and cyclization of plasmid pPPV2-VP1-5147 can reach 3.1g / 1000ml reaction, and the production process meets the requirements.

[0168] (5) Circular mRNA HPLC purification: A certain amount of circular mRNA was taken and purified by Capto TM Core 700 (GE Healthcare Life Science, USA) column was used for separation, and the main peak components were collected.

[0169] (6) Concentration of purified circular mRNA: The circular mRNA tube was concentrated to the desired concentration using a TFF capsule (LV Centramate, PALL). The membrane area of ​​the TFF capsule was 0.02 m 2 , flow rate is 120mL / min, and the ultrafiltered liquid is transferred to a new sample bottle.

[0170] Through the above process, a circular mRNA with the following sequence structure was successfully prepared:

[0171] The circular mRNA prepared from DNA plasmid pEVE-VP1-1501 was named EVE-VP1-1501, and the sequence is shown in SEQ ID NO.15.

[0172] The circular mRNA prepared from DNA plasmid pPPV-VP1-5147 was named PPV-VP1-5147, and its sequence is shown in SEQ ID NO.16.

[0173] The circular mRNA prepared from DNA plasmid pPPV2-VP1-5147 was named PPV2-VP1-5147, and the sequence is shown in SEQ ID NO.17.

[0174] 5.2 mRNA testing

[0175] Circular mRNA was tested according to Analytical Procedures for mRNA Vaccine Quality 2rd USP, and all indicators met the requirements.

[0176] (1) Identification: Sequence the mRNA and the sequence is consistent with the target sequence.

[0177] (2) Concentration analysis: The concentration of mRNA was measured by UV spectrophotometer. The results are shown in Table 6.

[0178] (3) Integrity analysis: The integrity of mRNA was determined by capillary gel electrophoresis (CGE), which was no less than 80%. The results are shown in Table 6.

[0179] (4) Purity analysis: The impurity residues were analyzed according to the prescribed method. The dsRNA content was less than 0.5%, the DNA template residue was less than 100 pg / mg RNA, the free nucleotide residue was less than 100 pg / mg RNA, and the T7 RNA polymerase residue was less than 50 ppm.

[0180] (5) Efficacy analysis: The cells were transfected according to the prescribed method, and the target protein expression was detected to meet the requirements. 48 hours after transfection, the target protein expression level of the PPV2-VP1-5147 group reached 107.4 ng / ml.

[0181] (6) Safety: Endotoxin was analyzed according to the prescribed method and the endotoxin content was less than 0.1 EU / ml.

[0182] (7) Others: Analyze the appearance and pH value according to the prescribed methods. The appearance inspection should be free of foreign matter and the pH value should be between 6.5 and 7.5.

[0183] Compared with the PPV-VP1-5147 group, the RNA yield of the PPV2-VP1-5147 group increased by 2.1 times, indicating that the optimization of the non-coding region is effective and necessary, and the yield of the circular RNA molecule was successfully increased. Therefore, the circular RNA molecule (numbered PPV2-VP1-5147), the plasmid DNA molecule (numbered pPPV2-VP1-5147) corresponding to the PPV2-VP1-5147 group, and the Escherichia coli strain containing the plasmid DNA molecule (numbered b-PPV2-VP1-5147) are preferred.

[0184] Table 6 CircRNA Molecular Yield and Purity

[0185]

[0186] Example 6: Comparison of the immune effects of various circular RNA molecules

[0187] 6.1 Commercial LNP Preparation

[0188] The circular RNA molecules (numbered EVE-VP1-1501, PPV2-VP1-5147) obtained in Example 5 and the control circular RNA molecules (numbered HM-VP1-5147) were prepared into RNA encapsulated by LNP using commercial LNPs to provide preparation for further animal experiments. The control circular RNA (numbered HM-VP1-5147) was prepared by using a commercially available circular RNA cloning and construction kit (Shanghai Kewei Chuang) to prepare the optimized nucleotide sequence (as shown in SEQ ID NO.14) of the truncated mutant protein of foot-and-mouth disease virus VP1 (29 amino acids truncated at the N-terminus and containing mutations L61P and K202Q, the amino acid sequence is shown in SEQ ID NO.13), and the specific steps are referred to the instructions. The method for preparing commercial LNP into RNA encapsulated by LNP is as follows:

[0189] (1) Test method

[0190] The commercial LNP-mRNA preparation reference is 10.1016 / j.cell.2022.03.044, and the specific process is as follows:

[0191] a) Dilute the circular RNA to a final concentration of 170 μg / mL using PNI formulation buffer (Precision NanoSystems, Cat. No. NWW0043).

[0192] b) On a NanoAssembler Ignite (Precision NanoSystems), commercial LNP (Precision NanoSystems) and circular RNA were mixed at a volume ratio of 1:3 using Ignite NxGenCartridge (Precision NanoSystems, Catalog No. NIT0002).

[0193] c) The buffer environment was replaced with PBS at pH 7.4 using tangential flow technology to remove ethanol, and LNP-mRNA was prepared, which were numbered as c-LNP / EVE-VP1-1501, c-LNP / PPV2-VP1-5147, and c-LNP / HM-VP1-5147, respectively.

[0194] d) Using Quant-iT TMRiboGreen TM RNA Assay Kit (Invitrogen TM The particle size, PDI and surface potential of LNP particles were measured by dynamic light scattering method on a Zeta potential laser particle size analyzer Malvern Zetasizer Nano-ZEN 3600 (Malvern).

[0195] (2) Test results

[0196] The mRNA-encapsulated LNPs prepared in this step passed the inspection: the particle size was small and uniformly distributed, the encapsulation effect was good, and the encapsulation rate was greater than 90%; the surface potential was electronegative, avoiding the cytotoxicity caused by the positive charge on the surface of the nanoparticles, and the biosafety was good.

[0197] 6.2 Mouse immunization and O-type antibody determination

[0198] (1) Experimental methods

[0199] Female C57BL / 6 mice aged 6 to 8 weeks were randomly divided into 4 groups, 10 mice in each group, and the three kinds of circular mRNAs (numbered c-LNP / EVE-VP1-1501, c-LNP / PPV2-VP1-5147, c-LNP / HM-VP1-5147) encapsulated by the commercial LNP prepared in this example were used for mouse immunization, and the inoculation dose was 30 μg / mouse. The mice inoculated with commercial LNP were negative controls, and the immunization was strengthened once on the 21st day after the initial immunization. Blood was collected 14 days after the second immunization to separate serum, and antibody detection was performed using a liquid phase blocking ELISA detection kit for foot-and-mouth disease O type antibody purchased from Lanzhou Veterinary Research Institute. At the same time, the antibody titer of each group against VP1 was detected using a commercial foot-and-mouth disease virus VP1 structural protein antibody enzyme-linked immunosorbent assay diagnostic kit (Shenlian Biopharmaceutical (Shanghai) Co., Ltd.), and the antibody titer was expressed as mean ± SD value.

[0200] (2) Experimental results

[0201] The results are shown in Table 7. The virus blocking antibody titer of the c-LNP / PPV2-VP1-5147 group was 8.3 times that of the c-LNP / EVE-VP1-1501, while the VP1 total antibody titer was significantly reduced, indicating that sequence optimization effectively reduced the consumption of immune resources mediated by immunodominant epitopes and successfully improved the effective antibody level. Furthermore, the circular RNA molecular skeleton provided in this study is superior to the commercial circular RNA molecular skeleton: the virus blocking antibody titer of the c-LNP / PPV2-VP1-5147 group was 1.4 times that of the c-LNP / HM-VP1-5147 group.

[0202] Table 7 VP1 antibody titers and virus blocking antibody titers induced in mice

[0203]

[0204] Example 7: LNP-mRNA production process research

[0205] This section studies the production process of LNP-encapsulated mRNA.

[0206] 7.1 Preparation of LNP-mRNA (1) Preparation of solution I: Dissolve the cationic lipid, cholesterol, neutral lipid, and PEG-modified lipid in ethanol to obtain solution I. (2) Preparation of solution II: Dissolve the preferred circular RNA (No. PPV2-VP1-5147) of Example 6 in 10 mM citric acid buffered saline solution at pH 4.0.

[0207] (3) Using microfluidics technology to quickly mix the two phases and using tangential flow technology to replace the buffer environment with PBS at pH 7.4 to remove ethanol, LNP-mRNA was prepared. In this section, the liposome preparation and vaccine preparation processes were optimized, specifically involving liposome components and the ratio of each component, and the ratio of LNP to mRNA, as follows:

[0208] 3.1) Screening of liposome components and ratio conditions: Solution I was prepared according to different components and different ratios, including cationic lipids (CMAX4 (4-[(3-{[3-({3-[bis(3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxypropyl)amino]propyl}(methyl)amino)propyl](3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxypropyl)amino}propoxy)oxy]butyl 2-octanoate) or SM-102 (1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate)) was set to 45% to 55%, cholesterol (5-cholesten-3β-ol) was set to 35% to 44%, and neutral lipids ( DSPC (distearylphosphatidylcholine) or DOPE (dioleoylphosphatidylethanolamine) was set to 3%-10%, and PEG-modified lipids (PEG-DMG (polyethylene glycol-dimyristyl glyceride) or PEG-DSG (bis-distearoyl-rac-glycerol-polyethylene glycol) or PEG-DPG (diphosphoglycerate polyethylene glycol))) were set to 0.8%-1.8%, wherein the cationic lipid CMAX4 was purchased from Suzhou Coremade Biopharmaceutical Technology Co., Ltd., and the remaining lipids were purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd., and LNP-encapsulated circular RAN molecules were prepared with a volume ratio of LNP / circular RNA (No. PPV2-VP1-5147) of 1:1, and the particle size and encapsulation efficiency of each group were compared.

[0209] The results showed that the composition and ratio of liposomes had a great influence on the results: for example, the combination of CMAX4 / cholesterol / DOPE / PEG-DPG at a ratio of 54.5:34.5:9.2:1.8 was ineffective, with an encapsulation efficiency of less than 10%.

[0210] Through experiments, the preferred liposome components were obtained to be CMAX4 / cholesterol / DSPC / PEG-DMG, and the preferred ratio of each component was 45.9:42.6:9.2:1.5. At this time, the molecular particle size was the smallest (65nm), the encapsulation rate reached 94%, and the production process met the requirements.

[0211] 3.2) Optimization of LNP / mRNA ratio: LNP and circular RNA (number PPV2-VP1-5147) were mixed in different proportions (volume ratio of solution I:II 1:1, 1:2, 1:3, 1:4, 1:5, 1:6), and the optimal LNP / mRNA ratio was verified by gel retardation test. The results showed that when the volume ratio of liposome nanomaterial (LNP) and circular mRNA molecule (number PPV2-VP1-5147) was greater than 1:3, the migration of PPV2-VP1-5147 could be completely blocked. Therefore, the volume ratio of LNP / circular RNA molecule was set to 1:3.

[0212] (4) The circular RNA molecule (numbered PPV2-VP1-5147) is encapsulated with LNP according to the above preferred conditions, and sterile filtered to obtain an RNA vaccine, numbered LNP / PPV2-VP1-5147.

[0213] (5) Using Quant-iT TM RiboGreen TM RNA Assay Kit (Invitrogen TM The encapsulation efficiency was determined by R11490, and the particle size, polydispersity index (PDI) and surface potential of the LNP particles were measured by dynamic light scattering on a Zeta potential-laser particle size analyzer Malvern Zetasizer Nano-ZEN 3600 (Malvern).

[0214] 7.2 Experimental Results

[0215] The results are shown in Table 8. The indicators of LNP-encapsulated mRNA were qualified: the particle size was less than 90nm, the PDI was less than 0.15, the encapsulation rate was greater than 90%, and the surface potential was electronegative. The overall particle size was small and uniformly distributed. The surface electronegativity avoided the cytotoxicity caused by the positive charge on the surface of the nanoparticles, and the biosafety was good, providing a feasibility basis for the large-scale preparation of vaccines.

[0216] Table 8 LNP-mRNA sample characterization

[0217] serial number Particle size (nm) PDI Surface potential (mV) Encapsulation rate (%) LNP / PPV2-VP1-5147 65 0.11 -6.0 94

[0218] Example 8 Evaluation of the immune effect of LNP-encapsulated RNA

[0219] For the vaccine prepared with the preferred LNP in Example 7, the immune effect was evaluated and the immune dose was determined

[0220] 8.1 Mouse immunization and O-type antibody determination

[0221] (1) Experimental methods

[0222] Female C57BL / 6 mice aged 6 to 8 weeks were randomly divided into 3 groups, 10 mice in each group, and the LNP / PPV2-VP1-5147 prepared with the preferred LNP in this example and the c-LNP / PPV2-VP1-5147 prepared with the commercial LNP in Example 6 were used for immunization of mice, with an inoculation dose of 30 μg / mouse. The mice inoculated with the commercial LNP were used as negative controls, and booster immunization was performed once on the 21st day after the initial immunization. Blood was collected 14 days after the second immunization to separate serum, and antibody detection was performed using a liquid phase blocking ELISA detection kit for foot-and-mouth disease type O antibodies purchased from Lanzhou Veterinary Research Institute, and the antibody titer was expressed as mean ± SD value.

[0223] (2) Experimental results

[0224] The results are shown in Table 9. The LNP prepared by the optimal process was superior to the commercial LNP and successfully further improved the virus blocking antibody titer in mice: the virus blocking antibody titer of the LNP / PPV2-VP1-5147 group was 1.3 times that of the c-LNP / PPV2-VP1-5147 group, providing a feasibility basis for further research.

[0225] Table 9 Induction of virus blocking antibody titers in mice

[0226] Group Mean SD value LNP / PPV2-VP1-5147 1059.2 356.2 c-LNP / PPV2-VP1-5147 842.1 103.1 LNP none none

[0227] 8.2 Pig Immunization and Vaccine Immunization Dosage Determination

[0228] (1) Test method

[0229] The screening criteria for experimental pigs are shown in Table 10 below, and a total of 28 piglets were screened.

[0230] Table 10 Screening criteria for experimental pigs

[0231]

[0232] The experimental animals were divided into 4 groups, with 7 animals in each group.

[0233] Experimental Group 1: LNP / PPV2-VP1-5147 prepared in Example 7 was inoculated into pigs (neck muscle), with an immunization dose of 60 μg / head, numbered PPV2-VP1-5147-60 μg.

[0234] Experimental Group 2: LNP / PPV2-VP1-5147 prepared in Example 7 was inoculated into pigs (neck muscle), with an immunization dose of 80 μg / head, numbered PPV2-VP1-5147-80 μg.

[0235] Experimental Group 3: LNP / PPV2-VP1-5147 prepared in Example 7 was inoculated into pigs (neck muscle), with an immunization dose of 100 μg / head, numbered PPV2-VP1-5147-100 μg.

[0236] Control group 1: immunized with PBS only, with an immunization dose of 1 ml / head, numbered PBS group.

[0237] The animals were boosted with a booster immunization on the 28th day after the first immunization. Serum was collected 21 days after the second immunization to prepare serum, and antibody detection was performed using a liquid-phase blocking ELISA test kit for foot-and-mouth disease type O antibodies purchased from Lanzhou Veterinary Research Institute.

[0238] Furthermore, 14 days after the second immunization, blood from the preferred group was collected to prepare serum, and the IFN-γ and IL-4 levels in the serum of each group were detected using the MILLIPLEX MAPPorcine Cytokine and Chemokine Magnetic Bead Panel-Immunology Multiplex Assay Kit (MERCK, Catalog No. PCYTMAG-23K-09) to preliminarily evaluate the cellular immune response. The specific operation was carried out according to the kit instructions.

[0239] (2) Test results

[0240] The results of virus blocking antibodies in pigs are shown in Table 11. No antibodies were produced in the PBS group, while the PPV2-VP1-5147 group was able to effectively induce high levels of virus blocking antibodies in pigs using low-dose immunization (80 μg), indicating that the vaccine is highly effective.

[0241] The levels of various cytokines in pig blood are shown in Table 12. Compared with the PBS group, the PPV2-VP1-5147 group can effectively increase the levels of IFN-γ and IL-4 in pig serum, suggesting that the cellular immune response is effectively activated, further improving the protective effect of the vaccine, and providing a basis for further research.

[0242] Table 11 Induced virus blocking antibody titer in pigs / Log 10 (Potency)

[0243]

[0244] Table 12 IFN-γ and IL-4 content in pig serum (mean ± SD value) (pg / ml)

[0245] Group IFN-γ IL-4 PPV2-VP1-5147-60μg 5352.9±1024.9 909.7±564.2 PPV2-VP1-5147-80 μg 13269.7±5425.1 1568.3±912.2 PPV2-VP1-5147-100 μg 15698.1±8332.9 1786.4±845.1 PBS 184.3±85.3 69.4±36.4

[0246] Example 9 Preparation of control linear RNA vaccine

[0247] To further compare the effects of circular RNA and linear RNA, this study used a kit to prepare linear RNA encoding foot-and-mouth disease virus (number linear-VP1-1051), which encodes the wild-type protein of foot-and-mouth disease virus VP1 (amino acid sequence see SEQ ID NO.3, nucleotide sequence see SEQ ID NO.4). The specific methods and kits mainly refer to the research paper (Development of O-type foot-and-mouth disease mRNA vaccine [D], 2022).

[0248] The control linear mRNA prepared above was prepared into a lipid nanoparticle / RNA composition, recorded as linear-VP1-1501 group. The specific operation was the same as the preferred conditions of Example 7, and it passed the inspection: the LNP particle size encapsulated mRNA was small and uniformly distributed, the encapsulation effect was good, and the encapsulation rate was greater than 90%; the surface potential was electronegative, and the biosafety was good.

[0249] Example 10: Study on pig immunity and vaccine effectiveness

[0250] 10.1 Experimental Animals.

[0251] The screening criteria for experimental pigs were the same as in Example 8, and a total of 28 piglets were screened.

[0252] 10.2 Immunity experiments.

[0253] The experimental animals were divided into 4 groups, with 7 animals in each group.

[0254] Group 1: Pigs (neck muscle) were inoculated with LNP / PPV2-VP1-5147 prepared in Example 7, with an immunization dose of 80 μg / head.

[0255] Group 2: Pigs (neck muscle) were inoculated with LNP / linear-VP1-1501 prepared in Example 10, with an immunization dose of 80 μg / head.

[0256] Group 3: A certain brand of commercially available O-type FMDV whole-virus inactivated vaccine, with an immunization dose of 2 ml.

[0257] Group 4: immunized with PBS only, with an immunization dose of 1 ml / head, numbered PBS group.

[0258] A booster vaccination was given on the 28th day after the initial vaccination.

[0259] 10.3 Virus blocking antibody detection

[0260] The serum was collected and prepared at 7 days, 28 days and 180 days respectively, and the antibody detection was carried out using the foot-and-mouth disease O type antibody liquid phase blocking ELISA detection kit purchased from Lanzhou Veterinary Research Institute.

[0261] The results are shown in Tables 13 to 15. The PBS group did not produce specific virus-blocking antibodies, while the PPV2-VP1-5147 group was superior to the linear RNA molecule (numbered linear-VP1-1051) in terms of antibody response speed, antibody titer, and antibody duration, reaching a level similar to that of the inactivated vaccine group: high levels of virus-blocking antibodies were rapidly produced 7 days after the second immunization and lasted until 180 days after the second immunization. The design and optimization of circular RNA vaccines around foot-and-mouth disease virus successfully improved the protective effect of the vaccine and provided a basis for the preparation of foot-and-mouth disease RNA vaccines.

[0262] Table 13 Induced virus blocking antibody titer in pigs (7 days after the second immunization) / Log 10 (Potency)Log 10 (Titer) reaching 1.8 or above is considered as immune standard

[0263]

[0264] Table 14 Induced virus blocking antibody titer in pigs (28 days after the second immunization) / Log 10 (Potency)Log 10 (Titer) reaching 1.8 or above is considered as immune standard

[0265]

[0266]

[0267] Table 15 Induced virus blocking antibody titer in pigs (180 days after the second immunization) / Log 10 (Potency)Log 10 (Titer) reaching 1.8 or above is considered as immune standard

[0268]

[0269] 10.4 Cellular Immune Response Detection

[0270] The cellular immune response stimulated by each vaccine group in the pigs was further detected. Blood PBMC cells were collected from each group immunized in this example 14 days after the second immunization, and the corresponding lymphocyte subtypes were detected by flow cytometry. The specific operation is as follows:

[0271] (1) 14 days after the second immunization, anticoagulated blood was collected from each group and single cell suspensions were prepared;

[0272] (2) Cell density adjustment and density counting: After filtering the above single cell suspension through a 100 μm cell sieve, take 18 μL and add it to a 200 μL EP tube, add 2 μL of trypan blue and mix well, then take 10 μL and add it to a counting plate for cell counting, and dilute it with an appropriate amount of PBS solution until the density of the single cell suspension is 1×10 6 / mL; observe the number of dead cells and living cells under a microscope. Antibody incubation can be carried out only after the cell density and cell viability of the single cell suspension meet the requirements.

[0273] (3) Antibody incubation:

[0274] Prepare single-stained tubes: take 0.5 mL of each group of single-cell suspensions into 3 1.5 mL EP tubes, add 1 μL of fluorescein isothiocyanate FITC-labeled mouse anti-swine CD3 antibody (BD Company), 1 μL of phycoerythrin PE-labeled mouse anti-swine CD4 antibody (BD Company), and 1 μL of allophycocyanin fluorescein APC-labeled mouse anti-swine CD8 antibody (BD Company), incubate at 4°C in the dark for 30 min, then centrifuge at 1000 r / min for 5 min, remove the supernatant, resuspend with 0.5 mL PBS, and place on ice. Add 2.5 μL 7-aminoactinomycin D (eBioscience Company) to each tube before loading.

[0275] Prepare sample tubes: take 1 mL of each group of single cell suspensions into 1.5 mL EP tubes, and add 2 μL of fluorescein isothiocyanate FITC-labeled mouse anti-swine CD3 antibody (BD Company), 5 μL of phycoerythrin fluorescein PE-labeled mouse anti-swine CD4 antibody (BD Company), and 5 μL of allophycocyanin fluorescein APC-labeled mouse anti-swine CD8 antibody (BD Company). Incubate at 4°C in the dark for 30 min, then centrifuge at 1000 r / min for 5 min, remove the supernatant, resuspend with 1 mL PBS, and place on ice. Add 5 μL of 7-aminoactinomycin D (eBioscience Company) to each tube before loading.

[0276] (4) Sorting on the flow cytometer: Sorting was performed on the flow cytometer (BD Aria SORP, BD). The above tubes were filtered into the flow tubes with a 100 μm cell sieve and marked. First, the blank tube was used to determine the location of lymphocytes, and then the single-stained tube was used to determine whether the antibody labeling was good. If the labeling was good (i.e., the antibody fluorescence signal value was 10 3The samples were placed in the sample tubes, and CD4-CD8- double negative cells, CD4+CD8+ double positive cells, CD4+CD8- single positive cells, and CD4-CD8+ single positive cells were circled and sorted. Unpaired t-test was used for statistical significance analysis, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0277] The results are as follows Figure 1 As shown, the LNP / linear-VP1-1501 group and the inactivated vaccine group can induce an increase in CD4+ cells in pigs, but the CD8+T cell immune response is insufficient (the increase ratio is less than 5% compared with the PBS group); while the proportion of CD8+T cells in the LNP / PPV2-VP1-5147 group is significantly higher than that of the other groups, and the increase of 12% compared with the PBS group indicates that the cellular immune response, especially the CD8+T cell immune response, is successfully activated. According to the literature (Chinese Veterinary Science 2012, 42(10): 1024-1030), the immune protection rate of the vaccine is well correlated with the CD8+T cell immune response, and an increase of ~5% in the CD8+T cell ratio can achieve an immune protection rate increase of more than 60%.

[0278] Example 11 Safety test of newborn piglets

[0279] This experiment further studied the safety of the swine foot-and-mouth disease virus circular RNA / lipid nanoparticle composition in pigs.

[0280] 11.1 Test methods

[0281] Sixteen 0-day-old foot-and-mouth disease-negative piglets (all foot-and-mouth disease virus antibodies were negative) were selected;

[0282] Group 1 is the vaccination group, in which 8 piglets were vaccinated with LNP / PPV2-VP1-5147 prepared in Example 7 (neck muscle), with an immunization dose of 80 μg / pig. ;

[0283] Group 1 was the control group, and 8 piglets were inoculated with sterile PBS at a dose of 1 ml / pig.

[0284] Afterwards, the clinical symptoms of each animal were continuously observed and recorded, body temperature was monitored, and weaning weight was recorded.

[0285] 11.2 Test results

[0286] The results are shown in Table 16. The vaccine is safe. No abnormal body temperature, other obvious side effects or abnormal clinical symptoms were observed within 14 days after vaccination. The weight of the LNP / PPV2-VP1-5147 group was not significantly different from that of the control group when weaned, indicating that the vaccine provided by the present invention has good safety in clinical application.

[0287] Table 16 Body temperature and weight of piglets after vaccination (mean ± SD value)

[0288]

[0289]

[0290] Example 12 Vaccine stability study

[0291] The stability of the vaccine was further evaluated as follows:

[0292] The LNP / PPV2-VP1-5147 prepared in Example 7 was aseptically dispensed into vials (5 ml / vial) after filtration and sterilization, and stored at -20°C. Samples were taken for inspection at 0 day, 1 month, 2 months, 3 months, and 6 months:

[0293] Using Quant-iT TM RiboGreen TM RNA Assay Kit (Invitrogen TM R11490) to determine the mRNA content and encapsulation efficiency. The particle size of LNP particles was measured using a Zeta potential-laser particle size analyzer Malvern Zetasizer Nano-ZEN 3600 (Malvern). The mRNA integrity was determined by capillary gel electrophoresis (CGE). pH and appearance inspection were performed.

[0294] The test results are shown in Table 17. The vaccine is stable when stored at -20°C for 6 months, with no significant changes in encapsulation efficiency, particle size, mRNA content, mRNA integrity, pH, and appearance, providing a basis for large-scale vaccine preparation and clinical application.

[0295] Table 17 Vaccine Storage Stability

[0296] Day 0 1 month 2 months 3 months 6 months Encapsulation rate / % 93.9 93.6 94.1 93.7 94.1 Particle size / nm 65.2 65.1 65.7 65.9 65.7 mRNA content / mg / ml 0.52 0.51 0.51 0.50 0.51 mRNA integrity / % 92.1 92.3 92.1 92.7 92.6 pH 7.12 7.13 7.15 7.11 7.11 Appearance No foreign matter found No foreign matter found No foreign matter found No foreign matter found No foreign matter found

[0297] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A foot-and-mouth disease vaccine, comprising a circular RNA molecule encoding a truncated plus mutant protein of VP1 of a foot-and-mouth disease virus and a pharmaceutically acceptable carrier.

2. The foot-and-mouth disease vaccine according to claim 1, characterized in that The amino acid sequence of the foot-and-mouth disease virus VP1 truncated and mutant protein is shown in SEQ ID No. 13 or its degenerate sequence.

3. The foot-and-mouth disease vaccine according to claim 1, characterized in that The nucleotide sequence of the truncated and mutant protein of foot-and-mouth disease virus VP1 is shown in SEQ ID No. 14 or its degenerate sequence.

4. The foot-and-mouth disease vaccine according to claim 1, characterized in that The nucleotide sequence of the circular RNA molecule is as shown in SEQ ID No.

17.

5. The foot-and-mouth disease vaccine according to claim 1, characterized in that The content of the circular RNA molecule in the foot-and-mouth disease vaccine is 30-200 μg / ml.

6. The foot-and-mouth disease vaccine according to claim 1, characterized in that The pharmaceutically acceptable carrier includes lipid nanoparticles; the volume ratio of lipid nanoparticles to circular RNA molecules in the foot-and-mouth disease vaccine is 1:1-1:

3.

7. The foot-and-mouth disease vaccine according to claim 1, characterized in that The raw material composition of the lipid nanoparticles includes: 45%-55% of cationic lipids, 35%-44% of cholesterol, 3%-10% of neutral lipids and 0.8%-1.8% of PEG-modified lipids; the cationic lipids are CMAX4 or SM-102; the cholesterol is 5-cholesten-3β-ol; the neutral lipids are DSPC or DOPE; the PEG-modified lipids are PEG-DMG, PEG-DSG or PEG-DPG.

8. The foot-and-mouth disease vaccine according to claim 7, characterized in that The cationic lipid is CMAX4; the cholesterol is 5-cholestene-3β-ol; the neutral lipid is DSPC; the PEG-modified lipid is PEG-DMG; the molar ratio of CMAX4:5-cholestene-3β-ol:DSPC:PEG-DMG is 45.9:42.6:9.2:1.

5.

9. A method for preparing a foot-and-mouth disease vaccine according to any one of claims 1 to 8, characterized in that: The method comprises the following steps: S1, synthesizing and inserting the truncated VP1 gene plus mutant protein and looping element of the foot-and-mouth disease virus into a cloning vector to obtain a recombinant vector containing the truncated VP1 gene plus mutant protein and looping element of the foot-and-mouth disease virus; S2, transforming or transducing a host with the recombinant vector containing the truncated VP1 plus mutant protein gene and the looping element in the foot-and-mouth disease virus obtained in step S1 to obtain a recombinant containing the recombinant vector; S3, culturing the recombinant obtained in step S2 to prepare a plasmid containing the truncated VP1 plus mutant protein gene and the circular element in the foot-and-mouth disease virus; S4, using the plasmid containing the truncated and mutant protein gene of VP1 in foot-and-mouth disease virus and the circular element obtained in step S3, to prepare a circular mRNA encoding the truncated and mutant protein of VP1 in foot-and-mouth disease virus; S5. Add the circular mRNA encoding the truncated and mutant protein of foot-and-mouth disease virus VP1 obtained in step S4 to a pharmaceutically acceptable carrier to obtain the foot-and-mouth disease vaccine.

10. Use of the foot-and-mouth disease vaccine according to any one of claims 1 to 8 in the preparation of a medicament for preventing and / or treating foot-and-mouth disease virus infection.

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