Rabies vaccine and preparation method thereof

Abstract

The invention discloses a rabies vaccine and a preparation method thereof. The rabies virus G truncated mutant protein comprises a circular RNA (Ribonucleic Acid) molecule for coding rabies virus G truncated mutant protein and a pharmaceutically acceptable carrier, wherein the amino acid sequence of the rabies virus G truncated mutant protein is as shown in SEQ ID No.13 or a degenerate sequence thereof, and the nucleotide sequence is as shown in SEQ ID No.14 or a degenerate sequence thereof; the nucleotide sequence of the circular RNA molecule is as shown in SEQ ID No. 20. At present, RNA vaccines for resisting rabies virus infection are not applied in batches, the optimized rabies virus G protein circular RNA molecules and the optimized lipid nanoparticles are used for preparing the vaccines, and the process can be industrially amplified. The animal can be induced to generate a high-level neutralizing antibody and a more lasting drug effect only by injecting a low-dose circular RNA vaccine (16 [mu] g / vaccine) once, the vaccine is good in safety and stability, and a basis is provided for industrial preparation and application of the vaccine.

Description

Technical Field

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

[0002] Rabies is a fatal neurological disease caused by the rabies virus (RV). It is one of the most deadly zoonotic diseases known to date. There is currently no specific drug for rabies. Once clinical symptoms appear, the disease is almost 100% fatal. It can be controlled through vaccination through pre-exposure prophylaxis (PrEP) and timely and effective post-exposure prophylaxis (PEP). Humans are infected with rabies mainly through bites and scratches from infected animals such as dogs and cats. Vaccinating animals that have close contact with humans in advance to prevent rabies is an important strategy to reduce human rabies infection from animals.

[0003] RABV belongs to the Lyssavirus genus of the Rhabdoviridae family. RABV virus particles are bullet-shaped under an electron microscope. The RABV genome is about 12,000 nt and is a single-stranded negative-sense RNA virus that encodes five structural proteins: phosphoprotein (P), nucleoprotein (N), glycoprotein (G), polymerase (L) and matrix protein (M). Among them, G protein is the only protein exposed on the surface of the viral envelope, the only ligand of the cell receptor, and the only structural protein known to induce the host to produce virus neutralizing antibodies (VNAs).

[0004] The most effective means of preventing rabies is still vaccination. Currently, the rabies vaccine on the market is mainly an inactivated rabies virus vaccine, which is made from live virus strains through cell culture amplification, inactivation, emulsification and other processes, and plays an important role in the prevention of rabies. However, this vaccine also has many disadvantages in practical applications: (1) The use of cell culture to produce viruses has the risk of virus strain leakage and incomplete inactivation, which poses certain safety hazards. (2) High quality control and process amplification requirements and high production costs. (3) The use of completely inactivated viruses as antigens requires large doses of immunization, and multiple immunizations are required to exert lasting protection. There is still room for breakthroughs in the development of new rabies virus vaccines that are lower in cost, more durable and more effective.

[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 rabies virus vaccines, including RNA vaccines, have entered clinical use worldwide, and breakthroughs are needed in areas such as rabies RNA molecular design and vaccine preparation technology. Summary of the invention

[0008] In view of the problems of inactivated rabies vaccines in clinical use and linear RNA vaccines under research, including incomplete inactivation of inactivated rabies vaccines, risk of virus strain leakage, high production cost, and the reported rabies linear RNA vaccines requiring multiple injections to maintain long-term protection (doi: 10.3389 / fimmu.2022.1099991), the present invention provides a rabies circular RNA vaccine and a preparation method thereof. The circular RNA vaccine uses an optimized circular RNA molecule of the rabies virus G protein and an optimized lipid nanoparticle preparation process, which can be industrially scaled up. Only one injection of a low-dose circular RNA vaccine (16 μg / animal) is required to induce animals to produce high levels of neutralizing antibodies and more lasting efficacy. The vaccine has good safety and stability, providing a basis for the industrial preparation and application of vaccines.

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

[0010] The invention provides a rabies vaccine, which comprises a circular RNA molecule encoding a truncated and mutant protein of rabies virus G and a pharmaceutically acceptable carrier.

[0011] As one embodiment of the present invention, the amino acid sequence of the truncated and mutant protein of rabies virus G is shown in SEQ ID No. 13 or its degenerate sequence.

[0012] As one embodiment of the present invention, the optimized nucleotide sequence of the truncated and mutant protein of rabies virus G is shown in SEQ ID No. 14 or its degenerate sequence.

[0013] As one embodiment of the present invention, the nucleotide sequence of the circular RNA molecule (LTE2-G-834) is as SEQ ID No.20.

[0014] As an embodiment of the present invention, the dosage of the circular RNA molecule (LTE2-G-834) in the rabies vaccine is 10-125 μg / animal, including 10-16 μg / animal, 16-25 μg / animal, 25-50 μg / animal, 50-75 μg / animal, 75-100 μg / animal, 100-125 μg / animal, preferably 60-100 μg / animal.

[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 rabies 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: in molar percentage, 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 by molar percentage.

[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 45.7:42.3:9.2:1.5.

[0024] The present invention also provides a method for preparing a rabies vaccine, the method comprising the following steps:

[0025] S1, synthesizing and cloning the truncated rabies virus G plus mutant protein gene and looping element into a recombinant vector to obtain a recombinant vector containing the truncated rabies virus G plus mutant protein gene and looping element;

[0026] S2, transforming or transducing a host with the recombinant vector containing the truncated rabies virus G plus mutant protein gene and the looping element 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 and mutant protein gene of rabies virus G and a circular element;

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

[0029] S5. Add the circular mRNA encoding the truncated and mutant protein of rabies virus G obtained in step S4 to a pharmaceutically acceptable carrier to obtain the rabies vaccine.

[0030] As one embodiment of the present invention, the loop-forming element of the present invention comprises intron fragments I and II, translation initiation element truncation fragments I and II, and an insertion element.

[0031] The present invention also provides an application of a rabies vaccine in preparing a medicine for preventing and / or treating rabies virus infection.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) Currently, no rabies virus RNA vaccine has been approved for use. The present invention uses an optimized circular RNA molecule encoding a truncated and mutant protein of the rabies virus G, an optimized RNA preparation process, and an optimized formulation process to prepare an anti-rabies circular RNA vaccine, which has many advantages such as simple process, safety, and long-term effect.

[0034] (2) Compared with the disclosed elements, the total yield of circular RNA molecules containing optimized elements and optimized nucleotide sequences is increased, the immune effect of vaccines prepared using the optimized formulation process is improved, and the process meets the requirements of large-scale production.

[0035] (3) Compared with linear RNA vaccines and commercially available inactivated vaccines, rabies circular RNA vaccine only requires a low dose (RNA content of 16 μg / rabies animal) for a single injection, which can induce higher levels of virus neutralizing antibodies and has a longer protection period.

[0036] (4) The rabies circular RNA vaccine provided by the present invention is safe for use in dogs.

[0037] (5) The rabies circular RNA vaccine prepared by the optimized process of the present invention is stable when stored at -20°C for 6 months, providing a basis for the industrial preparation and application of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] 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:

[0039] Figure 1 The duration of virus neutralizing antibodies after dog immunization. DETAILED DESCRIPTION

[0040] 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.

[0041] 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".

[0042] 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.

[0043] In the present invention, the word "may" includes both performing a certain process and not performing a certain process.

[0044] 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.

[0045] 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 expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are modified by "about".

[0046] 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).

[0047] 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".

[0048] 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".

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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).

[0057] 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 are completely suppressed. Suffering from a disease means: the body has symptoms of the disease.

[0058] 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.

[0059] 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.

[0060] <circular RNA>

[0061] The present invention provides a circular RNA, wherein the circular RNA comprises a coding element, wherein the coding element encodes a truncated and mutant protein of rabies virus G; the amino acid sequence of the truncated and mutant protein of rabies virus G is shown in SEQ ID No. 13 or its degenerate sequence; the optimized nucleotide sequence of the truncated and mutant protein of rabies virus G is shown in SEQ ID No. 14 or its degenerate sequence; the nucleotide sequence of the circular RNA molecule (numbered LTE2-G-834) is shown in SEQ ID No. 20.

[0062] 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.3), translation initiation element truncated fragment II (numbered LTE-II, nucleotide sequence such as SEQ ID NO.9), protein coding element (numbered G-834, nucleotide sequence such as SEQ ID NO.14), insertion element (numbered B-polyAC, nucleotide sequence such as SEQ IDNO.16), translation initiation element truncated fragment I (numbered LTE-I, nucleotide sequence such as SEQ ID NO.10), intron fragment I (numbered BI, nucleotide sequence such as SEQ ID NO.4).

[0063] 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):

[0064] (c) translation initiation elements and coding elements;

[0065] (d) Translation initiation elements, coding elements and insertion elements.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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 factor; 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.16.

[0072] 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. 17 to 20.

[0073] <Recombinant Nucleic Acid Molecule>

[0074] The present invention provides a recombinant nucleic acid molecule, which forms the above-mentioned linear RNA after transcription.

[0075] 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.

[0076] <Nucleic acid lipid nanoparticle composition>

[0077] The present invention provides a nucleic acid lipid nanoparticle composition, which comprises lipid nanoparticles and the above-mentioned circular RNA.

[0078] 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 rabies vaccines.

[0079] <Medical Use>

[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 prevent and / or treat rabies.

[0081] 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 rabies.

[0082] The present invention provides a method for preventing and / or treating rabies, 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.

[0083] 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.

[0084] 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.

[0085] Example 1: Gene design and synthesis

[0086] Different vector DNA plasmids were designed for rabies virus G protein (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.

[0087] Table 1 Vector DNA design and construction

[0088]

[0089] Among them, intron fragment II (numbered A-II), translation initiation element truncated fragment II (numbered EVE-II or CKV-II), insertion element (A-polyAC), translation initiation element truncated fragment I (numbered EVE-I or CKV-I), and intron fragment I (numbered AI) are designed with reference to patent CN 114574483 A, wherein the translation initiation element CKV is a mutant sequence obtained by mutation of G at position 80 to T, A at position 81 to C, and G at position 82 to T in the Caprinekobuvirus IRES sequence, and the translation initiation element EVE is a mutant sequence obtained by mutation of G at position 13 to T, T at position 19 to C, and G at position 20 to T in the Echovirus E29 IRES sequence. Intron fragment II (numbered B-II), intron fragment I (numbered BI), translation initiation element truncated fragment II (numbered LTE-II), translation initiation element truncated fragment I (numbered LTE-I), and insertion element (B-polyAC) are obtained through design optimization screening, and the specific information is as follows:

[0090] The nucleotide sequence of intron fragment II (numbered A-II) is shown in SEQ ID NO.1.

[0091] The nucleotide sequence of intron fragment I (numbered as AI) is shown in SEQ ID NO.2.

[0092] The nucleotide sequence of intron fragment II (numbered B-II) is shown in SEQ ID NO.3.

[0093] The nucleotide sequence of intron fragment I (numbered BI) is shown in SEQ ID NO.4.

[0094] The nucleotide sequence of the translation initiation element truncation fragment II (numbered as EVE-II) is shown in SEQ ID NO.5.

[0095] The nucleotide sequence of the translation initiation element truncation fragment I (numbered as EVE-I) is shown in SEQ ID NO.6.

[0096] The nucleotide sequence of the translation initiation element truncation fragment II (numbered as CKV-II) is shown in SEQ ID NO.7.

[0097] The nucleotide sequence of the translation initiation element truncation fragment I (numbered as CKV-I) is shown in SEQ ID NO.8.

[0098] The nucleotide sequence of the translation initiation element truncation fragment II (numbered as LTE-II) is shown in SEQ ID NO.9.

[0099] The nucleotide sequence of the translation initiation element truncation fragment I (numbered as LTE-I) is shown in SEQ ID NO.10.

[0100] The protein coding element (No. G-501) includes: a nucleotide sequence (as shown in SEQ ID NO.12) encoding the wild-type G protein of rabies virus (the amino acid sequence is shown in SEQ ID NO.11), 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).

[0101] The protein coding element (No. G-834) includes: an optimized nucleotide sequence (as shown in SEQ ID NO.14) encoding a rabies virus G truncated plus mutant protein (N-terminal truncated 19 amino acids / C-terminal truncated 13 amino acids / including mutation W33Q / including mutation E356I, 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).

[0102] The nucleotide sequence of the insertion element (A-polyAC) is shown in SEQ ID NO.15.

[0103] The nucleotide sequence of the insertion element (B-polyAC) is shown in SEQ ID NO.16.

[0104] 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.

[0105] The corresponding linear RNA molecules are transcribed from the above vectors, and the mechanism of the formation of circular RNA molecules from linear RNA molecules is as follows: the ribozyme recognizes the connection position between the translation initiation element truncated fragment I and the intron fragment I to first generate a break, releasing the intron fragment I; then the ribozyme recognizes the connection position between the translation initiation element truncated fragment II and the intron fragment II to generate a break, releasing the intron fragment II. The 3' end of the translation initiation element truncated fragment I is connected to the 5' end of the translation initiation element truncated fragment II to form a circular molecule.

[0106] Example 2: Construction of recombinant bacteria

[0107] 2.1 Construction of original seeds. The above-mentioned vector DNA molecules pCKV-G-501, pEVE-G-501, pLTE-G-834, and pLTE2-G-834 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-pCKV-G-501, b-pEVE-G-501, b-pLTE-G-834, and b-pLTE2-G-834) as original seed banks.

[0108] 2.2 Identification of original seeds.

[0109] (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.

[0110] (2) Culture characteristics: It grows on LB solid medium to form round, shiny, milky white, smooth colonies with neat edges and raised areas.

[0111] (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.

[0112] (4) Identification: Sequencing analysis was performed and the nucleotide sequence was consistent with the target sequence.

[0113] Example 3: Fermentation of recombinant bacteria

[0114] The b-pCKV-G-501, b-pEVE-G-501, b-pLTE-G-834, and b-pLTE2-G-834 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.

[0115] Example 4 Plasmid DNA preparation and testing

[0116] 4.1 Bacterial lysis

[0117] (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.

[0118] (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.

[0119] (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.

[0120] (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.

[0121] 4.2 Ultrafiltration concentration

[0122] 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.

[0123] 4.3 Ion exchange chromatography

[0124] Plasmid was purified by AKTA purifier TM 100 purification system, Capto TM DEAE (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.

[0125] 4.4 Gel filtration chromatography

[0126] 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.

[0127] 4.5 Ultrafiltration and Diafiltration

[0128] 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.

[0129] 4.6 Plasmid DNA yield and yield / impurity removal effect

[0130] For plasmid DNA (pLTE2-G-834), 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.

[0131] 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.

[0132] The quality inspection results are shown in Table 2. For the pLTE2-G-834 plasmid, the optimized process was used, and the yields of lysis, ultrafiltration concentration, ion exchange chromatography, gel filtration chromatography, and ultrafiltration diafiltration reached 79%, 91%, 87%, 93%, and 85%, respectively, and the total yield reached 49.7%. The production process met the requirements.

[0133] Table 2 Plasmid DNA yield (pLTE2-G-834)

[0134]

[0135] 4.7 Plasmid testing

[0136] The plasmids (pCKV-G-501, pEVE-G-501, pLTE-G-834, pLTE2-G-834) were tested according to Analytical Procedures for mRNA Vaccine Quality 2rd USP, and all indicators met the requirements.

[0137] (1) Identification: The plasmid was sequenced and analyzed, and the sequence was consistent with the target sequence.

[0138] (2) Concentration analysis: The concentration of the plasmid was measured by UV spectrophotometer. The results are shown in Table 3.

[0139] (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.

[0140] (4) Safety: Endotoxin test was performed according to the method specified by the regulations, and the endotoxin content was less than 10EU / ml.

[0141] (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.

[0142] Table 3 DNA plasmid yield and purity

[0143]

[0144] Example 5: Preparation and testing of circular RNA

[0145] 5.1 CircRNA Preparation

[0146] (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.

[0147] (2) In vitro transcription: The transcription system is configured as shown in Table 4 below:

[0148] Table 4

[0149] Reagents volume 10× Reaction Buffer 100ml <![CDATA[ATP (containing 4% m 6 ATP)]]> 100ml CTP 100ml <![CDATA[N 1 P]]> 100ml GTP 100ml Linearized plasmid 30ml T7 RNA Polymerase 100ml RNase inhibitor (40U / μl) 100ml Nuclease free water Total 1000ml

[0150] 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.

[0151] 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 pLTE2-G-834 to obtain the optimal transcription conditions:

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 4) Optimization of enzymatic reaction temperature: The enzymatic reaction temperature was set to 20°C, 30°C, 37°C, and 40°C, respectively.

[0156] 5) Optimization of enzymatic reaction duration: The enzymatic reaction duration was set to 1 h, 2 h, 3 h, and 4 h respectively.

[0157] The RNA content was determined by UV spectrophotometry, and the integrity of the RNA was analyzed by capillary electrophoresis.

[0158] 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 pLTE2-G-834 is only 0.5g / 1000ml reaction, which contains a lot of incomplete RNA.

[0159] 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 / 32mM / 8mM / 8mM, the final concentration of T7 RNA polymerase is 1000KU, the enzymatic reaction temperature is 37°C, and the enzymatic reaction time is 2h. Under these conditions, the RNA content obtained by in vitro transcription of plasmid pLTE2-G-834 can be increased to 3.6g / 1000ml reaction, and the production process meets the requirements.

[0160] (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.

[0161] (4) mRNA cyclization and concentration: The cyclization system and conditions are shown in Table 5 below:

[0162] Table 5

[0163]

[0164] 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.

[0165] 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 pLTE2-G-834 to obtain the optimal circularization conditions:

[0166] (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.

[0167] (2) Optimization of magnesium ion concentration: The final magnesium ion concentrations in the system were set to 5 mM, 8 mM, 10 mM, 12 mM, and 15 mM, respectively.

[0168] (3) Optimization of cyclization reaction temperature: the reaction temperatures were set to 48°C, 52°C, 55°C, 58°C, and 62°C, respectively.

[0169] (4) Optimization of cyclization reaction time: set the reaction time to 8 min, 10 min, 12 min, 15 min, and 20 min, respectively.

[0170] 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 10mM, the cyclization reaction temperature was 52℃, and the cyclization reaction time was 15min. Under these conditions, the content of circular RNA obtained by in vitro transcription and cyclization of plasmid pLTE2-G-834 can reach 3.2g / 1000ml reaction, and the production process meets the requirements.

[0171] (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.

[0172] (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, the flow rate was 120 mL / min, and the ultrafiltered liquid was transferred to a new sample bottle after sterile filtration.

[0173] Through the above optimization process, a circular mRNA with the following sequence structure was successfully prepared:

[0174] The circular mRNA prepared from DNA plasmid pCKV-G-501 was named CKV-G-501, and its sequence is shown in SEQ ID NO.17.

[0175] The circular mRNA prepared from DNA plasmid pEVE-G-501 was named EVE-G-501, and its sequence is shown in SEQ ID NO.18.

[0176] The circular mRNA prepared from DNA plasmid pLTE-G-834 was named LTE-G-834, and the sequence is shown in SEQ ID NO.19.

[0177] The circular mRNA prepared from DNA plasmid pLTE2-G-834 was named LTE2-G-834, and its sequence is shown in SEQ ID NO.20.

[0178] 5.2 mRNA testing

[0179] Circular mRNA was tested according to Analytical Procedures for mRNA Vaccine Quality 2rd USP, and all indicators met the requirements.

[0180] (1) Identification: Sequence the mRNA and the sequence is consistent with the target sequence.

[0181] (2) Concentration analysis: The concentration of mRNA was measured by UV spectrophotometer. The results are shown in Table 6.

[0182] (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.

[0183] (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.

[0184] (5) Efficacy analysis: 293T cells were transfected according to the prescribed method, and the expression of the target protein met the requirements. 48 hours after transfection, the target protein expression levels of CKV-G-501, EVE-G-501, LTE-G-834, and LTE2-G-834 groups were 62.4 ng / ml, 70.3 ng / ml, 89.2 ng / ml, and 117.4 ng / ml, respectively. The protein expression level of LTE2-G-834 group was significantly higher than that of CKV-G-501, EVE-G-501, and LTE-G-834, indicating that the optimization of coding elements, translation initiation elements, and insertion elements was effective in improving the expression of the target protein.

[0185] (6) Safety: Endotoxin was analyzed according to the prescribed method and the endotoxin content was less than 0.1 EU / ml.

[0186] (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.

[0187] The mRNA molecule yield of the LTE2-G-834 group was significantly higher than that of the other groups (see Table 6). Compared with the LTE-G-834 group encoding the same protein sequence, the RNA yield of the LTE2-G-834 group increased by 1.5 times, indicating that the optimization of the non-coding region was effective and necessary and successfully increased the yield. Therefore, among the two groups, the circular RNA molecule (numbered LTE2-G-834), plasmid DNA molecule (numbered pLTE2-G-834) and Escherichia coli strain containing the plasmid DNA molecule (numbered b-pLTE2-G-834) corresponding to the LTE2-G-834 group were preferred.

[0188] Table 6 CircRNA Molecular Yield and Purity

[0189]

[0190] Example 6: Comparison of the immune effects of various circular RNA molecules

[0191] 6.1 Commercial LNP Preparation

[0192] The candidate circular RNA molecules (numbered CKV-G-501, EVE-G-501, LTE2-G-834) obtained in Example 5 are prepared into RNA wrapped in LNP using commercial LNP, and a control circular RNA molecule (numbered HM-G-834) is set to provide preparation for further animal experiments. Wherein the control circular RNA (numbered HM-G-834) is a truncated mutant protein of rabies virus G (19 amino acids truncated at the N-terminus / 13 amino acids truncated at the C-terminus / containing mutation W33Q / containing mutation E356I (amino acid sequence see SEQ ID NO.13, nucleotide sequence see SEQ ID NO.14)) prepared by a commercially available circular RNA cloning and construction kit (Shanghai Kewei Chuang), and the specific steps are referred to the instructions. The method for preparing RNA wrapped in LNP using commercial LNP is described in reference 10.1016 / j.cell.2022.03.044, as follows:

[0193] (1) Test method

[0194] a) Dilute the circular RNA to a final concentration of 170 μg / mL using PNI formulation buffer (Precision NanoSystems, Cat. No. NWW0043).

[0195] 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).

[0196] c) using tangential flow technology to replace the buffer environment with PBS at pH 7.4 to remove ethanol, and prepare LNP-mRNA, which are numbered as c-LNP / CKV-G-501, c-LNP / EVE-G-501, c-LNP / LTE2-G-834, and c-LNP / HM-G-834, respectively.

[0197] d) Using Quant-iT TM RiboGreen 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).

[0198] (2) Test results

[0199] The mRNA-encapsulated LNPs prepared in this step passed the inspection: the particle size was small (~72nm) 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.

[0200] 6.2 Mouse immunization and antibody assay

[0201] (1) Experimental methods

[0202] Female C57BL / 6 mice aged 6 to 8 weeks were randomly divided into 5 groups, 5 mice in each group, and 4 groups of LNP-encapsulated circular mRNA vaccines prepared in this example (numbered c-LNP / CKV-G-501, c-LNP / EVE-G-501, c-LNP / LTE2-G-834, and c-LNP / HM-G-834, respectively) were immunized with mice muscle at a dose of 3 μg / mouse. At the same time, mice vaccinated with LNP were used as negative controls. Blood was collected on the 21st day after immunization to separate serum, and rabies virus G protein IgG titer and virus neutralizing antibody were detected, respectively. The specific operation method mainly refers to the literature (DOI: 10.3389 / fimmu.2022.1099991).

[0203] (2) Experimental results

[0204] The results are shown in Table 7. No neutralizing antibodies were produced in the LNP group, while the neutralizing antibody levels in each RNA group were significantly affected by the target protein nucleotide sequence and translation initiation element: the neutralizing antibody level in the c-LNP / LTE2-G-834 group was better than that in other groups, indicating that the optimization was effective and necessary. In addition, it was observed that the increase in the neutralizing antibody titer was positively correlated with the decrease in the titer of G protein-specific antibodies, which may be related to the improvement of the target protein expression efficiency after optimization, the destruction of the dominant epitope to better expose the effective epitope, and the more uniform expression of the protein structure. In addition, the neutralizing antibody level in the LTE2-G-834 group was higher than that in the HM-G-834 group, indicating that the circular RNA molecules prepared in this study are superior to the circular RNA molecules prepared using the commercial circular RNA construction kit.

[0205] Table 7 Antibody response in mice

[0206]

[0207] Example 7: LNP-mRNA production process research

[0208] This section studies the production process of LNP-encapsulated mRNA.

[0209] 7.1 LNP-mRNA preparation

[0210] (1) Preparation of solution I: Dissolve the cationic lipid, cholesterol, neutral lipid, and PEG-modified lipid in ethanol to obtain solution I.

[0211] (2) Preparation of Solution II: Dissolve the preferred circular RNA (No. LTE2-G-834) of Example 6 in a 10 mM citric acid buffered saline solution at pH 4.0.

[0212] (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:

[0213] 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 were set to 5% to 60%. The content of PEG-modified lipids (DSPC (distearoylphosphatidylcholine) or DOPE (dioleoylphosphatidylethanolamine)) was set to 3%-10%, and the content of PEG-modified lipids (PEG-DMG (polyethylene glycol-dimyristyl glyceride) or PEG-DSG (bis-distearoyl-rac-glycerol-polyethylene glycol) or PEG-DPG (diphosphoglycerate polyethylene glycol))) was set to 0.8%-1.8%. 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. The LNP-encapsulated circular RNA molecules were prepared with a LNP / circular RNA (No. LTE2-G-834) volume ratio of 1:1, and the particle size and encapsulation efficiency of each group were compared.

[0214] 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 54.5:36.5:9.2:1.8 was ineffective, with an encapsulation efficiency of less than 10%.

[0215] Through experiments, the preferred liposome components were obtained to be CMAX4 / cholesterol / DSPC / PEG-DMG, and the preferred ratio of each component was 45.7:42.3:9.2:1.5. At this time, the molecular particle size was the smallest (70nm), the encapsulation rate reached 93%, and the production process met the requirements.

[0216] 3.2) Optimization of LNP / mRNA ratio: LNP and circular RNA (numbered LTE2-G-834) were mixed in different ratios (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 (numbered LTE2-G-834) was greater than 1:3, the migration of LTE2-G-834 could be completely blocked. Therefore, the volume ratio of LNP / circular RNA molecule was set to 1:3.

[0217] (4) The circular RNA molecule (numbered LTE2-G-834) was encapsulated with LNP according to the above preferred conditions and sterile filtered to obtain an RNA vaccine, numbered LNP / LTE2-G-834.

[0218] (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).

[0219] 7.2 Experimental Results

[0220] The results are shown in Table 8. The indicators of LNP-encapsulated mRNA were qualified: the particle size was less than 80nm, 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.

[0221] Table 8 LNP-mRNA sample characterization

[0222] serial number Particle size (nm) PDI Surface potential (mV) Encapsulation rate (%) LNP / LTE2-G-834 70 0.12 -5.4 93

[0223] Example 8 Evaluation of the immune effect of LNP-encapsulated RNA

[0224] For the vaccine prepared with the preferred LNP in Example 7, the immune effect was evaluated and the immune dose was determined

[0225] 8.1 Mouse immunization and antibody determination

[0226] (1) Experimental methods

[0227] Female C57BL / 6 mice aged 6 to 8 weeks were randomly divided into 3 groups, 5 mice in each group, and LNP / LTE2-G-834 prepared with the preferred LNP in this example and c-LNP / LTE2-G-834 prepared with commercial LNP in Example 6 were used to immunize mice at a dose of 3 μg / mouse. At the same time, mice vaccinated with LNP were used as negative controls. Blood was collected on the 21st day after immunization to separate serum, and rabies virus G protein IgG titer and virus neutralizing antibody were detected.

[0228] (2) Experimental results

[0229] The results are shown in Table 9. The LNP prepared by the optimal process was superior to the commercial LNP and successfully increased the neutralizing antibody titer in mice: the neutralizing antibody titer of the LNP / LTE2-G-834 group was 1.6 times that of the c-LNP / LTE2-G-834 group, providing a feasibility basis for further research.

[0230] Table 9 Antibody response in mice

[0231]

[0232] 8.2 Canine Immunization and Immunization Routes and Immunization Dosage Determination

[0233] (1) Test method

[0234] Thirty-five rabies antibody-negative beagles (~3 months old, 5-6 kg) were randomly divided into 7 groups, 5 in each group, and vaccinated by different routes and doses:

[0235] Group 1 to Group 3: LNP / LTE2-G-834 was injected intramuscularly into the lateral thigh of the hind limb, with the vaccination doses of 12μg / animal, 16μg / animal, and 20μg / animal, respectively.

[0236] Groups 4 to 6: LNP / LTE2-G-834 was intradermally injected into the lateral thigh of the hind limb, with the inoculation doses of 12 μg / animal, 16 μg / animal, and 20 μg / animal, respectively.

[0237] Group 7: PBS control group, inoculated with sterile PBS, the dose was 1 ml / mouse.

[0238] After one administration, blood was collected on the 21st day to prepare serum for virus neutralizing antibody detection. The test results are shown in Table 10. There was no neutralizing antibody in the PBS group, and the intradermal immunization effect of the LTE2-G-834 group was significantly better than that of the muscle immunization, indicating that it is a better route of administration. In terms of dosage, intradermal immunization can produce high levels of neutralizing antibodies through a low-dose single administration (16μg / mouse), which is significantly better than 12μg / mouse, reaching a level similar to the conventional application dose of 20μg / mouse, indicating that the vaccine is highly effective.

[0239] Table 10 Virus neutralizing antibody levels in dogs at different immunization routes and immunization doses (IU / ml) VNT values ​​above 0.5 IU / ml are considered to meet the immunization standard

[0240]

[0241] Example 9 Preparation of control linear RNA vaccine

[0242] In order to compare the effects of circular RNA and linear RNA, this study prepared a linear RNA encoding rabies virus G protein (numbered linear-G, nucleotide sequence see SEQ ID NO.21) with reference to the literature (doi: 10.1186 / s12985-022-01919-7), and the specific steps included in vitro transcription and purification process. The control linear mRNA prepared above was prepared into a lipid nanoparticle / RNA composition, recorded as the linear-G group, and the specific operation was the same as the preferred conditions of Example 7. After inspection, it was qualified: the LNP particle size distribution of the mRNA encapsulated was uniform, the encapsulation effect was good, and the encapsulation rate was greater than 90%; the surface potential was electronegative, and the biological safety was good.

[0243] Example 10: Canine Immunization and Antibody Duration Monitoring

[0244] Twenty-five rabies antibody-negative beagles (~3 months old, 5-6 kg) were randomly divided into 5 groups, 5 in each group, and immunized: LNP / LTE2-G-834 was injected intramuscularly on the outer thigh of the hind limb, with a vaccination dose of 16 μg / animal (100 μL). LNP / LTE2-G-834 was injected intradermally on the thigh of the hind limb, with a vaccination dose of 16 μg / animal (100 μL). LNP-encapsulated linear-G was injected intramuscularly on the thigh of the hind limb, with a vaccination dose of 16 μg / animal (100 μL). Commercially available inactivated rabies vaccine (containing the inactivated rabies virus HCP-SAD strain, which was not less than 10 μg / animal before inactivation) was administered. 6.3 FAID 50 / ml), injected into the lateral thigh muscle of the hind limb, with a dose of 1ml / mouse. The negative control group was inoculated with sterile PBS, with a dose of 1ml / mouse.

[0245] After one dose, neutralizing antibodies and antibody persistence tests were performed: at 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 ​​weeks, and 52 weeks after administration, blood was collected to prepare serum and the virus neutralizing antibodies were tested by conventional methods.

[0246] Test results are shown in Figure 1 . Compared with commercial inactivated vaccines and linear RNA molecule corresponding vaccines (linear-G group), the circular RNA molecule corresponding vaccine prepared in this study produces higher levels of neutralizing antibodies, and only a low dose (16μg / mouse) is required for a single administration to keep the neutralizing antibodies above the standard within 1 year. In contrast, the effective antibodies of the control linear RNA vaccine group (linear-G group) can only be maintained until the 32nd week after a single administration of a low dose (16μg / mouse). If more lasting antibodies are needed, multiple administrations or a single high-dose administration may be required. Achieving longer-lasting protection with a single administration can effectively reduce the cost of vaccine use, improve vaccination compliance, and is beneficial to the long-term prevention and control of rabies and virus purification.

[0247] Example 11 Vaccine Safety Test

[0248] The safety of LNP / LTE2-G-834 was further preliminarily evaluated. 24 beagles (~3 months old, 5-6 kg) were randomly divided into 3 groups, 8 in each group, and divided into blank control group, single-dose group and double-dose group. The blank control group did not receive any treatment; the single-dose group was vaccinated by intradermal injection of the hind thigh on day 0, with a vaccination dose of 16 μg / animal; the double-dose group was vaccinated by intradermal injection of the hind thigh on days 0 and 21, respectively, with a vaccination dose of 16 μg / animal. The clinical manifestations of the experimental dogs were observed every day, and the routine blood test, urine test, blood gas and biochemical indicators were tested on day 0 (before administration) and day 28 after the last immunization:

[0249] (1) Clinical manifestations of experimental dogs: Observe the clinical manifestations of the dogs, including body temperature, mental state, appetite, drinking desire, vomiting and diarrhea.

[0250] (2) Blood routine test: Blood samples were collected and blood routine was tested using an optoelectronic blood cell analyzer (model MEK-7222K, Nihon Kohden Industries, Ltd.).

[0251] (3) Urinalysis test: Collect urine samples and use a urine routine analyzer (model US-200, Changchun Manteno Medical Instrument Co., Ltd.) to test urine pH, urine specific gravity, urine ketones, urine bilirubin, urine protein, and urine sugar.

[0252] (4) Blood gas test: Blood samples were collected and blood electrolyte analyzer (model vetstat, IDEXX Biotech, USA) was used to test blood gas ions and the concentrations of potassium, calcium, sodium, and chloride in the blood.

[0253] (5) Detection of relevant biochemical indicators: Blood samples were collected and incubated at 4°C and 3500 r / min to obtain the supernatant. The concentrations of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, glucose, total bilirubin, creatinine, and urea nitrogen in serum were detected using a biochemical analyzer (model VetTest8008, IDEXX Biotech, USA).

[0254] All the test dogs had no abnormal body temperature during the test, were all lively, had normal appetite and no other obvious abnormal behaviors. The results of blood routine tests are shown in Table 11. The blood routine values ​​of all the test dogs during the test were within the normal range. The results of urine routine tests are shown in Table 12. No abnormal values ​​were found in the urine routine values ​​of each group. The results of blood gas tests are shown in Table 13. During the test, the potassium, calcium, sodium and chloride ion contents of all the test dogs were within the normal range. The results of biochemical index tests are shown in Table 14. No abnormalities were found in the biochemical index values ​​of all the test dogs, and the biochemical index values ​​were all within the normal range. The above results preliminarily indicate that the vaccine provided by the present invention is safe in the animal dog.

[0255] Table 11 Routine blood test (mean ± SD)

[0256]

[0257]

[0258] Table 12 Urinalysis (mean ± SD)

[0259]

[0260] Table 13 Blood gas electrolytes (mean ± SD values)

[0261]

[0262] Table 14 Biochemical indices (mean ± SD values)

[0263]

[0264]

[0265] Example 12 Vaccine stability study

[0266] The stability of the vaccine was further evaluated as follows:

[0267] The LNP / LTE2-G-834 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:

[0268] 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.

[0269] The test results are shown in Table 15. 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.

[0270] Table 15 Vaccine storage stability

[0271] Day 0 1 month 2 months 3 months 6 months Encapsulation rate / % 93.2 94.1 93.7 93.5 93.3 Particle size / nm 71.2 69.8 70.4 71.1 70.9 mRNA content / mg / ml 0.17 0.16 0.17 0.16 0.16 mRNA integrity / % 92.4 92.3 92.5 92.7 92.4 pH 7.11 7.16 7.11 7.12 7.15 Appearance No foreign matter found No foreign matter found No foreign matter found No foreign matter found No foreign matter found

[0272] 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 rabies vaccine, characterized in that: The rabies vaccine comprises a circular RNA molecule encoding a truncated and mutant protein of rabies virus G and a pharmaceutically acceptable carrier.

2. The rabies vaccine according to claim 1, characterized in that The amino acid sequence of the truncated mutant protein of rabies virus G is shown in SEQ ID No. 13 or its degenerate sequence.

3. The rabies vaccine according to claim 1, characterized in that The nucleotide sequence of the truncated mutant protein of rabies virus G is shown in SEQ ID No. 14 or its degenerate sequence.

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

20.

5. The rabies vaccine according to claim 1, characterized in that When the rabies vaccine is administered, the dosage of the circular RNA molecule is 10-150 μg / animal.

6. The rabies 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 rabies vaccine is 1:1-1:

3.

7. The rabies vaccine according to claim 6, characterized in that In terms of molar percentage, 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; and the PEG-modified lipids are PEG-DMG, PEG-DSG or PEG-DPG.

8. The rabies 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 45.7:42.3:9.2:1.

5.

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

10. Use of the rabies vaccine according to any one of claims 1 to 9 in the preparation of a medicament for preventing and / or treating rabies virus infection.

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