Vaccine composition comprising norovirus gii mRNA

Abstract

The present invention relates to a vaccine composition comprising mRNA encoding structural protein VP1 of a norovirus GI genotype. An mRNA vaccine composition was prepared by deriving a consensus sequence of norovirus GI genotype VP1 antigen, and the immunogenicity of the vaccine composition was confirmed. Therefore, the vaccine composition can be effectively used to prevent and treat norovirus infection.

Description

Vaccine composition comprising norovirus GII mRNA

[0001] The present invention relates to a vaccine composition comprising mRNA encoding structural protein VP1 of norovirus GII genotype.

[0002] Norovirus is a type of virus that causes nonbacterial acute gastroenteritis. It is estimated that norovirus-related illnesses infect 700 million people worldwide annually, and that more than 200,000 people die annually from norovirus infection. Compared to other infectious diseases such as rotavirus or shingles, norovirus has a significantly higher mortality rate and treatment burden among middle-aged and older adults, making it a major public health threat in most countries.

[0003] Norovirus, a member of the Caliciviridae family, is a non-enveloped virus measuring approximately 30–40 nm in diameter. It consists of a 7.6-kbp single-stranded (+) RNA and has three open reading frames (ORFs). ORF2 encodes VP1, the major structural protein that forms the norovirus morphology, while ORF3 encodes VP2, which is not directly involved in structural formation. VP1, with a total size of 59 kDa, forms a dimer. Ninety of these dimers self-assemble, forming a total of 180 VP1 units in a single virus particle. ORF2, which encodes VP1, and ORF1, which encodes RNA-dependent RNA polymerase (RdRp), are adjacent to each other, and slipped strand mispairing at this junction can lead to amino acid mutations, which lead to the classification of various genotypes. VP1, the main immunogen of norovirus, has a slightly different amino acid sequence depending on the type of RdRp.

[0004] Noroviruses are classified based on the phylogenetic clustering of the amino acid sequence of their structural protein, the VP1 capsid protein. Seven genotypes (GI-GVII) have been reported to date, of which three genotypes (GI, GII, and GIV) are known to be pathogenic viruses that cause acute gastroenteritis in humans.

[0005] Norovirus GI and GII are very different in terms of infectiousness, molecular biology, epidemiology, and phylogenetics. To date, norovirus GI and GII have been further subdivided into 31 genotypes based on the base sequence of the capsid gene. Among them, GII has been found to be the most prevalent genotype in gastroenteritis outbreaks worldwide, and the GII.4 variant is responsible for most infections. The capsid of norovirus is covered with morphological protrusions that frequently change shape, making it difficult for immune attack or therapeutic substances to bind, making it difficult to develop antiviral agents to treat norovirus infection or vaccines to prevent infection.

[0006] Advances in biotechnology are leading to the development of mRNA-based therapies that address the root causes of diseases, going beyond treatment to preventative measures. Compared to traditional vaccines, mRNA vaccines can be mass-produced in a cell-free environment using in vitro transcription (IVT), enabling rapid development. The production process is simple and cost-effective. Furthermore, they eliminate the need for integration into the host cell genome, offering safety advantages. Furthermore, they eliminate the risk of host mutations due to the absence of DNA interaction. Despite these numerous advantages, mRNA remains unstable and easily degraded by ribonucleases, RNA-degrading enzymes found in human bodily fluids (tears, saliva, mucus, sweat, etc.), making their development as therapeutics challenging. However, structural research into mRNA has led to the development of modified forms of mRNA with increased protein translation efficiency and mRNA half-life, enabling the development of mRNA-based therapies.

[0007] Accordingly, the present inventors analyzed various subtypes of the VP1 gene encoding the major capsid protein of ORF2 (open reading frame 2) of norovirus genotype GII, analyzed the antigenicity, allergenicity, and toxicity of the derived sequences, and derived conserved sequences through multiple sequence analysis of the screened sequences, and confirmed that this can be developed into an mRNA vaccine useful for norovirus infectious diseases, thereby completing the present invention.

[0008] The purpose of the present invention is to provide a norovirus vaccine composition.

[0009] Another object of the present invention is to provide a method for inducing an immune response against norovirus in a subject.

[0010] Another object of the present invention is to provide a method for producing a norovirus vaccine composition.

[0011] To achieve the above purpose,

[0012] The present invention provides a norovirus vaccine composition comprising mRNA encoding structural protein VP1 of norovirus GII genotype or an immunogenic fragment thereof.

[0013] Additionally, the present invention provides a method for inducing an immune response against norovirus in a subject, comprising administering to the subject an effective amount of the vaccine composition.

[0014] In addition, the present invention provides a method for producing the vaccine composition, which comprises a step of in vitro transcribing mRNA encoding structural protein VP1 of norovirus GII genotype or an immunogenic fragment thereof.

[0015] The present invention relates to a vaccine composition comprising mRNA encoding a structural protein VP1 of the norovirus GII genotype. An mRNA vaccine composition was prepared by deriving a common sequence of the norovirus GII genotype VP1 antigen, and the immunogenicity of the vaccine composition was confirmed, and thus it can be usefully used to prevent and treat norovirus infection.

[0016] Figure 1 is a diagram showing a workflow for designing a norovirus GII mRNA vaccine.

[0017] Figure 2 is a diagram showing the final arrangement of the vaccine structure.

[0018] Figure 3a is a diagram showing the results of a three-dimensional structural analysis of seven mRNA vaccine structures (V1.1, V2.1, V3.1, V4.1, V6.1, V7.1, V_NA1.1) using the AlphaFold program.

[0019] Figure 3b is a diagram showing the results of a three-dimensional structural analysis of seven mRNA vaccine structures (V1.1, V2.1, V3.1, V4.1, V6.1, V7.1, V_NA1.1) using the I-TASSE program.

[0020] Figure 3c is a diagram showing the results of a three-dimensional structural analysis of seven types of mRNA vaccine structures (V1.1, V2.1, V3.1, V4.1, V6.1, V7.1, V_NA1.1) using the Robetta program.

[0021] Figure 4a is a vector map showing GII.4-1.1 subcloned into the final vector (6,917 bp).

[0022] Figure 4b is a vector map showing GII.4-3.1 subcloned into the final vector (7,295 bp).

[0023] Figure 4c is a vector map showing the subcloning of GII.4-4.1 into the final vector (7,664 bp).

[0024] Figure 4d is a vector map showing the subcloning of GII.4-2003 into the final vector (7,159 bp).

[0025] Figure 4e is a vector map showing the subcloning of GII.17-2015 into the final vector (7,159 bp).

[0026] Figure 5a shows the results of RNA gel electrophoresis after performing poly(A) tailing on mRNA synthesized by IVT of the GII.4-1.1 sequence.

[0027] Figure 5b shows the results of RNA gel electrophoresis after performing poly(A) tailing on mRNA synthesized by IVT of the GII.4-3.1 sequence.

[0028] Figure 5c shows the results of RNA gel electrophoresis after performing poly(A) tailing on mRNA synthesized by IVT of the GII.4-4.1 sequence.

[0029] Figure 5d shows the results of RNA gel electrophoresis after performing poly(A) tailing on mRNA synthesized by IVT of the GII.4-2003 sequence.

[0030] Figure 5e shows the results of RNA gel electrophoresis after performing poly(A) tailing on mRNA synthesized using IVT with the GII.17-2015 sequence.

[0031] Figure 6a is a diagram showing the results of protein expression after introducing 2.5 ㎍, 5 ㎍, and 10 ㎍ of norovirus GII.4-1.1 and 2.5 ㎍ of GII.4-2003.

[0032] Figure 6b is a diagram showing the results of quantitative analysis of the results of Figure 6a.

[0033] Figure 6c is a diagram showing the results of protein expression and quantitative analysis after introducing 2.5 ㎍, 5 ㎍, and 10 ㎍ of norovirus GII.4-3.1 and 2.5 ㎍ of GII.4-2003.

[0034] Figure 6d is a diagram showing the results of quantitative analysis of the results of Figure 6c.

[0035] Figure 6e is a diagram showing the results of protein expression after introducing 2.5 ㎍ and 5 ㎍ of norovirus GII.4-4.1 and 2.5 ㎍ and 5 ㎍ of GII.17-2015.

[0036] Figure 6f is a diagram showing the results of quantitative analysis of the results of Figure 6e.

[0037] Figure 6g is a diagram showing the results of protein expression after introducing 2.5 ㎍ and 5 ㎍ of norovirus GII.4-2003 and 2.5 ㎍ and 5 ㎍ of GII.17-2015.

[0038] Figure 6h is a diagram showing the results of quantitative analysis of the results of Figure 6g.

[0039] Figure 7a is a diagram showing the results of introducing GII.4-1.1, GII.4-3.1, GII.4-4.1, GII.4-2003, and GII.17-2015 into mammalian cells using Lipofectamine™ MessengerMAX™ reagent, treating the supernatant with human peripheral blood mononuclear cells, and confirming the results with an ICS assay.

[0040] Figure 7b is a diagram showing the results of quantitative analysis of the results of Figure 7a.

[0041] Figure 8a is a photograph of a culture in which a synthesized norovirus GII mRNA vaccine candidate sequence was introduced into mammalian cells using Lipofectamine™ MessengerMAX™ reagent and the supernatant was treated with human peripheral blood mononuclear cells.

[0042] Figure 8b is a diagram quantitatively representing the ELISpot assay results of Figure 9a.

[0043] Figure 9 is a diagram showing the results of an ICS assay using splenocytes obtained by lysing the spleen after intramuscular injection of a norovirus GII mRNA vaccine into 6-week-old BALB / c mice.

[0044] Hereinafter, the present invention will be described in detail.

[0045] The present invention provides a norovirus vaccine composition comprising mRNA encoding structural protein VP1 of norovirus GII genotype or an immunogenic fragment thereof.

[0046] The mRNA encoding the structural protein VP1 or an immunogenic fragment thereof of the above-mentioned norovirus GII genotype is derived from a common sequence of two or more strains of norovirus. The common sequence can be derived by aligning and comparing the nucleotide sequences of two or more noroviruses classified into the same genotype and genotype.

[0047] Nucleic acid and protein sequences for several norovirus isolates are known. Representative, non-limiting sequences, including those of ORF1, ORF2, and ORF3, and their encoded polypeptides from norovirus isolates, are listed in the National Center for Biotechnology Information (NCBI) database.

[0048] The mRNA encoding the structural protein VP1 of the norovirus GII genotype may typically be an mRNA having at least one open reading frame that can be translated by a cell or organism provided with the mRNA. The product of this translation is an antigen, preferably a peptide or protein that can act as an immunogen. The product may also be a fusion protein composed of two or more immunogens, for example, a fusion protein composed of two or more epitopes, peptides, or proteins derived from the same or different viral proteins, wherein the epitopes, peptides, or proteins may be linked by a linker sequence.

[0049] Furthermore, the mRNA encoding the structural protein VP1 of the norovirus GII genotype may be understood as an artificial mRNA, i.e., an mRNA molecule that does not occur naturally. An artificial mRNA molecule may be understood as a non-natural mRNA molecule. Such mRNA molecules may be non-natural due to individual sequences (that do not occur naturally) and / or other non-naturally occurring modifications, such as structural modifications of nucleotides. An artificial mRNA molecule may be designed and / or produced by genetic engineering methods corresponding to a desired artificial sequence of nucleotides (a heterologous sequence). That is, the mRNA encoding the structural protein VP1 of the norovirus GII genotype differs from the wild-type sequence by at least one nucleotide. Here, 'wild-type' may be understood as a naturally occurring sequence.

[0050] In one embodiment of the present invention, the mRNA may encode the amino acid sequence of SEQ ID NO: 2. In one embodiment of the present invention, the mRNA encoding the amino acid sequence of SEQ ID NO: 2 may include the base sequence of SEQ ID NO: 3.

[0051] The mRNA encoding the structural protein VP1 of the norovirus GI genotype used in the present invention is interpreted to also include a sequence showing substantial identity with the sequence described in SEQ ID NO: 3. The above substantial identity means that when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art, at least 60% homology (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%), more preferably 70% homology (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%), even more preferably 80% homology (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), and most preferably A sequence exhibiting 90% homology (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) is meant. All integers greater than or equal to 70% and less than or equal to 100%, and decimals therebetween, are included within the scope of the present invention with respect to % homology.

[0052] In the present invention, "mRNA" refers to messenger ribonucleic acid. Typically, such mRNA encodes a polypeptide and is translated into the protein it encodes in a target cell. Furthermore, mRNA may exhibit modifications that increase resistance to in vivo degradation (e.g., degradation by exo- or endo-nucleases) and / or in vitro degradation (e.g., during the manufacturing process prior to vaccine administration, such as during the preparation of the vaccine solution to be administered). RNA stabilization can be achieved, for example, by providing a poly-A tail, a 3'-untranslated region (UTR), a 5'-untranslated region, or any other modification. Furthermore, mRNA stabilization can be achieved by chemical modification or by altering the G / C content of the nucleic acid. Various other methods are known in the art and can be applied to the present invention.

[0053] In the present invention, the "untranslated region" refers to a region upstream of the start codon of an mRNA and downstream of the stop codon, which is an untranslated sequence. The UTR located upstream of the start codon of an mRNA is called the 5'UTR, and the UTR located downstream of the stop codon of an mRNA is called the 3'UTR. The untranslated region of an mRNA plays a pivotal role in regulating both mRNA stability and mRNA translation.

[0054] In the present invention, a poly-A tail is added to the sequence to increase mRNA stability.

[0055] The mRNA of the present invention further comprises a poly-A tail having a length of 20 to 150 nt. In specific embodiments, the poly-A tail may be, but is not limited to, 20 to 150 nt, 20 to 120 nt, 20 to 100 nt, 20 to 90 nt, 20 to 80 nt, 20 to 70 nt, 20 to 60 nt, 20 to 50 nt, or 20 to 40 nt, preferably about 30 nt.

[0056] In one embodiment of the present disclosure, the mRNA may additionally comprise an RNA having an open reading frame (ORF) encoding a signal peptide fused to the structural protein VP1 of the norovirus GII genotype. Signal peptides, which comprise 10-60 amino acids at the N-terminus of a protein, are typically required for translocation across membranes in the secretory pathway and thus universally control the entry of most proteins into the secretory pathway in both eukaryotes and prokaryotes. In eukaryotes, the signal peptide of the early precursor protein (pre-protein) guides ribosomes to the rough endoplasmic reticulum (ER) membrane and initiates transport of the growing peptide chain across the membrane for processing. ER processing produces the mature protein, and the signal peptide is typically cleaved from the precursor protein by an ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor. Signal peptides can also facilitate targeting of proteins to cell membranes.

[0057] Signal peptides can be 10-60 amino acids long. For example, signal peptides are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, It can be 57, 58, 59, or 60 amino acids long.

[0058] The composition may further comprise a liposome or a lipid nanoparticle (LNP). Specifically, the mRNA encoding the structural protein VP1 of the norovirus GI genotype included in the vaccine composition of the present disclosure may be adsorbed or associated to the outside of the liposome or lipid nanoparticle, or encapsulated or encapsulated inside the liposome or lipid nanoparticle. A key factor in the development of RNA-based vaccines and therapeutics is the efficient delivery of RNA into cells without degradation, and lipid nanoparticles are used as mRNA delivery vehicles for this purpose. In the present invention, 'liposome' refers to a material used to stably deliver a physiologically active ingredient (e.g., mRNA) and maximize the penetration effect. In the present invention, 'lipid nanoparticle' refers to a particle having at least one size of about nanometers (e.g., 1 to 1,000 nm) and comprising one or more lipids. These lipid nanoparticles may comprise one or more excipients selected from cationic lipids, neutral lipids, charged lipids, steroids, and polymer-conjugated lipids.

[0059] The lipid nanoparticles described above are not limited to any particular form, and should be construed to include any form formed when a cationic lipid or an ionic lipid, and optionally one or more additional lipids, are combined in an aqueous environment and / or in the presence of a nucleic acid compound. For example, lipid complexes, lipoplexes, etc. are included within the scope of lipid nanoparticles.

[0060] In the present invention, the mRNA encoding the structural protein VP1 of the norovirus GII genotype or a portion thereof may be adsorbed to the lipid portion of the liposome or lipid nanoparticle, or encapsulated in an aqueous space surrounded by part or all of the lipid portion of the liposome or lipid nanoparticle, so that the mRNA or a portion thereof may be protected from enzymatic degradation or other undesirable effects induced by mechanisms of the host organism or cell, such as an adverse immune response.

[0061] The above composition may further include an immunostimulant. In the present invention, an adjuvant means something that can maximize and maintain a protective immune response to an antigen used as a vaccine for a long time. Alexander Glenny discovered that aluminum salt increases the immune response, and it was approved as the first immunostimulant applied to human vaccines in 1932 and is currently used in the largest number of vaccines. Depending on the type, an immunostimulant induces Th1 or Th2 activity, and in particular, when it contains an immunostimulant that activates pathogen recognition receptors (PRRs), it induces Th1 activity through IFN-γ when activating macrophages, thereby inducing cellular immunity. An immunostimulant that can be administered together with the vaccine composition of the present invention to enhance an immune response includes any of a variety of immunostimulants, and is a drug, substance, or combination of substances used to enhance an immune response, including but not limited to, toll-like receptor (TLR) agonists, monophosphoryl lipid A (MPL), synthetic lipid A, lipid A mimetic or analogues, aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharides (LPS) of Gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly(lactide-co-glycolide) (PLG) microparticles, poloxamer particles, microparticles, liposomes, oil-in-water emulsions, MF59, and squalene. The immunostimulants may be administered simultaneously with the vaccine composition or sequentially with an interval of time.

[0062] The vaccine may further comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier, including a pharmaceutically acceptable diluent or excipient, includes any pharmaceutical substance that does not induce a harmful immune response in a subject receiving the vaccine and can be administered without excessive toxicity. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic buffer, and combinations thereof.

[0063] The above vaccine composition may additionally include a solvent, excipients, etc. The solvent includes physiological saline solution, distilled water, etc., and the excipient includes aluminum phosphate, aluminum hydroxide, aluminum potassium sulfate, etc., but is not limited thereto, and may further include materials commonly used in vaccine production in the field to which the present invention pertains.

[0064] The vaccine composition of the present invention can be manufactured by a method commonly used in the technical field to which the present invention pertains. The vaccine composition of the present invention can be manufactured as an oral or parenteral preparation, and is preferably manufactured as an injection solution as a parenteral preparation, and can be administered via the dermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, or epidural route.

[0065] The vaccine composition of the present invention can be administered to a subject in an immunologically effective amount. The “immunologically effective amount” above means an amount sufficient to exhibit a norovirus preventive effect and an amount that does not cause side effects or serious or excessive immune responses, and the effective dosage level can be determined according to factors including the type and severity of the subject, age, sex, type of infected virus, activity of the drug, sensitivity to the drug, administration time, administration route, excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field. The vaccine of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0066] Additionally, the present invention provides a method for inducing an immune response against norovirus in a subject, comprising administering to the subject an effective amount of the vaccine composition.

[0067] In the present invention, "immune response" refers to the ability to induce a humoral and / or cell-mediated immune response. A humoral immune response includes a B cell-mediated antibody response. A cellular immune response includes a T cell-mediated immune response, including but not limited to CD4+ T cells and CD8+ T cells. The ability of an antigen to induce an immune response is called immunogenicity, which may be a humoral and / or cell-mediated immune response. The immune response of the present invention is preferably an immune response against norovirus, and even more preferably an immune response against a norovirus infection in a subject.

[0068] In the present invention, the term "subject" refers to an animal, preferably a mammal, such as a mouse, rat, guinea pig, hamster, rabbit, dog, cat, or primate. Preferably, the subject is a human. However, the term "subject" also includes cells, preferably mammalian cells, and even more preferably human cells. Such cells may be immune cells, preferably lymphocytes.

[0069] The route of administration of the above mutant norovirus vaccine composition is not limited, but includes intradermal, intramuscular, subcutaneous, or intranasal administration. The preventive effect against the mutant SARS-CoV-2 virus resulting from administration of the above vaccine composition can be obtained at least 7, 8, 9, 10, 11, 12, 13, or 14 days after administration.

[0070] The above administration frequency may be 1, 2, 3, 4 or more times, but it is possible to obtain sufficient preventive effect with 1 administration.

[0071] In one embodiment of the present disclosure, the vaccine composition is formulated in an effective amount to produce an antigen-specific immune response (e.g., production of antibodies specific for a norovirus antigen) in a subject. An "effective amount" is a dose of RNA effective to produce an antigen-specific immune response.

[0072] In addition, the present invention provides a method for producing a vaccine composition comprising a step of in vitro transcribing mRNA encoding structural protein VP1 of norovirus GII genotype or an immunogenic fragment thereof.

[0073] The above mRNA may be produced through in vitro transcription.

[0074] In the present invention, “in vitro transcription” means DNA-dependent RNA synthesis in vitro, outside of a cell. Template DNA may be linearized with a suitable restriction enzyme prior to in vitro transcription. Reagents used for RNA in vitro transcription are typically polymerases such as bacteriophage-encoded RNA polymerases (T7, T3, SP6 or Syn5); ribonucleotide triphosphates (NTPs) for four bases (adenine, cytosine, guanine and uracil), and optionally, but are not limited to, cap analogs; modified nucleotides; RNase inhibitors, etc.

[0075] The mRNA encoding the structural protein VP1 of the norovirus GII genotype, and / or liposomes or lipid nanoparticles may be provided in a lyophilized powder form, or may be provided dissolved in an appropriate solution or buffer. When the mRNA encoding the structural protein VP1 of the norovirus GII genotype, and / or liposomes or lipid nanoparticles are provided in a lyophilized form, they may be used by dissolving them in an appropriate solution or buffer.

[0076] Hereinafter, the present invention will be described in more detail through the following examples.

[0077] These examples are intended to illustrate the present invention in detail, and the scope of the present invention is not limited by these examples.

[0078] <Example 1> Derivation of norovirus GII mRNA vaccine candidate sequence

[0079] To develop an effective norovirus mRNA vaccine, we explored mRNA vaccine candidate sequences.

[0080] Derivation of common sequence

[0081] In order to analyze various subtypes of the VP1 gene encoding the major capsid protein, 15 conserved sequences were selected from 222 GIIs obtained through the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) by selecting 138 sequences with antigenicity ≥ 0.5 and no allergenicity or toxicity. Among them, the epitope part that can be effective as a vaccine was selected as the target sequence as the GII consensus sequence as shown in Table 1 below.

[0082]

[0083] Composition of vaccine structure

[0084] A structurally stable vaccine construct was constructed by arranging domains recognized as antigens by immune cells [HTL (Helper T lymphocyte), CTL (Cytotoxic T lymphocyte), BL (B cell), etc.], a specific adjuvant, Pan HLA DR-binding epitope (PADRE), and MHC class I trafficking signal (MITD) and a linker sequence. That is, along with three sets of epitopes, a potential linker, Pan HLA DR-binding epitope (PADRE), a universal peptide that activates antigen-specific CD4+ T cells that can be used as an agonist adjuvant in the development of immunotherapeutic vaccines, and an MHC class I trafficking signal (MITD) sequence, a sequence that directs peptides to the ER and Golgi apparatus for efficient secretion and presentation by MHC molecules, were added. Epitopes were linked and analyzed using various linkers, and finally EAAAK, AAY, GPGPG, and (EAAAK)2 were finalized and used for epitope linkage, and the final arrangement is as shown in Figure 2. A total of eight combinations of vaccine constructs were generated, and the antigenicity of each vaccine construct was evaluated using VexiJen and AntigenPro. When toxicity was evaluated using ToxinPred2, all constructs were non-toxic except for V8 and V8.1. Allergenicity was investigated using AlgPred, AllerTop, and AllergenFP, and as a result of the evaluation, V5 and V5.1 were confirmed to be allergenic substances, and the rest were non-allergenic. Solubility, stability, heat resistance, etc. were all within the acceptable range. Therefore, excluding the sequences confirmed to be toxic and allergenic, the results are as shown in Table 2 below.

[0085]

[0086] Among them, 7 mRNA vaccine structures (V1.1, V2.1, V3.1, V4.1, V6.1, V7.1, V_NA1.1) were subjected to 3D structural analysis using 3 programs: AlphaFold, I-TASSE, and Robetta, and a full-length structural model was created by repeatedly assembling the fragments. The quality of the vaccine structures created in this way was evaluated, and the deviation in the energy distribution and non-bonded atomic interactions generated in the structure were measured to calculate the quality factor, and an evaluation was conducted to determine whether the structure did not exceed the range of natural proteins.

[0087] Using the evaluated sequences, immune simulations of each vaccine construct were also performed using the C-IMMSIM program. The evaluation items included immunoglobulin production, immune cell (B cells, helper T cells, cytotoxic T cells, macrophages, dendritic cells) or immune cell population responses after mRNA vaccine administration, and cytokine and interleukin secretion stimulation. Cellular and humoral immune responses were evaluated in silico for the mRNA vaccine constructs.

[0088] In addition, binding energy was evaluated to measure the binding affinity of the mRNA vaccine constructs to TLR2, TLR4, and TLR8, and amino acid interactions were evaluated to select human norovirus GII mRNA vaccine candidate sequences. Finally, codon optimization and complexity were calculated to select three human norovirus GII.4 mRNA vaccine candidates (V1.1, V3.1, V4.1).

[0089] Codon optimization

[0090] Finally, codon optimization was performed for each vaccine construct as shown in Table 3 to exhibit a high GC content ratio, and the mRNA vaccine construct was completed.

[0091]

[0092] <Example 2> Norovirus GII mRNA vaccine candidate sequence mRNA synthesis and poly A tailing

[0093] The norovirus GII.4 mRNA vaccine candidate sequence derived in Example 1 was synthesized and inserted into the pUCIDT plasmid. To produce the norovirus GII.4 mRNA vaccine candidate sequences GII.4-1.1, GII.4-3.1, GII.4-4.1, GII.4-2003, and GII.17-2015, the forward primer 5'-GCGATATCGTGAGAAGCTGCTGCTAAAGCCG-3' (SEQ ID NO: 38) for GII.4-1.1, the reverse primer 5'-GCCTCGAGAAGCAGTCAAAGATACGTCTGACCCTTG-3' (SEQ ID NO: 39) for GII.4-3.1, the forward primer 5'-GCGATATCGTGAGAGGCGGCGGC-3' (SEQ ID NO: 40) for GII.4-3.1, the reverse primer 5'-GCCTCGAGAGGCAGTAAGCGACACGTCTG-3' (SEQ ID NO: 41) for GII-4.1 were used. 5'-GCGATATCGTGAGAAGCTGCTGCTAAAGCCG-3' (SEQ ID NO: 42), reverse primer 5'-GCCTCGAGACGCGGTTAAAGATACGTCCGAGC-3' (SEQ ID NO: 43), forward primer 5'-GCGATATCGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGA-3' (SEQ ID NO: 44) for GII.4-2003 and GII.17-2015, reverse primer 5'-GCCTCGAGGTAATGCACGTCTACGCCCCG-3' (SEQ ID NO: 45) for GII.4-2003, reverse primer 5'- GCCTCGAGGCTGAGCCCTCCTTCGCC-3' (SEQ ID NO: 46) for GII.17-2015, and polymerase chain reaction was performed at 98°C for 30 seconds with platinum SuperFi Taq, a high fidelity Taq polymerase. Denaturation was performed and 25 cycles of 98°C for 10 seconds, 60-62°C for 30 seconds, and 72°C for 60-90 seconds were repeated, followed by PCR at 72°C for 5 minutes to amplify the DNA. The PCR product was purified and treated with restriction enzymes to produce pcDNA3.Subcloning was performed into the 1 V5 / His C vector (Figures 4a to 4e).

[0094] The vector containing the genes of the recombinant norovirus GII mRNA vaccine candidates, GII.4-1.1, GII.4-3.1, GII.4-4.1, GII.4-2003, and GII.17-2015, was linearized using PmeI restriction enzyme. Linearization was confirmed by agarose gel electrophoresis, and linearized DNA was obtained by phenol / chloroform / isoamyl alcohol purification and ammonium acetate precipitation. The concentration and purity were confirmed, and in vitro transcription experiments were performed. 75 ㎍ of linearized DNA was transcribed in vitro at 37°C for 4 h using T7 RNA polymerase, CleanCap AG, NTPs, RNase inhibitor, and yeast inorganic pyrophosphatase. The template DNA was then removed using DNase I. The synthesized mRNA was recovered using LiCl and dissolved in RNase- and DNase-free DW, and the concentration and purity of the mRNA were measured. Using some of these, RNA gel electrophoresis was performed to confirm whether mRNA of the correct size was synthesized without denaturation. After confirming the results, poly(A) tailing was performed with E. coli poly(A) polymerase at 37°C for 30 minutes using 100 μg of synthetic mRNA and ATP, and RNA gel electrophoresis was performed again to confirm a band with a size shifted from the mRNA.

[0095] As a result, as shown in FIGS. 5a to 5e, mRNAs of the correct size were synthesized for all of the vaccine candidate sequences GII.4-1.1, GII.4-3.1, GII.4-4.1, GII.4-2003, and GII.17-2015, and the Poly(A) tailed RNA was approximately 100 bp larger than the mRNA, confirming that Poly(A) tailing was successful.

[0096] It was performed using Lipofectamine™ MessengerMAX™ reagent from ific. 293T cells were cultured at 1X10 6 After seeding cells / well in a 6-well plate, the medium was replaced with serum-free medium the next day and stabilized for 1 hour before intracellular introduction of poly(A) mRNA. Lipofectamine™ MessengerMAX™ reagent was mixed with serum-free medium and incubated at room temperature for 10 minutes. After mixing the poly(A) RNA of each norovirus GII mRNA vaccine candidate sequence (GII.4-1.1, GII.4-3.1, GII.4-4.1, GII.4-2003, and GII.17-2015) in serum-free medium at different concentrations, the solution was mixed well with Lipofectamine™ MessengerMAX™ reagent serum-free medium and incubated at room temperature for 5 minutes. After spraying dropwise onto 293T cells and shaking to distribute evenly, the cells were incubated in a 37℃ incubator for 12 hours. After incubation, the supernatant was aspirated, the cells were washed with cold PBS, and the cells were lysed using a protein lysis reagent and the proteins were extracted. The extracted protein quantitation was mixed with sample buffer, electrophoresed on an SDS-PAGE gel, blotted on a PVDF membrane, blocked with skim milk, and then attached with anti-V5 tag antibody, anti-6XHis tag antibody, and anti-β-actin antibody. The protein expression of the introduced G1 mRNA vaccine candidate sequence was confirmed by luminescence using ECL solution, and the amount of protein used for electrophoresis was corrected with β-actin.

[0097] As a result, as shown in Figs. 6a and 6b, the GII.4-1.1 candidate sequence showed that a protein was expressed with a size of 52 kDa, and the GII.4-2003 candidate sequence showed a size of 62 kDa. The expression amount of GII.4-1.1 increased depending on the amount of mRNA introduced in a concentration-dependent manner, but it was confirmed that the expression efficiency was not higher than that of the actual original sequence, GII.4-2003. In addition, as shown in Figs. 6c and 6d, the GII.4-3.1 candidate sequence showed a size of 65 kDa and showed an expression efficiency that was about 1.3 times higher than that of GII.4-2003. In addition, as shown in FIGS. 6e and 6h, the GII.4-4.1 candidate sequence had a size of 78 kDa and showed an expression efficiency similar to that of GII.4-2003, and the GII.4-2003 and GII.17-2015 candidate sequences had a size of 62 kDa, with no significant difference in size.

[0098] <Example 4> Immunogenicity evaluation of norovirus GII mRNA vaccine candidate sequence (in vitro)

[0099] After intracellular introduction of mRNA of the human norovirus GII mRNA vaccine candidate sequence, the supernatant was treated with human peripheral blood mononuclear cells to evaluate immunogenicity.

[0100] Specifically, intracellular introduction of poly(A) mRNA of the human norovirus GII mRNA vaccine candidate sequence was performed in the same manner as protein expression using Lipofectamine™ MessengerMAX™ reagent from Thermofisher Scientific. Four hours after intracellular introduction, the cells were washed with serum-free medium, the Lipofectamine™ MessengerMAX™ reagent was removed, and fresh serum-free medium was added. Incubation was performed in a 37°C incubator for 8 hours. The supernatant was then collected, centrifuged at 3,000 rpm for 10 minutes, and 800 μL was used for the ICS assay and 100 μL for the ELISpot assay, respectively.

[0101] For the ICS Assay, the supernatant containing the human norovirus GII mRNA vaccine candidate protein was incubated with 1X10 human peripheral blood mononuclear cells suspended in X-VIVO 15 medium. 6After mixing with dogs and seeding in 6-well plates, culture was performed in an incubator at 37℃ for 48 hours. mAb CD3-2 was used at a 1:1000 dilution as a positive control. Brefeldin A was added 12 hours before harvesting human peripheral blood mononuclear cells to ensure that the proteins and cytokines produced within the cells remain intact in the human peripheral blood mononuclear cells. After 12 hours, the cells were harvested and centrifuged to collect only human peripheral blood mononuclear cells. The harvested human peripheral blood mononuclear cells were immediately fixed with 1% paraformaldehyde for 20 minutes at room temperature and washed with PBS by centrifugation. The fixed cells were permeabilized with 0.3% triton X-100 for 10 minutes at room temperature, and then PBS (FACS buffer) containing 2% FBS was added and centrifuged to wash the permeabilized human peripheral blood mononuclear cells. Anti-CD3-FITC antibody, anti-CD4-PE antibody, anti-CD8-PE cy7 antibody, and anti-IFNγ-APC antibody were added to washed human peripheral blood mononuclear cells, and staining was performed on ice for 1 hour. Afterwards, the stained human peripheral blood mononuclear cells were washed with FACS buffer and centrifuged, and flow cytometry analysis was performed using FACS.

[0102] As a result, as shown in Fig. 7a and Fig. 7b, in the group treated with the supernatant containing the mixed proteins of GII.4-1.1, GII.4-3.1, GII.4-2003, and GII.17-2015, both the CD3+ / CD4+ / IFNγ+ population, which is described as helper T cells in human peripheral blood, and the CD3+ / CD8+ / IFNγ+ population, which is cytotoxic T cells, increased by about 2-fold or more compared to the control group, and only the GII.4-4.1 treatment group showed a small increase, and in all groups including the positive control, a phenomenon in which cytotoxic T cells increased slightly more than helper T cells was observed.

[0103] To confirm the immunogenicity of the vaccine by measuring the degree of T cell activation, the efficacy of the mRNA vaccine was evaluated by counting cells secreting IFN-γ in human peripheral blood mononuclear cells treated with the substance using ELISpot analysis.

[0104] Specifically, the ELISpot assay was performed using the ELISpot Pro: Human IFN-γ (ALP) kit from MABTECH. The 96-well plate coated with anti-human IFN-γ antibody was removed from the package, washed four times with 200 ㎕ of PBS, and then 200 ㎕ of X-VIVO-15 medium for the ELISpot assay was added and incubated at room temperature for more than 30 minutes. After incubation, the medium was removed from the 96-well plate and 2.5X10 human peripheral blood mononuclear cells were added. 5 cells / well were evenly distributed, and 100 μl of the supernatant containing the protein of the human norovirus GI mRNA vaccine candidate sequence was dispensed into each well. As a positive control, mAb CD3-2 provided in the kit was used at a 1:1000 dilution. After all distribution, the wells were incubated in a 37℃ incubator for 48 hours. After 48 hours, the supernatant was removed, and the 96-well plate was washed five times with 200 μl of PBS. Then, 7-B6-ALP was diluted 1:200 in PBS containing 0.5% FBS, 100 μl was added to each well, and the wells were incubated for 2 hours at room temperature. After washing five times with 200 μl of PBS, 100 μl of the filtered BCIP / NBT-plus solution was added to the wells to develop the color, and after the reaction was terminated with distilled water, the plate was dried, and a photograph was taken to analyze the number of spots.

[0105] As a result, as shown in FIGS. 8a and 8b, it was observed that the activity increased in the group treated with the protein supernatants of the mRNA vaccine candidates GII.4-1.1, GII.4-3.1, GII.4-4.1, GII.4-2003, and GII.17-2015 compared to the vehicle, confirming that the proteins produced by the introduction of GII.4-1.1, GII.4-3.1, GII.4-4.1, GII.4-2003, and GII.17-2015 mRNA into cells have immunogenicity that induces a cellular immune response that activates T cells.

[0106] <Example 5> Evaluation of the efficacy of a norovirus GII mRNA vaccine candidate (in vivo)

[0107] An experiment was conducted to evaluate the efficacy of a norovirus GII mRNA vaccine candidate using BALB / c mice.

[0108] Specifically, after administering a norovirus GII mRNA vaccine candidate into the muscle of 6-week-old female BALB / c mice, T cell activity of spleen cells was measured. The test substance was dissolved on ice on the day of administration without a separate preparation process, mixed by hand tapping, and spun down briefly before use. The test substance was administered intramuscularly at a dose of 20 μg / head at 40-50 μl / head, and the test substance was administered intramuscularly at a dose of 20 μg / head at 40-50 μl / head. The test groups were divided into six groups: the placebo luciferase2 mRNA vaccine group, the GII.4-1.1 mRNA vaccine group, the GII.4-3.1 mRNA vaccine group, the GII.4-4.1 mRNA vaccine group, the GII.4-2003 mRNA vaccine group, and the GII.17-2015 mRNA vaccine group. Three weeks after administration of the norovirus GII mRNA vaccine, the spleen was removed, weighed, crushed to isolate splenocytes, and T cell activity was measured.

[0109] Splenocytes obtained by lysing the spleen 3 weeks after mRNA vaccine administration were subjected to Intracellular Cytokine Staining (ICS) using fluorescently conjugated antibodies (anti-CD3-FITC antibody, anti-CD4-PE antibody, anti-CD8-PE-Cy7 antibody, and anti-IFN-r-APC antibody), and confirmed by FACS.

[0110] As a result, as shown in Fig. 9, compared to the placebo group, the luciferase 2 mRNA vaccine treatment group, an increase in CD3+ / CD8+ / IFN-r+ and CD3+ / CD4+ / IFN-r+ cells was observed in the groups administered the GII.4-3.1, GII.4-4.1, and GII.4-2003 mRNA vaccines. This shows that the ratio of Cytotoxic T cells labeled as CD3+ / CD8+ / IFN-r+ and Helper T cells labeled as CD3+ / CD4+ / IFN-r+ is increased by the norovirus mRNA vaccine.

[0111] Furthermore, the mRNA vaccine of the present invention stimulated cytotoxic T cells more than helper T cells at three weeks after administration, confirming an increase in cellular immunity. This result indirectly demonstrates the efficacy of the norovirus GII mRNA vaccine candidate in activating T cells. However, GII.4-1.1 and GII.17-2015 were observed to have lower immune cell activation effects compared to other mRNA vaccine candidates.

Claims

1. A norovirus vaccine composition comprising mRNA encoding structural protein VP1 of norovirus GII genotype or an immunogenic fragment thereof.

2. In paragraph 1, A vaccine composition, characterized in that the structural protein VP1 or an immunogenic fragment thereof is derived from a consensus sequence comprising the amino acid sequence of SEQ ID NO:

1.

3. In paragraph 1, A vaccine composition, characterized in that the mRNA encodes an amino acid sequence represented by sequence number 2.

4. In paragraph 1, A vaccine composition, characterized in that the mRNA comprises a base sequence represented by sequence number 3.

5. In paragraph 1, A norovirus vaccine composition, characterized in that the mRNA further comprises a poly-A tail, a 3'-untranslated region, and a 5'-untranslated region.

6. In paragraph 1, A vaccine composition, characterized in that the composition further comprises liposomes or lipid nanoparticles.

7. In paragraph 1, A vaccine composition characterized in that the composition is used to prevent or treat norovirus infection in a subject.

8. In paragraph 1, A vaccine composition, characterized in that the composition further comprises an immunostimulant.

9. In paragraph 1, A vaccine composition, characterized in that the composition further comprises a pharmaceutically acceptable carrier.

10. A method for inducing an immune response against norovirus in a subject, characterized by comprising a step of administering to the subject an effective amount of a vaccine composition according to any one of claims 1 to 9.

11. A method for producing a vaccine composition according to any one of claims 1 to 9, comprising a step of in vitro transcription (IVT) of mRNA encoding structural protein VP1 of norovirus GII genotype or an immunogenic fragment thereof.

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