A pre-fusion conformation mutant of lyssavirus gpc protein, recombinant vector and vaccine

Abstract

The application discloses a pre-fusion conformation mutant of lyssavirus GPC protein, a recombinant vector and a vaccine, and relates to the technical field of lyssavirus vaccine research and development. The pre-fusion conformation mutant of lyssavirus GPC protein is obtained by replacing the enzyme cleavage site 'RKLM' site with a G4S linker, performing site-directed mutagenesis on a G290P of a GP2 subunit, and adding a T4 trimerization motif at a C terminal, and has the advantages of being capable of being efficiently expressed in a eukaryotic system, having high conformation stability, and completely retaining neutralizing epitopes in a pre-fusion conformation. After animals are immunized with the mutant, high-level neutralizing antibodies can be induced.

Description

Technical Field

[0001] This invention relates to the field of Luyovirus vaccine development technology, and more specifically, to a pre-fusion conformational mutant of the Luyovirus GPC protein, a recombinant vector, and a vaccine. Background Technology

[0002] Lujo virus (LUJV) belongs to the genus Arenavirus of the family Arenaviridae. It is a highly pathogenic pathogen that causes Lujo Hemorrhagic Fever (LHF). The virus was first discovered in Zambia, Africa in 2008 and quickly spread within hospitals in South Africa, exhibiting clinical characteristics of high mortality and posing a serious potential threat to global public health security.

[0003] Luyo hemorrhagic fever (LUJV) is characterized by acute fever, bleeding tendency, and multiple organ failure. The disease progresses rapidly and has an extremely high mortality rate. Currently, there are no FDA-approved specific treatments, preventative vaccines, or effective interventions, and it is classified as a newly emerging, highly virulent viral infectious disease requiring close monitoring. As a typical zoonotic pathogen, the natural reservoir host of LUJV is not fully understood. Its cross-species transmission routes, human-to-human transmission risks, and regional epidemic trends remain unclear. Once imported cases or local outbreaks occur, the epidemic can easily spread and is difficult to control quickly.

[0004] The World Health Organization (WHO) has included Luyo hemorrhagic fever (LUJV) in its list of priority diseases for research and development, explicitly requiring accelerated emergency research and development of vaccines, targeted drugs, and diagnostic reagents. Although vaccine development against arenaviruses has made some progress in recent years—for example, the Lassa fever vaccine, based on vesicular stomatitis virus (VSV), has successfully entered clinical trials, and the live attenuated vaccine Candid#1 for preventing Junin virus infection has been approved for use in relevant epidemic areas and has shown some protective efficacy—vaccine research against LUJV is still in the early exploratory stage. No candidate vaccines have yet entered clinical trials or been approved for use, and the core technological bottlenecks have remained unresolved for a long time.

[0005] LUJV is an enveloped, single-stranded, negative-sense RNA virus. The viral particles are pleomorphic, predominantly spherical, with a diameter of approximately 80-150 nm. They consist of a nucleocapsid and a lipid envelope. The genome contains two RNA segments: a large (L) segment and a small (S) segment. The S segment encodes the viral nucleoprotein NP and the glycoprotein complex GPC. GPC is the only structural antigen on the LUJV envelope surface and a key molecule mediating host cell recognition, viral invasion, and membrane fusion. It is also the only effective target for inducing the production of neutralizing antibodies and exerting immune protection.

[0006] After being cleaved by the host protease, the GPC precursor protein of LUJV forms two functional subunits, GP1 and GP2: GP1 is responsible for recognizing and binding to the host cell surface receptor, initiating the viral adsorption and invasion process; GP2 mediates the fusion of the viral envelope with the host cell membrane and is the core element for the virus to complete the infection cycle.

[0007] In its natural state, LUJV GPCs are located on the viral envelope surface as trimers. Their structure is highly unstable, readily and spontaneously transitioning rapidly from the pre-fusion conformation (high energy level) to the post-fusion conformation (low energy level). Recombinantly expressed wild-type GPCs readily dissociate into monomers, leading to the destruction or masking of key neutralizing epitopes and exposing numerous non-neutralizing dominant epitopes located within the trimer. This induces immune bias, significantly reducing immunogenicity and protective efficacy. Furthermore, the complex glycosylation modifications of GPCs greatly weaken immunogenicity, further increasing the difficulty of designing stable and efficient vaccine antigens, becoming a key scientific problem hindering the development of LUJV vaccines.

[0008] In view of this, the present invention is proposed. Summary of the Invention

[0009] The purpose of this invention is to provide a pre-fusion conformation mutant of Luyovirus GPC protein, a recombinant vector, and a vaccine, thereby enabling the Luyovirus GPC protein to remain stably in the pre-fusion conformation and fully retain key neutralizing epitopes.

[0010] This invention is implemented as follows: In a first aspect, the present invention provides a pre-fusion conformational mutant of the Luyovirus GPC protein, the Luyovirus GPC protein comprising GP1 subunit and GP2 subunit; comprising the following mutations: (1) Replace the enzyme cleavage site “RKLM” between the GP1 subunit and the GP2 subunit in the GPC protein with (GGGGS)3 flexible linker; (2) The extracellular region of GP2 was extracted, and a G69P mutation was introduced at position 69 of the extracellular amino acid sequence of wild-type GP2; (3) Add a T4 trimerization motif to the C-terminus of the GPC protein; the amino acid sequence of the T4 trimerization motif is shown in SEQ ID NO.7; The amino acid sequence of wild-type GP1 is shown in SEQ ID NO.8, and the amino acid sequence of the extracellular region of wild-type GP2 is shown in SEQ ID NO.11.

[0011] Secondly, the present invention provides a nucleic acid molecule that encodes a pre-fusion conformational mutant of the Luyovirus GPC protein described above.

[0012] Thirdly, the present invention provides a recombinant vector comprising the aforementioned nucleic acid molecules.

[0013] Fourthly, the present invention provides a recombinant cell or recombinant bacteria comprising the above-described nucleic acid molecule or the above-described recombinant vector.

[0014] Fifthly, the present invention provides the use of a pre-fusion conformational mutant of Luyovirus GPC protein, nucleic acid molecule, recombinant vector, recombinant cell, or Luyovirus chimeric recombinant virus prepared by the above methods in the preparation of a drug or vaccine for the treatment or prevention of Luyo hemorrhagic fever.

[0015] In a sixth aspect, the present invention provides the use of a pre-fusion conformational mutant of the Luyovirus GPC protein, a nucleic acid molecule, a recombinant vector, a recombinant cell, or a Luyovirus chimeric recombinant virus prepared by the above methods in the preparation of a vaccine or drug for inducing a protective immune response against arenavirus.

[0016] In a seventh aspect, the present invention provides a vaccine or drug comprising: a pre-fusion conformational mutant of the Luyovirus GPC protein described above, the nucleic acid molecule or recombinant vector described above or a Luyovirus chimeric recombinant virus prepared by the above method, and pharmaceutically acceptable excipients.

[0017] The present invention has the following beneficial effects: This invention, based on the GPC structure (PDB: 8P4T) of Luyovirus and antigen design principles, involves a triple modification: replacing the "RKLM" cleavage site with a G4S linker, performing a site-directed mutation at G69P in the extracellular region of GP2, and adding a T4 trimerization motif at the C-terminus. This yields a pre-fusion conformational mutant of the Luyovirus GPC protein that is efficiently expressed in eukaryotic systems, exhibits high conformational stability, and fully retains the pre-fusion conformational neutralizing epitopes. Immunization of animals with this mutant induces high levels of neutralizing antibodies, demonstrating significant potential as a safe and highly effective candidate antigen for a Luyo hemorrhagic fever vaccine. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 For GPC L Schematic diagram of the variant sequence; Figure 2 For GPC L Figure showing the alignment results of the variant sequence with Lassa virus (strain Mouse / Sierra Leone / Josiah / 1976) (LASV) Uniport: P08669; Figure 3 For GPC L A schematic diagram of the three-dimensional structure of the variant; Figure 4 For GPC L Images of SDS-PAGE and Western Blot results for variant detection; Figure 5 The diagram shows the binding activity analysis results of the immunized mouse procedure and immune serum. Figure 6 The graph shows the results of the neutralizing activity analysis of serum from immunized mice. Figure 7 The figure shows the results of the broad-spectrum neutralizing activity analysis of serum from immunized mice. Detailed Implementation

[0020] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0021] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0023] Definition of noun In this invention, variants and mutants are synonymous.

[0024] As used herein, the term "prevention" means reducing the risk of disease in an individual or group, or mitigating the severity of disease or delaying its progression, through a series of proactive interventions. Specifically, in this invention, "prevention" refers to administering the drug of this application prior to Luyovirus infection, which can alleviate Luyo hemorrhagic fever following Luyovirus infection. Luyo hemorrhagic fever is characterized by acute fever, bleeding tendency, and multiple organ failure as its main clinical manifestations. Disease prevention is a proactive health management concept; by "intervening" to avoid or mitigate the harm of disease, its value far exceeds that of treatment after the onset of disease.

[0025] As used herein, the term "treatment" refers to a series of medical actions that involve intervention through medical means, techniques, or methods to address an existing disease, with the aim of eliminating the cause, alleviating symptoms, controlling disease progression, promoting tissue repair, restoring function, or reducing pain. Specifically, in this application, "treatment" refers to administering the drug of this invention after Luyovirus infection, which can improve acute fever, bleeding tendency, and multiple organ failure.

[0026] In this invention, "subject" refers to an individual who voluntarily participates in and receives research interventions (such as drugs, vaccines, medical devices, surgery, or behavioral interventions) or serves as an observation subject in clinical trials or biomedical research.

[0027] In a first aspect, the present invention provides a pre-fusion conformational mutant of the Luyovirus GPC protein, the Luyovirus GPC protein comprising GP1 subunit and GP2 subunit; comprising the following mutations: (1) Replace the enzyme cleavage site “RKLM” between the GP1 subunit and the GP2 subunit in the GPC protein with (GGGGS)3 flexible linker; (2) The extracellular region of GP2 was extracted, and a G69P mutation was introduced at position 69 of the extracellular amino acid sequence of wild-type GP2; (3) Add a T4 trimerization motif to the C-terminus of the GPC protein; the amino acid sequence of the T4 trimerization motif is shown in SEQ ID NO.7; The amino acid sequence of wild-type GP1 is shown in SEQ ID NO.8, and the amino acid sequence of the extracellular region of wild-type GP2 is shown in SEQ ID NO.11.

[0028] This invention provides a pre-fusion conformational mutant of the Luyovirus (LUJV) GPC protein. The wild-type Luyovirus GPC protein comprises three subunits: SPP, GP1, and GP2. The wild-type GPC protein structure is extremely unstable, severely affecting the immunogenicity of vaccine antigens. The LUJV GPC protein pre-fusion conformational mutant of this invention is based on the wild-type Luyovirus GPC protein (SEQ ID NO.1) with key mutation design: replacing the S1P / SKI-1 restriction site “RKLM” between GP1 and GP2 with three consecutive G4S flexible linker peptides, and introducing a proline site-directed mutation in the conformationally unstable loose region of the amino acid sequence at positions 288-295 of the GP2 subunit (specifically, at position 290 of the wild-type Luyovirus GPC protein shown in SEQ ID NO.1, introducing a G290P mutation; the amino acid sequence information shown in SEQ ID NO.1 is from NCBI Reference Sequence: NC_012776.1), truncating the extracellular region of GP2. The above-mentioned G290P mutation corresponds to the introduction of a G69P mutation at position 69 of the extracellular region amino acid sequence of wild-type GP2.

[0029] Finally, a T4 trimerization motif (nucleotide sequence shown in SEQ ID NO.4) was added to the C-terminus of GP2 to enable the variant protein to efficiently assemble into a stable trimer, completely mimicking the natural antigen morphology on the viral envelope surface, thus obtaining the Luyovirus GPC trimer pre-fusion conformational antigen variant.

[0030] Replacing the S1P restriction site “RKLM” between GP1 and GP2 with three consecutive G4S flexible linker peptides helps prevent the dissociation of the GP1 and GP2 subunits.

[0031] The 290th position is a non-complex glycosylation modification region. Site-directed mutagenesis at this position avoids affecting the normal function of GP2 and improves the stability of the pre-fusion conformation.

[0032] Adding a T4 trimerization motif (foldon) to the C-terminus of the GPC protein promotes protein assembly into the native trimer. Using foldons can "lock" the otherwise unstable protein into its native, pre-fusion trimer conformation, which helps to stimulate highly effective neutralizing antibodies.

[0033] In a preferred embodiment of the present invention, the Luyovirus GPC protein further includes a tPA signal peptide (Tissue Plasminogen Activator (tPA) Signal Peptide, human tissue plasminogen activator) at the N-terminus of the GP1 subunit, the amino acid sequence of which is shown in SEQ ID NO. 10.

[0034] The inventors discovered that the SPP subunit of the wild-type Luyovirus GPC protein cannot secrete GPC protein mutants outside the cell. Replacing the SPP subunit with the tPA signal peptide (Tissue Plasminogen Activator (tPA) Signal Peptide, a human tissue plasminogen activator) can promote the secretion of large quantities of GPC protein mutants outside the cell after synthesis, which helps to facilitate the efficient expression of recombinant antigens.

[0035] In a preferred embodiment of the present invention, the amino acid sequence of the pre-fusion conformational mutant of the Luyovirus GPC protein is shown in SEQ ID NO. 3. SEQ ID NO. 3 comprises, from N-terminus to C-terminus, the following: tPA signal peptide (Tissue Plasminogen Activator (tPA) Signal Peptide, human tissue plasminogen activator), GP1, (GGGGS)3 flexible linker, GP2 (extracellular segment), and T4 trimerization motif.

[0036] In a preferred embodiment of the present invention, the pre-fusion conformational mutant of the Luyovirus GPC protein can be a monomer or a multimer. The multimer is formed by the polymerization of multiple pre-fusion conformational mutant polypeptide single chains of the Luyovirus GPC protein. This includes, but is not limited to, trimers, hexamers, etc.

[0037] Secondly, the present invention provides a nucleic acid molecule that encodes a pre-fusion conformational mutant of the Luyovirus GPC protein described above.

[0038] As used herein, the term "nucleic acid molecule" refers to a sequence of nucleoside or nucleotide monomers composed of natural bases, sugars, and interglycosylation (backbone) bonds. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid molecules of this invention can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences and may contain natural bases, including adenine, guanine, cytosine, thymine, and uracil. Modified bases may also be present. Examples of these modified bases include nitrogenous and denitrogenated adenine, guanine, cytosine, thymine, and uracil; and xanthine and hypoxanthine.

[0039] In a preferred embodiment of the present invention, the nucleotide sequence is shown in SEQ ID NO. 6. SEQ ID NO. 6 is a codon-optimized nucleotide sequence, which helps to improve eukaryotic expression efficiency.

[0040] Thirdly, the present invention provides a recombinant vector comprising the aforementioned nucleic acid molecules.

[0041] The term "vector" is used herein in its most common sense and includes any intermediate medium for nucleic acids that enables the introduction of said nucleic acids, for example, into prokaryotic, eukaryotic cells, or viruses. Vectors of this type are preferably replicated and / or expressed in cells. The term "vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. The term "plasmid," as used herein, generally refers to a construct of extrachromosomal genetic material, typically a circular double-stranded DNA that can replicate independently of chromosomal DNA. Any plasmid and vector can be used as long as it can replicate and remain stable within a host.

[0042] In one alternative embodiment, the vector is an expression vector, and an important feature of an expression vector is that it typically contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0043] In a preferred embodiment of the present invention, the recombinant vector is a eukaryotic expression vector or a recombinant viral vector; the eukaryotic expression vector is, for example, pcDNA 3.1 or pcDNA4.

[0044] In a preferred embodiment of the present invention, the recombinant viral vector is selected from vesicular stomatitis virus vector.

[0045] In a preferred embodiment of the present invention, the base vector of the vesicular stomatitis virus vector is a VSV plasmid that lacks its own G protein encoding gene. In a preferred embodiment of the present invention, a Kozak sequence is positioned upstream of the start codon ATG sequence of the recombinant vector. Adding a tPA sequence at the N-terminus and introducing a Kozak sequence near ATG facilitates the efficient expression of the pre-fusion conformational mutant of the Luyovirus GPC protein.

[0046] Fourthly, the present invention provides a recombinant cell or recombinant bacteria comprising the above-described nucleic acid molecule or the above-described recombinant vector.

[0047] The term "recombinant cell" refers to any cell that can be transformed or transfected with exogenous nucleic acids. According to the invention, the term "recombinant cell" includes prokaryotic (e.g., *Escherichia coli*) or eukaryotic cells (e.g., mammalian cells, particularly human cells, yeast cells, and insect cells). Mammalian cells, such as those derived from humans, mice, hamsters, pigs, goats, or primates, are particularly preferred. Cells can originate from multiple tissue types and comprise primary cells and cell lines. Nucleic acids may be present in the host cell in single-copy or two or more copies, and in one embodiment, are expressed in the recombinant cell.

[0048] In one alternative implementation, the recombinant cells are eukaryotic cells.

[0049] In one alternative implementation, the recombinant cells are mammalian cells.

[0050] In one alternative implementation, the recombinant cells are eukaryotic cells such as 293T, Expi 293F, and BHK-21, which can achieve efficient expression and purification of recombinant antigens or be used for recombinant virus packaging.

[0051] Fifthly, the present invention provides the use of pre-fusion conformational mutants of Luyovirus GPC proteins, nucleic acid molecules, recombinant vectors, recombinant cells or recombinant bacteria in the preparation of drugs or vaccines for the treatment or prevention of Luyo hemorrhagic fever; In a preferred embodiment of the present invention, the application includes any of the following: (1) Preparation of protein vaccines based on pre-fusion conformational mutants of Luyovirus GPC protein; (2) Preparation of DNA vaccines based on nucleic acid molecules; (3) Prepare RNA vaccines based on mRNA molecules obtained by transcription of nucleic acid molecules.

[0052] In a preferred embodiment of the present invention, the vaccine further includes an adjuvant. In a preferred embodiment of the present invention, the adjuvant is selected from at least one of Freund's complete adjuvant, Freund's incomplete adjuvant, Toll-like receptor agonists, RIG-I-like receptor agonists, NOD-like receptor agonists, C-type lectin receptors, STING agonists, bacterial toxins and their derivatives, saponins, cytokines, and other adjuvants; other adjuvants are selected from at least one of heat shock proteins, A151, GTP-GDP, sodium fluoride, alkyl polyacrylate polymers, and dimethyl dioctadecyl quaternary ammonium bromide (DDA).

[0053] In a preferred embodiment of the present invention, the Toll-like receptor agonist is selected from at least one of CpG-ODN, CpG 1018, peptidoglycan, lipoteichoic acid, MPLA (monophospholipid A), imiquimod, remiquimod, bacterial flagellin, and Poly I:C (polyinosinic-cytotoxic protein). Furthermore, the Toll-like receptor agonist includes, but is not limited to, agonists of TLR1, TLR2, TLR4, TLR5, and TLR9.

[0054] This invention successfully prepared a pre-fusion conformational mutant of Luyovirus GPC by expression in Expi 293F cells and subsequent purification. Mice were immunized with this mutant after emulsification with either Freund's complete adjuvant or Freund's incomplete adjuvant, and the binding and neutralizing activities of the serum were periodically measured. Experimental results showed that this pre-fusion conformational mutant effectively induced a humoral immune response and produced high levels of neutralizing antibody activity; even at a 640-fold dilution, the inhibition rate against viral infection remained at approximately 50%.

[0055] This invention provides the application of the above-mentioned mRNA in the preparation of Luyovirus mRNA vaccines. Through multiple sequence engineering modifications such as 5'-UTR / 3'-UTR optimization, codon bias optimization, mRNA secondary structure stabilization, and PolyA tail modification, highly stable and efficiently expressed optimized mRNA sequences can be obtained. Furthermore, efficient delivery vectors such as liposomes and nanoparticles are used to encapsulate and deliver the optimized mRNA into the host, thereby effectively stimulating a protective immune response.

[0056] In a sixth aspect, the present invention provides the use of pre-fusion conformational mutants of Luyovirus GPC proteins, nucleic acid molecules, recombinant vectors, recombinant cells or recombinant bacteria in the preparation of vaccines or pharmaceuticals for inducing a protective immune response against arenavirus.

[0057] A protective immune response refers to a series of specific immune responses produced by the host's immune system after encountering a pathogen (such as a virus). These responses effectively eliminate the pathogen, limit its replication in the body, and prevent reinfection. The core result is that the individual acquires immunity against the pathogen, which is usually manifested as no disease or significantly reduced symptoms upon re-exposure to the same pathogen.

[0058] In a preferred embodiment of the present invention, induction includes: administering a drug to a subject in need; In a preferred embodiment of the present invention, the method of administering the drug to the subject in need is selected from any of the following: oral, intramuscular injection, or electrotransmission.

[0059] In a seventh aspect, the present invention provides a vaccine or drug comprising: the pre-fusion conformational mutant of the Luyovirus GPC protein described above, the nucleic acid molecule or recombinant vector described above, and pharmaceutically acceptable excipients. In a preferred embodiment of the present invention, the vaccine or drug is an injectable or oral formulation.

[0060] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0061] Example 1 This embodiment describes the design, construction, and expression of a pre-fusion conformational variant (or mutant) of the GPC protein trimer.

[0062] In order to design a soluble expression (recombinant protein secreted into the extracellular space without aggregation and precipitation, existing in a dissolved state, usually corresponding to correct folding and biological activity) and structurally stable pre-fusion GPC mutant antigen, this invention extracts the extracellular region sequence of wild-type GPC (SEQ ID NO. 1) and introduces mutations using the crystal structure of the pre-fusion conformation GPC (PDB: 8P4T) as a model.

[0063] (1) To improve stability and avoid dissociation of GP1 and GP2, the S1P / SKI-1 site (Site1Protease) “RKLM” is replaced with GGGGSGGGGSGGGGS flexible linker.

[0064] (2) To stabilize the extracellular region of the soluble GPC in the pre-fusion conformation, we introduced the G290P mutation at position 290 of the wild-type Luyovirus GPC protein shown in SEQ ID NO.1 and introduced a proline mutation at position 288-295 of the loosely bound region G290, which is conformationally unstable, to spatially hinder the folding process inside GP2. The amino acid sequence information shown in SEQ ID NO.1 is from NCBI Reference Sequence: NC_012776.1.

[0065] SEQ ID NO.2 is the mutant amino acid sequence lacking the tPA and T4 trimerization motifs, containing only the extracellular amino acid sequences of GP1 and GP2. The mutated G290 corresponds to position 242 in SEQ ID NO.2 or position 265 in SEQ ID NO.3. SEQ ID NO.3 is the amino acid sequence including the extracellular segments of tPA, GP1, and GP2, and the T4 trimerization motif.

[0066] The extracellular amino acid sequence of wild-type GP2 is shown in SEQ ID NO.11. The G290P mutation mentioned above corresponds to the introduction of a G69P mutation at position 69 of the extracellular amino acid sequence of wild-type GP2. The extracellular region of GP2 was truncated and fused. The amino acid sequence of wild-type GP2 is shown in SEQ ID NO.9 (including the extracellular and intracellular regions).

[0067] (3) In addition, to maintain the GPC trimer structure on the viral membrane surface, a T4 trimerization motif was added to the C-terminus, enabling the GPC monomer to form a natural trimer structure. This GPC mutant was named GPC. L (SEQ ID NO. 3). The nucleotide sequence of the T4 trimerized motif is shown in SEQ ID NO. 4 (codon-optimized sequence).

[0068] Structural diagrams of the pre-fusion GPC mutant and the pre-fusion GPC wild type are shown below. Figure 1 As shown. GPC L See the diagram for the results of the variant sequence comparison. Figure 2 As shown, the three-dimensional structure is referenced. Figure 3 As shown.

[0069] A eukaryotic expression vector based on pcDNA3.1 encodes the cDNA of a GPC variant. Codon optimization was performed for expression and synthesis (the optimized nucleotide sequence is shown in SEQ ID NO. 6, with the GP1, GP2 extracellular, and T4 trimerization motifs from 5'-3'). The unoptimized nucleotide sequence is shown in SEQ ID NO. 5. The expression vector was transiently transfected into Expi293F cells using PEI MAX 40K transfection reagent. Cells were cultured in shake flasks at 37°C and 5% CO2 at 120 rpm. After 3 days of transfection culture, the sample was centrifuged at 5000×g for 10 min to remove the cell pellet; the supernatant was filtered through a 0.22 μm filter and then purified using Strep Tag II affinity packing material.

[0070] Coomassie Brilliant Blue Staining: Take the purified protein solution, add 6× protein loading buffer according to the ratio, mix thoroughly, and denature in a boiling water bath at 100 ℃ for 10 min; pipette 20 μL of sample for gel loading. Electrophoresis is initially run at a constant voltage of 80 V until the sample is concentrated and aggregated in the stacking gel; after the bromophenol blue indicator front enters the separating gel, increase the voltage to 120 V and continue electrophoresis until the indicator migrates to the bottom of the gel. After electrophoresis, peel off the gel and place it in Coomassie Brilliant Blue R-250 staining solution, and stain with a horizontal shaker at room temperature for about 30 min. The staining time can be adjusted flexibly according to the staining depth of the gel. Recover the staining solution, transfer the gel to double-distilled water (ddH2O) and destain with a shaker until the protein bands are clear and the background is transparent; finally, use a gel imaging system to acquire imaging data.

[0071] Western blot: Purify the protein solution, add 6× protein loading buffer according to the ratio, mix thoroughly, and incubate in a boiling water bath at 100 ℃ for 10 min for denaturation; pipette 20 μL of sample for gel loading. Electrophoresis is initially run at a constant voltage of 80 V until the sample is concentrated and aggregated in the stacking gel; after the bromophenol blue indicator front enters the separating gel, increase the voltage to 120 V and continue electrophoresis until the indicator migrates to the bottom of the gel.

[0072] Prepare the transfer buffer in advance and pre-cool it at 4 ℃. After activating the PVDF membrane with methanol for 30 s, immerse it together with the filter paper in the transfer buffer for equilibration. After peeling off the gel, cut off the stacking gel and excess gel edges and immerse it in the transfer buffer. When assembling the transfer sandwich, stack the filter paper, PVDF membrane, gel and filter paper in that order from bottom to top, removing air bubbles layer by layer. Transfer the membrane at a constant current of 1.6 A for 15 min. After the transfer was completed, the PVDF membrane was removed and blocked with 5% skim milk powder on a shaker at room temperature for 2 h. It was then washed 5 times with TBST for 5 min each time. A mouse Strep II-Tag monoclonal primary antibody (Aibotek, AE066) diluted 1:5000 was added and incubated overnight on a shaker at 4 ℃. It was then washed with TBST as above. Finally, it was incubated with HRP-labeled goat anti-mouse secondary antibody (Aibotek, AS003) diluted 1:5000 at room temperature for 1 h. After repeated washing with TBST, it was chemiluminescently developed using Millipore Immobilon ECLUltra chromogenic solution (WBULS0100).

[0073] GPC L The protein molecular weight (~62.3 kDa) results are shown in Figure 4. SDS-PAGE and Western Blot analysis results indicate that GPC... LThe protein bands were single and of good purity; after mutation of the restriction sites between GP1 and GP2, no cleavage occurred between GP1 and GP2 (Figure 4).

[0074] Example 2 This embodiment analyzes the binding and neutralizing activity of serum from immunized mice.

[0075] 1. To assess humoral immune responses, BALB / c mice (n=6) were injected intramuscularly at days 0, 14, 28, and 42 with an antigen (the pre-fusion conformational variant of the GPC protein trimer expressed in Example 1) fully emulsified with Freund's complete adjuvant (for primary immunization) or Freund's incomplete adjuvant (for booster immunization) at a volume ratio of 40 μg per mouse. Blood samples were collected periodically after the second immunization for the detection of binding and neutralizing activities.

[0076] The titer of GPC-specific binding antibodies in the serum of immunized mice was detected using an indirect ELISA method. Figure 5 The results showed that on day 56, the serum antibody titer reached 5.75 (Log10), which was the same as the level on day 42 and significantly higher than that on day 28 (5.00 Log10). In this embodiment, titer was defined as: serum sample OD 450 When the nm value is ≥ 2.1 times the OD value of the negative control, it is the reciprocal of the highest dilution factor of the corresponding serum.

[0077] 2. Construct Luyovirus recombinant VSV virus (i.e. Luyovirus chimeric recombinant virus) and perform neutralization assays to analyze the neutralizing antibody titers in immune serum.

[0078] Specifically, a recombinant Luyovirus based on VSV as the backbone and carrying an eGFP reporter gene was constructed, and the neutralizing antibody titer of immune serum was analyzed using a neutralization assay with this recombinant virus.

[0079] (1) Using vesicular stomatitis virus carrying the eGFP reporter gene as the backbone vector, replace its envelope protein G with Luyo virus GPC to construct Luyo virus recombinant virus (i.e. Luyo virus chimeric recombinant virus).

[0080] The process began with molecular cloning, inserting the Luyovirus (LUJV) GPC gene into a VSV backbone plasmid (pVSV-ΔG-eGFP, purchased from addgene) that lacked its own G protein gene. Subsequently, this recombinant genome plasmid and support plasmids expressing VSV N, P, G, and L proteins (pBS-VSV G, pBS-VSV N, pBS-VSV P, and pBS-VSV L) were co-transfected into BHK-21 cells (purchased from ATCC) already infected with poxvirus vTF7-3 to provide T7 RNA polymerase, thereby rescuing the original virus.

[0081] To promote the effective assembly and release of viral particles, the harvested viral fluid was used to infect another batch of BHK-21 cells expressing exogenous VSVG protein (the VSVG G vector used was pCAGGS).

[0082] Finally, the rescue was confirmed by observing the cytopathic effect and the expression of the green fluorescent reporter gene (GFP). The recombinant virus was harvested and amplified. Its genome and replication ability are derived from the VSV backbone, while the surface of the viral particles displays the GPC protein of LUJV, thus mimicking the entry characteristics of LUJV.

[0083] (2) To assess humoral immune response, BALB / c mice (n=6) were injected intramuscularly on days 0, 14, 28 and 42 with a dose of 40 μg per mouse. The antigen was fully emulsified with Freund's complete adjuvant (for primary immunization) or Freund's incomplete adjuvant (for booster immunization) at a volume ratio of 1:1. Blood samples were collected periodically after the second immunization for the detection of antibody neutralizing activity.

[0084] First, heat-inactivated serum was serially diluted 1:20. The diluted serum samples were then mixed with an equal volume of recombinant virus solution at MOI=0.1 and incubated at 37 ℃ for 1 h to prepare a serum-virus mixture. This mixture was used to infect Vero E6 cells for 1 h, after which the supernatant was removed, and the cells were cultured in DMEM medium containing 2% FBS for 24 h. Cell samples were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then incubated with DAPI (4′,6-diamidindole-2-phenylindole) in the dark for nuclear fluorescence staining. After staining, multi-field fluorescence images were acquired using a high-content automated cell imaging system, and cell counting was performed using the accompanying analysis software. eGFP represented positive cells, characterizing the degree of viral infection in the host cells. The inhibition rate was calculated as: 1 - (number of eGFP cells in the experimental group / number of DAPI cells in the experimental group) / (number of eGFP cells in the infected group / number of DAPI cells in the infected group) × 100%.

[0085] Figure 6 shows that the higher the dilution factor of the heat-inactivated serum, the lower the level of residual neutralizing antibodies, the weaker the neutralizing ability against the virus, the fewer viruses are neutralized, and the higher the proportion of virus infection. Therefore, the serum dilution factor is positively correlated with the eGFP fluorescence intensity. Under a 640-fold dilution, the serum still maintained an inhibition rate of approximately 50% against viral infection.

[0086] Example 3 This embodiment uses recombinant Lassa virus (LASVrv) and recombinant Guanareto virus (GTOVrv) to verify the broad-spectrum neutralizing activity of Luyo virus immune serum. The method for constructing the recombinant virus is as described in Example 2; Figure 7 Broad-spectrum neutralization assays showed that mouse serum immunized with LUJV glycoprotein complex (GPC) did not exhibit cross-neutralization against other arena viruses.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pre-fusion conformational mutant of the Luyovirus GPC protein, characterized in that, Luyovirus GPC protein includes GP1 and GP2 subunits; it includes the following mutations: (1) Replace the enzyme cleavage site "RKLM" between the GP1 and GP2 subunits in the GPC protein with (GGGGS)3 flexible linker; (2) The extracellular region of GP2 was extracted, and a G69P mutation was introduced at position 69 of the extracellular amino acid sequence of wild-type GP2; (3) Add a T4 trimerization motif to the C-terminus of the GPC protein; the amino acid sequence of the T4 trimerization motif is shown in SEQ ID NO.7; The amino acid sequence of wild-type GP1 is shown in SEQ ID NO.8, and the amino acid sequence of the extracellular region of wild-type GP2 is shown in SEQ ID NO.

11.

2. The pre-fusion conformational mutant of the Luyovirus GPC protein according to claim 1, characterized in that, The Luyovirus GPC protein also includes a tPA signal peptide at the N-terminus of the GP1 subunit, the amino acid sequence of which is shown in SEQ ID NO.

10. Preferably, the amino acid sequence of the pre-fusion conformational mutant of the Luyovirus GPC protein is shown in SEQ ID NO.

3.

3. A nucleic acid molecule, characterized in that, It encodes a pre-fusion conformational mutant of the Luyovirus GPC protein as described in any one of claims 1-2.

4. The nucleic acid molecule according to claim 3, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

6.

5. A recombinant vector, characterized in that, It includes the nucleic acid molecule as described in any one of claims 3-4; Preferably, the recombinant vector is a eukaryotic expression vector or a recombinant viral vector; Preferably, the recombinant viral vector is selected from vesicular stomatitis virus vector; Preferably, the base vector of the vesicular stomatitis virus vector is a VSV plasmid that lacks its own G protein encoding gene; Preferably, the recombinant vector has a Kozak sequence upstream of the start codon ATG sequence.

6. A recombinant cell or recombinant bacterium, characterized in that, It includes the nucleic acid molecule as described in any one of claims 3-4 or the recombinant vector as described in claim 5.

7. The use of the pre-fusion conformational mutant of the Luyovirus GPC protein as described in any one of claims 1-2, the nucleic acid molecule as described in any one of claims 3-4, the recombinant vector as described in claim 5, and the recombinant cell or recombinant bacterium as described in claim 6 in the preparation of a drug or vaccine for the treatment or prevention of Luyo hemorrhagic fever.

8. The application according to claim 7, characterized in that, The application includes any of the following: (1) A protein vaccine was prepared based on the pre-fusion conformational mutant of the Luyovirus GPC protein; (2) Prepare a DNA vaccine based on the nucleic acid molecule; (3) Prepare an RNA vaccine based on the mRNA molecule obtained by transcription of the nucleic acid molecule; Preferably, the vaccine further includes an adjuvant.

9. The use of the pre-fusion conformational mutant of Luyovirus GPC protein as described in any one of claims 1-2, the nucleic acid molecule as described in any one of claims 3-4, the recombinant vector as described in claim 5, or the recombinant cell or recombinant bacterium as described in claim 6 in the preparation of a vaccine or drug for inducing a protective immune response against Arenavirus; Preferably, the induction includes: Administer the drug to subjects who require it; Preferably, the method of administering the drug to the subject in need is selected from any of the following: oral, intramuscular injection, or electrotransfer.

10. A vaccine or drug, characterized in that, It includes: The pre-fusion conformational mutant of Luyovirus GPC protein according to any one of claims 1-2, the nucleic acid molecule according to any one of claims 3-4, or the recombinant vector according to claim 5, and pharmaceutically acceptable excipients; Preferably, the vaccine or drug is an injectable or oral formulation.

Images