Lassa virus GPC protein pre-fusion conformation mutant and application thereof

Abstract

The invention provides a prefusion conformational variant of lasa virus GPC protein, which is characterized in that on the basis of a wild type, an S1P site between GP1 and GP2 is replaced by a flexible connecting peptide, proline mutation is introduced to a loose region with unstable conformation in GP2, and a trimeric motif T4 fib is added to the C-terminal of GP2, so that the prefusion conformational variant of the lasa virus GPC protein is obtained. The trimer of the prefusion conformation variant of the lassa virus GPC protein retains important neutralizing antibody epitopes of prefusion conformation, can induce efficient and long-term humoral immune and cellular immune responses, and protects mice from lassa pseudovirus infection. The invention also provides application of the prefusion conformational variant of the lasa virus GPC protein in preparation of drugs or vaccines for treating and preventing lasa fever.

Description

Technical Field

[0001] The present invention relates to the technical field of Lassa virus vaccines, and in particular to a Lassa virus GPC pre-fusion conformation mutant protein and an application thereof.

[0002] This application requests the priority of Chinese invention patent application 202411712983.1, filed on November 27, 2024. Background Art

[0003] Lassa virus (LASV) is the pathogen that causes Lassa fever (LF), which is mainly prevalent in West Africa. Lassa fever is a viral hemorrhagic fever that can cause severe multi-organ failure, bleeding, and even death in infected people, causing great harm to the health of people in areas where the virus is prevalent. An estimated 100,000 to 300,000 people are infected with LASV each year, of which 5,000 to 10,000 die from the disease. There is currently no FDA-approved vaccine or antiviral drug specifically for the treatment of Lassa fever (Nat Rev Microbiol 21(2):87-96(2023)).

[0004] The research and development of Lassa fever vaccines has been included in the WHO's priority disease research and development plan for urgent research and development. Although some progress has been made in the research of Lassa fever vaccines, it is still in the stage of continuous exploration. At present, there are many different forms of Lassa fever candidate vaccines that have shown a certain protective effect against viral infection in animal models, including DNA vaccines (Hum Vaccines Immunotherapy 2019. 15:2066–2074), RNA vaccines (Nat Communication 2023.14(1):5603), live attenuated vaccines and viral vector vaccines (npj Vaccines 4:8(2019)), but no vaccine has been approved for use.

[0005] Lassa virus belongs to the Arenavirus genus of the Arenaviridae family. Virus particles are usually round or oval with a diameter of about 80-150 nm. Virus particles are composed of a nucleocapsid and an envelope. The nucleocapsid has a helical symmetric structure and contains the genetic material of the virus. The envelope originates from the host cell membrane and has virus-encoded glycoproteins embedded on the surface. The genetic material of Lassa virus is a single-stranded negative-sense RNA, which is divided into two segments, L (large) and S (small). The L segment encodes proteins such as the virus's RNA polymerase, and the S segment encodes the virus's nucleoprotein NP and glycoprotein complex GPC (Glycoprotein Protein complex). GPC is the only antigen on the surface of the Lassa virus membrane and the only target for inducing neutralizing antibodies. The glycoproteins on the Lassa virus envelope are divided into two subunits, GP1 and GP2. GP1 is responsible for binding to host cell receptors and mediating the adsorption and invasion of the virus; GP2 is involved in the fusion process between the virus and the host cell membrane.

[0006] In its natural state, GPC is displayed on the virus surface as a trimer. GPC is structurally unstable and tends to spontaneously change from a high-energy pre-fusion conformation to a low-energy post-fusion conformation. According to multiple GPC structural studies, most known Lassa virus neutralizing antibodies target conformational epitopes across multiple discontinuous domains. Recombinantly expressed GPC antigens rapidly dissociate into monomers, resulting in the destruction of neutralizing antibody epitopes and the exposure of multiple immunodominant non-neutralizing antibody epitopes within the trimer. GPC is structurally unstable, prone to conformational changes, has complex surface glycosylation, and there are multiple Lassa virus strains, which poses certain difficulties in the design of a broad-spectrum Lassa fever vaccine antigen structure (Nature 603:174–179 (2022); Science 356:923–928 (2017)).

[0007] The object of the present invention is to provide a GPC variant, which can provide a stable GPC pre-fusion conformation and display broad-spectrum neutralizing antibody epitopes. Summary of the invention

[0008] Based on the above purpose, the present invention first provides a pre-fusion conformation variant of Lassa virus GPC protein. Lassa virus GPC protein includes two subunits GP1 and GP2. The structure of wild-type GPC protein is unstable, which affects the effect of immunogen. The mutation positions of the pre-fusion conformation variant of Lassa virus GPC protein described in the present invention based on the wild-type Lassa virus GPC protein (SEQ ID NO.1) include: replacing the S1P site between GP1 and GP2 with a (G4S) 3 flexible connecting peptide, and introducing a proline mutation at the loose region of 326-333 in the GP2 amino acid sequence where the conformation is unstable.

[0009] In a specific embodiment of the present invention, the amino acid sequence of the pre-fusion conformation variant of the Lassa virus GPC protein is shown in SEQ ID NO: 2, and the GPC mutant is named "GPCv1".

[0010] Based on the GPCv1 mutation, the mutation position of the pre-fusion conformation variant of the Lassa virus GPC protein described in the present invention also includes adding a trimerization motif T4 fib (sequence as shown in SEQ ID NO.6) to the C-terminus of GP2, thereby providing a Lassa virus GPC trimer pre-fusion conformation antigen variant, and the mutant exists in the form of a trimer or a polymer.

[0011] In a specific embodiment of the present invention, the amino acid sequence of the pre-fusion conformation variant of the Lassa virus GPC protein is shown in SEQ ID NO:4, and the mutant is named "GPCv2" in the present invention.

[0012] Secondly, the present invention provides a DNA molecule encoding the pre-fusion conformation variant of the above-mentioned Lassa virus GPC protein. In an optional embodiment, the DNA molecule is a codon-optimized DNA molecule, wherein the sequence of GPCv1 is shown in SEQ ID NO:3, and the sequence of GPCv2 is shown in SEQ ID NO:5.

[0013] Third, the present invention provides an mRNA molecule obtained by transcribing the above-mentioned DNA molecule.

[0014] Fourth, the present invention provides a vector containing the above-mentioned DNA molecule, and the vector is used to load, replicate, and express the DNA molecule. The vector can be a prokaryotic cell expression vector, a eukaryotic cell expression vector, or a viral vector. In a specific embodiment of the present invention, the vector is a eukaryotic cell expression vector pcDNA 3.1, and a specific construction strategy of the present invention is: adding a tPA sequence to the N-terminus of the amino acid sequence encoding the GPC mutant, adding a Kozak sequence near the 5' start codon ATG of the DNA sequence encoding the GPC mutant, and connecting the gene to the eukaryotic expression vector pcDNA 3.1 through the restriction site.

[0015] In an optional embodiment of the present invention, the vector is a lentiviral vector. The present invention obtains a Lassa virus pseudovirus carrying full-length GPC in an Env-deficient, luciferase-expressing, replication-incompetent human immunodeficiency virus (HIV)-1 backbone by using a lentiviral vector packaging system.

[0016] Fifth, the present invention provides a host cell containing the above DNA molecule. In a specific embodiment of the present invention, the eukaryotic expression vector pcDNA 3.1 constructed above is transformed into eukaryotic cells Expi293F to express and purify antigenic proteins, thereby obtaining Lassa virus GPC trimer pre-fusion conformation variants.

[0017] In another specific embodiment of the present invention, the vector pDC316-LASV-GPC containing the Lassa virus GPC encoding gene and the helper plasmid pNL4-3.Luc-RE were co-transfected into 293T cells using the above-mentioned lentiviral packaging system, and Lassa virus pseudoviruses carrying the full-length GPC were harvested at 48 and 72 hours.

[0018] Sixth, the present invention provides the use of monomers or polymers of the above-mentioned pre-fusion conformational variants of the Lassa virus GPC protein in the preparation of Lassa fever treatment, prevention drugs or vaccines.

[0019] The present invention successfully prepared the pre-fusion conformation variant of Lassa virus GPC protein through expression in Expi293F cells and purification. The mutant was combined with aluminum adjuvant and CpG to immunize mice, and serum antibody detection was performed. The experimental results showed that the trimer mutant GPCv2 could significantly improve the level of neutralizing antibodies against Lassa virus GPC compared with the mostly monomeric mutant GPCv1.

[0020] The results of the evaluation after immunizing mice with aluminum adjuvant and CpG showed that the antibody titer of GPCv2 after immunization can reach 10 6 , which is about 100 times the antibody titer stimulated by GPCv1. The antibody subclasses IgG1 and IgG2a were also significantly higher than those of the GPCv1 group after two injections. In combination with other adjuvants, the antibodies against GP1 were also significantly higher than those of the control GPCv1.

[0021] In the application, the pre-fusion conformational variant of the Lassa virus GPC protein can also be used alone or with other small chemical molecules, biological macromolecules to form a composition, or a conjugate for the preparation of a Lassa fever treatment drug, and the drug can be prepared in the form of injections, sprays, tablets and other common pharmaceutical dosage forms. Compared with GPCv1, the GPC modified antigen GPCv2 of the present invention can significantly improve the neutralizing antibody level of immunized animals against Lassa virus. The antigen preparation method of the present invention is simple and has good application prospects.

[0022] In a technical solution of a specific vaccine prepared according to the above application, the vaccine further includes an aluminum adjuvant and / or a CpG adjuvant.

[0023] Seventh, the present invention provides an application of a molecule containing the above-mentioned polynucleotide in the preparation of a nucleic acid vaccine. In a specific embodiment of the present invention, on the basis of the above-mentioned polynucleotide molecule as shown in SEQ ID NO: 3 or 5 provided by the present invention, the application can be provided by using conventional technical means in the art. For example, using eukaryotic expression vectors such as pcDNA3.1 as a skeleton, adding a Kozak sequence before the base of the ATG initiation codon of the gene of the above-mentioned polynucleotide molecule, a Lassa fever DNA vaccine is constructed, and the host is immunized by muscle / electroporation / a combination of the two to exert an immune protection effect.

[0024] Finally, the present invention provides the use of the above mRNA in the preparation of Lassa fever mRNA vaccine. For example, based on the mRNA sequence transcribed from the DNA molecule shown in SEQ ID NO: 3 or 5, the selection of 5'UTR and 3'UTR, optimization of the secondary structure of mRNA, optimization of the PolyA tail, optimization of mRNA codons, selection of modified nucleotides, etc. are performed respectively to obtain the optimized sequence with stable expression, and the mRNA vaccine is delivered to the host in combination with an efficient delivery system such as liposomes to exert an immune protection effect.

[0025] Based on the experience of antigen design and the structural information of Lassa virus GPC, the present invention carried out the following antigen modification based on the wild-type GPC prefusion conformation (PDB: 7PUY; 5VK2): (1) proline mutation at position 328 of the amino acid sequence; (2) replacing the S1P restriction site with a flexible G4S Linker; (3) adding a T4 phage trimer sequence to the C-terminus of the protein. The modified mutant GPCv2 can be efficiently expressed in the Expi293F expression system and retains the important neutralizing antibody epitopes of the prefusion conformation. The binding activity and affinity of GPCv2 to the neutralizing antibody 37.2D of the GPC-B group are greatly improved. Mice produced neutralizing antibodies after two immunizations with GPCv2, and the antibodies lasted until the 120th day after immunization. Subsequent experiments showed that immunization with GPCv2 can protect mice from Lassa virus pseudovirus infection. These findings indicate that the prefusion conformation variant GPCv2 of the Lassa virus GPC protein has the potential to be an effective candidate antigen for LASV vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 . Schematic diagram of sequence variation of GPCv1 and GPCv2 variants; Figure 2 . Schematic diagram of the three-dimensional structure of GPCv1 and GPCv2 variants; Figure 3 .SDS-PAGE profiles of GPCv1 and GPCv2 variants; Figure 4 . HPLC analysis of GPCv1 and GPCv2 variants; Figure 5 . ELISA analysis of the binding activity of neutralizing antibodies to GPCv1 and GPCv2 proteins; Figure 6 . SPR analysis of the binding activity of neutralizing antibodies to GPCv1 protein; Figure 7 . SPR analysis of the binding activity of neutralizing antibodies to GPCv2 protein; Figure 8 . ELISA was used to detect the serum GPC-specific binding antibody titer of immunized mice at different time points; Fig. 9 . Analysis of serum antibody subclasses in immunized mice; Fig.10 . Analysis of specific antibodies against the receptor binding subunit GP1 in immunized mice; Fig.11 .Analysis of neutralizing activity of serum antibodies in immunized mice; Fig.12 . Fluorescence analysis of the protective effect of GPCv2 on pseudovirus-challenged mice; Fig.13 .Statistical graph of the fluorescent protein Fluc values ​​in mice after Lassa pseudovirus attack; Fig.14 .ELISA was used to detect the specific antibody levels induced by GPCv2 and different adjuvants; Fig.15 .Analysis of cellular immunity level of GPCv2. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0028] Example 1. Design, construction and expression of GPC protein trimer pre-fusion conformation variants In order to design a pre-fusion GPC mutant antigen that can be expressed soluble and has a stable structure, the present invention intercepted the extracellular region sequence of the wild-type GPC (Josiah Strain) (SEQ ID NO.1) and introduced mutations based on the crystal structure of the pre-fusion conformation GPC (PDB: 7PUY; 5VK2). (1) In order to improve stability and avoid the dissociation of GP1 and GP2, the S1P site (site 1protease) "RRLL" was replaced with a flexible connection of GGGGSGGGGSGGGGS. (2) In order to stabilize the soluble GPC extracellular region in the pre-fusion conformation, we introduced a proline mutation at A328 in the loose region A328 of the GP2 amino acid sequence 326-333, which is unstable in conformation, to spatially hinder the refolding process inside GP2. The GPC mutant was named GPCv1 (SEQ ID NO.2).

[0029] Based on GPCv1, in order to restore the GPC trimer structure on the viral membrane surface, we added a T4 fib (GGYIPEAPRDGQAYVRKDGEWVLLSTFLTGG) trimerization motif to the C-terminus of GP2 to make the GPC monomer form a natural trimer structure, and named this GPC mutant GPCv2 (SEQ ID NO.4) (see Figure 1 and Figure 2 ).

[0030] Eukaryotic expression vectors based on pcDNA 3.1 encoding GPC variant cDNAs were codon-optimized for expression and synthesized (optimized codons for GPCv1 are shown as SEQ ID NO.3 and optimized codons for GPCv2 are shown as SEQ ID NO.5). All expression vectors were transiently transfected into Expi293F cells (Thermo Fisher Scientific, subcultured in Expi293™ Expression Medium) using ExpiFectamine 293 transfection reagent (Thermo Fisher Scientific). Cells were cultured in shake flasks at 37°C and 5% CO2 at 120 rpm. Six days after transfection, cells were harvested by centrifugation at 3500×g for 15 minutes. The supernatant was filtered through a 0.22 μm filter and stored at 4°C until purification. The GPC mutant was purified from the culture supernatant using a StrepTrap™ affinity chromatography column (Cytiva, Sweden) and further purified using a Superdex 200Increase 10 / 300 column (Cytiva, Sweden). The protein molecular weight of GPCv2 is Figure 3 As shown, SDS-PAGE showed that GPCv2 was almost entirely a trimer band with a molecular weight of approximately 300 kDa ( Figure 3 ).

[0031] Example 2. Structural analysis of GPCv2 After affinity purification with Strep-Tag and streptomycin, high performance liquid chromatography was used for analysis. The running buffer was PBS, and the analytical column used was G5000PWXL (TSK, Japan). After the instrument was degassed and rinsed, the column was connected and PBS was run at a flow rate of 1 mL / min. After the baseline was stable, the sample information and running time were set in the software, and the sample was added to the HPLC sample bottle, the lid was closed, and the bottle was placed in the corresponding position of the machine turntable. The program was started to run the experiment. HPLC analysis showed that both GPCv1 and GPCv2 had two protein conformations, monomer and trimer, but the trimer component of GPCv2 was higher ( Figure 4 ).

[0032] Example 3. ELISA and SPR analysis of neutralizing antibody epitopes displayed by GPCv2 To verify whether the designed GPC mutants maintain the pre-fusion trimer conformation, we verified the antigenicity of GPCv2 by enzyme-linked immunosorbent assay and surface plasmon resonance binding assay.

[0033] According to the reported grouping of neutralizing antibodies 37.7H, 37.2D, 25.1C, 12.1F, and 8.9F (Natcommunication 7:1 1544 (2016)), we took 1-2 neutralizing antibodies from each group of GPC-A, GPC-B, GPC-C, and GP1-A, and first evaluated their binding to GPCv2 by ELISA. The specific operation was as follows: 1 µg / mL of Lassa virus GPCv2 protein was coated on a 96-well high binding microplate (Corning, USA) in carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. Then, the plate was blocked in PBS containing 2% BSA at 37°C for 1 hour and washed with PBST (PBS + 0.1% Tween-20). Mouse sera serially diluted in dilution buffer (PBS containing 0.2% BSA) were added to the plate and incubated for 1 hour at room temperature. HRP-conjugated goat anti-mouse IgG (Abcam, UK) was diluted 10,000 times and added to the plate. Then, the plate was incubated for 1 hour and washed with PBST. Color was developed using 100 µL of TMB substrate solution (Solaibao Technology Co., Ltd., Beijing, China) for 5 minutes in the dark at room temperature, and then 50 µL of stop solution (Solaibao Technology Co., Ltd., Beijing, China) was added to stop the reaction, and the emission light was subsequently measured at 450 nm (SPECTRA MAX 190). The endpoint titer was defined as the reciprocal of the highest serum dilution factor that produced an absorbance ≥ 2.1 times the negative control serum value. The ELISA results showed that all groups of monoclonal antibodies, except for the neutralizing antibody 8.9F in the GPC-C group, were able to stably bind to GPCv2. The binding of the neutralizing antibody 37.2D in the GPC-B group spanned the region between two monomers on GPC, which belongs to the conformational epitope. We observed that GPCv2 had significantly improved binding activity to 37.2D (~10-fold increase in affinity) compared to monomeric GPCv1, suggesting that it may better represent the neutralizing antibody epitope of the GPC-B group ( Figure 5 ).

[0034] Secondly, monoclonal antibody binding analysis was performed on Biacore 3000 (GE Healthcare, UK). Cytiva sensor chip Protein A with 0.5 μg / ml Lassa virus neutralizing antibody was used. First, Lassa virus neutralizing antibody concentrations between 50 (0) and 500 (Rmax) "Response" were captured for interaction analysis. The buffer used was HEPES-EP buffer (pH 7.5). After coupling the antibody, Lassa virus GPC variants with different molar concentrations were injected, ranging from 100 nM to 0.78125 nM. After the experiment was completed, the kinetic parameters and affinity were determined using a 1:1 antigen-antibody affinity curve fit. The SPR experimental results also showed that the affinity of GPCv2 and 37.2D was significantly improved (GPCv1 affinity was 5.06nM, GPCv1 was 0.379 nM, an increase of about 10 times). This result indicates that GPCv2 better restores the bottom side of the trimer before fusion conformation, and the GPC-B family neutralizing antibody epitope across GP1 and GP2 ( Figure 6-Figure 7 The binding kinetic parameters of the six antibodies to GPCv1 and GPCv2 are shown in Table 1.

[0035] Table 1. Binding kinetic parameters of 6 antibodies to GPCv2

[0036]

[0037] Example 4. Analysis of antibody binding activity in sera of immunized mice To evaluate humoral immune responses, BALB / c mice (n = 6 per group) were immunized intramuscularly with GPCv1 and GPCv2 at a dose of 20 µg per mouse on days 0, 14, and 28. Serum was collected 14, 28, 42, and 120 days after the first immunization for the determination of Lassa virus GPC-specific antibody and Lassa virus neutralizing antibody titers.

[0038] The titer of GPC-specific binding antibody in serum after immunization was detected by ELISA. After the first immunization, the titer of GPC-specific binding antibody IgG induced by GPCv2 was significantly higher than that of GPCv1 ( Figure 8 After the first booster, it was also observed that GPCv2 induced higher GPC-specific binding antibody IgG titers ( Figure 8 After the second injection, the antibody titers induced by GPCv2 and GPCv1 reached their peak values, and there was no significant difference between the two groups ( Figure 8Serum antibody subclass analysis showed that the antibody titers of IgG1 and IG2a were comparable ( Fig. 9 ).

[0039] In addition, we also found that on the 28th day after the primary immunization, mice in the GPCv2 group produced higher levels of receptor binding subunit GP1-specific antibodies ( Fig.10 ) (GPCv1 average antibody titer 452, GPCv2 average antibody titer 77840), indicating that GPCv2 effectively induced the production of antibodies targeting GP1.

[0040] Example 5. Neutralizing activity analysis of serum antibodies in immunized mice We constructed a lentivirus-based Lassa virus pseudovirus and analyzed the neutralizing antibody titers of immune sera using this pseudovirus neutralization assay.

[0041] Lassa virus pseudoviruses carrying full-length GPC were generated in an Env-deficient, replication-incompetent human immunodeficiency virus (HIV)-1 backbone expressing luciferase. Briefly, pDC316-LASV-GPC and pNL4-3.Luc-RE were co-transfected into 293T cells using TurboFect (Thermo Fisher Scientific). Supernatants containing pseudoviruses were harvested at 48 and 72 h and replenished with fresh medium. Lassa virus pseudoviruses were filtered through a 0.45 μm filter, aliquoted, and stored at −80°C. Heat-inactivated serum and titrated pseudoviruses were serially diluted, incubated at 37°C for 60 min, and added to 96-well microplates along with 293T cell suspensions. Luciferase activity was measured using a Luciferase Assay System (Promega, Madison, USA). The half-maximal inhibitory concentration (IC50) neutralization titer of each mouse serum sample was calculated using the Reed–Muench method. After the primary immunization and the first booster, no neutralizing antibody response was detected in any of the experimental groups. However, after the second booster, neutralizing antibody responses were detected in all six mice in the GPCv2 group, with IC50 values ​​of 20-80, and the neutralizing antibody response lasted until 120 days, while no obvious neutralizing antibody response was detected in the PBS group and the GPCv1 group ( Fig.11 ).

[0042] Example 6. Evaluation of the protective ability of immune GPCv2 against Lassa virus infection Mice were challenged with Lassa virus pseudoviruses, which entered cells and expressed the Fluc reporter gene through the interaction of GPC on the pseudovirus surface with viral receptors on the cell surface. Mice (n=4) were immunized with 20μg protein, 20μg CpG, and 100μg aluminum adjuvant on days 0, 14, and 28. Control animals (n=4) were immunized with the same volume of PBS three times. Two weeks after the third vaccination, mice were injected intraperitoneally with pseudoviruses.

[0043] The expression of the Fluc gene carried by the pseudovirus can be detected from the first to the seventh day after infection ( Fig.12 The experimental results showed that on the 7th day after the challenge, the average fluorescence value of mice in the PBS group was 127600, the average fluorescence value of mice in the GPCv1 group was 98950, and the average fluorescence value of mice in the GPCv2 group was 78550, indicating that the GPCv2 immunization group provided better protection than the PBS group and the GPCv1 group ( Fig.12 ), the numerical statistical analysis of fluorescent protein is shown in Figure 13.

[0044] Example 7. Analysis of antibody levels after GPCv2 antigen combined with different adjuvants The results of GPC-specific antibody detection levels on days 14 and 28 when GPCv2 antigen was combined with different adjuvants are as follows Fig.14 As shown. On the 14th day, the antibody level produced by Al + CpG adjuvant was the highest, reaching 4.73, followed by Addvax (3.70), R848 (3.41), and AS01 (3.27), while the antibody level of the PBS immunization group was the lowest, at 2.02. On the 28th day, the Al + CpG1826 adjuvant still maintained the highest antibody level of 6.30, the antibody levels of Addvax and AS01 were similar, at 6.09 and 5.20, respectively, the antibody level of R848 was 4.70, and the antibody level of the PBS group was still the lowest, at 2.10. From the statistical analysis results, on the 14th day, the antibody level of the Al + CpG group was significantly different from that of other groups (p < 0.05). On the 28th day, the Al + CpG group was still significantly different from that of other groups (p < 0.05), and there was no significant difference between the Al + CpG group and the Addvax group (p > 0.05). Overall, Al + CpG adjuvant produced the highest antibody levels at both time points and was statistically significantly higher than some adjuvant groups, while the antibody level in the PBS group remained low throughout the experiment.

[0045] Example 8. Analysis of cellular immunity in mice after immunization with GPCv2 As shown in Figure 15, it can be observed from the ELISpot plate images of different treatment groups (PBS, GPCv1 and GPCv2) that the number of spots in the GPCv1 and GPCv2 groups is significantly greater than that in the PBS group, indicating that a stronger cellular immune response was induced. The bar graph on the right quantitatively displays these results, with 2×10 5 The number of spots in the PBS group was relatively small as the control group. The number of GPCv1 and GPCv2 was significantly higher than that in the PBS group. This suggests that GPCv2 has potential advantages in stimulating cellular immunity and is worthy of further study on its application value as an immunogen.

[0046] Statistical analysis in the examples of the present invention: All assays were repeated at least three times independently and data are presented as mean ± standard error. Statistical analysis was performed in GraphPad Prism (version 9.0). Statistical significance between groups was assessed using a common one-way ANOVA with Dunnett's multiple comparison test or Tukey's multiple comparison test. The statistical tests used are indicated in the relevant figure legends: * p < 0.05, **p < 0.01, *** p < 0.001, * *** p < 0.0001.

Claims

1. A prefusion conformation variant of the Lassa virus GPC protein, characterized in that: The mutation positions of the pre-fusion conformation variant of the Lassa virus GPC protein based on the amino acid sequence of the wild-type Lassa virus GPC protein shown in SEQ ID NO.1 include: replacing the S1P site between GP1 and GP2 with a (G4S)3 flexible connecting peptide, and introducing a proline mutation in the loose region 326-333 in the GP2 amino acid sequence where the conformation is unstable.

2. The pre-fusion conformation variant of the Lassa virus GPC protein according to claim 1, characterized in that: The amino acid sequence of the pre-fusion conformation variant of the Lassa virus GPC protein is shown in SEQ ID NO:

2.

3. The pre-fusion conformation variant of the Lassa virus GPC protein according to claim 1, characterized in that The mutation positions of the pre-fusion conformation variant of the Lassa virus GPC protein also include adding a trimerization motif at the C-terminus of GP2.

4. The pre-fusion conformation variant of the Lassa virus GPC protein according to claim 3, characterized in that The amino acid sequence of the pre-fusion conformation variant of the Lassa virus GPC protein is shown in SEQ ID NO:

4.

5. A DNA molecule encoding a pre-fusion conformational variant of the Lassa virus GPC protein according to claim 2 or 4, characterized in that: The sequence of the DNA molecule is shown in SEQ ID NO: 3 or 5.

6. An mRNA molecule obtained by transcribing the DNA molecule according to claim 5.

7. A vector comprising the DNA molecule according to claim 5.

8. The carrier according to claim 7, characterized in that The vector is a lentiviral vector.

9. A host cell containing the vector according to claim 7.

10. Use of the pre-fusion conformation variant of the Lassa virus GPC protein according to claim 2 or 4 in the preparation of a Lassa fever treatment, prevention drug or vaccine.

11. Use of the DNA according to claim 5 in the preparation of a Lassa fever DNA vaccine.

12. Use of the mRNA according to claim 6 in the preparation of a Lassa fever mRNA vaccine.

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