A Lassa virus GPC protein pre-fusion conformation mutant and its application

Abstract

The present invention provides a prefusion conformational variant of the Lassa virus GPC protein. Based on the wild-type, the S1P site between GP1 and GP2 is replaced with a flexible linker peptide, a proline mutation is introduced in the conformationally unstable loose region of GP2, and a trimerization motif T4 fib is added to the C-terminus of GP2. The trimer of the prefusion conformational variant of the Lassa virus GPC protein retains important neutralizing antibody epitopes of the prefusion conformation, can induce efficient and long-term humoral and cellular immune responses, and protect mice from Lassa pseudovirus infection. The present invention also provides the use of the prefusion conformational variant of the Lassa virus GPC protein in the preparation of a drug or vaccine for the treatment or prevention of Lassa 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 applications thereof.

[0002] This application claims priority to 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 prevalent primarily in West Africa. Lassa fever is a viral hemorrhagic fever that can cause severe multi-organ failure, bleeding, and even death in infected individuals, posing a significant health threat to people in areas where the virus is prevalent. An estimated 100,000 to 300,000 people are infected with LASV annually, with 5,000 to 10,000 dying from the disease. Currently, there are no FDA-approved vaccines or antiviral drugs specifically for the treatment of Lassa fever (Nat Rev Microbiol 21(2):87-96(2023)).

[0004] The research and development of a Lassa fever vaccine has been included in the WHO's priority disease research and development program 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. Currently, 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)). However, no vaccine has been approved for use.

[0005] Lassa virus belongs to the Arenaviridae family, genus Arenavirus. Viruses are typically round or oval, with a diameter of approximately 80-150 nm. Virions consist of a nucleocapsid and an envelope. The nucleocapsid has a helically symmetrical structure and contains the viral genetic material. The envelope, derived from the host cell membrane, is embedded with virus-encoded glycoproteins. The genetic material of Lassa virus consists of single-stranded negative-sense RNA (S) fragments, divided into two segments: L (large) and S (small). The L fragment encodes viral proteins such as RNA polymerase, while the S fragment encodes the viral nucleoprotein (NP) and the glycoprotein complex (GPC). GPC is the sole antigen on the Lassa virus membrane and the sole target for 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, mediating viral attachment and entry; GP2 participates in the fusion process between the virus and the host cell membrane.

[0006] In its natural state, GPC is displayed on the viral surface as a trimer. GPC is structurally unstable and prone to spontaneously transition from a high-energy prefusion conformation to a low-energy postfusion conformation. Multiple GPC structural studies have revealed that most known Lassa virus neutralizing antibodies target conformational epitopes spanning multiple discrete domains. Recombinantly expressed GPC antigens rapidly dissociate into monomers, destroying neutralizing epitopes while exposing multiple immunodominant non-neutralizing epitopes within the trimer. GPC's unstable structure, prone to conformational changes, complex surface glycosylation, and the existence of multiple Lassa virus strains present significant challenges in designing a broad-spectrum Lassa fever vaccine antigen (Nature 603:174–179 (2022); Science 356:923–928 (2017)).

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

[0008] To achieve the above objectives, the present invention first provides a prefusion conformational variant of the Lassa virus GPC protein. The Lassa virus GPC protein consists of two subunits, GP1 and GP2. The wild-type GPC protein is structurally unstable, which affects the effectiveness of the immunogen. The prefusion conformational variant of the Lassa virus GPC protein described herein, based on the wild-type GPC protein (SEQ ID NO. 1), includes mutations at the following positions: replacing the S1P site between GP1 and GP2 with a (G4S)3 flexible linker peptide, and introducing a proline mutation at amino acid positions 326-333 in the GP2 sequence, a loose region known to be 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 prefusion 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 prefusion conformation antigen variant, which exists in the form of a trimer or a multimer.

[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 transcription from the above-mentioned DNA molecule.

[0014] Fourth, the present invention provides a vector containing the aforementioned DNA molecule, which is used to carry, replicate, and express the DNA molecule. The vector can be a prokaryotic expression vector, a eukaryotic expression vector, or a viral vector. In a specific embodiment of the present invention, the vector is the eukaryotic expression vector pcDNA 3.1. A specific construction strategy of the present invention is to add a tPA sequence to the N-terminus of the amino acid sequence encoding the GPC mutant, add a Kozak sequence near the 5' start codon ATG of the DNA sequence encoding the GPC mutant, and ligate the gene to the eukaryotic expression vector pcDNA 3.1 via restriction enzyme cleavage sites.

[0015] In an alternative embodiment of the present invention, the vector is a lentiviral vector. The present invention obtains a Lassa virus pseudovirus carrying the 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 host cells containing the aforementioned DNA molecules. In a specific embodiment of the present invention, the eukaryotic expression vector pcDNA 3.1 constructed above was transformed into eukaryotic cells Expi293F to express and purify the antigenic protein, thereby obtaining a Lassa virus GPC trimer prefusion conformation variant.

[0017] In another specific embodiment of the present invention, the above-mentioned lentiviral packaging system was used to co-transfect the vector pDC316-LASV-GPC containing the Lassa virus GPC encoding gene and the helper plasmid pNL4-3.Luc-RE into 293T cells, 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 a prefusion conformational variant of the Lassa virus GPC protein through expression in Expi293F cells and subsequent purification. This variant was used in combination with aluminum adjuvant and CpG to immunize mice, and serum antibody testing was performed. The experimental results showed that the trimer mutant GPCv2 significantly increased neutralizing antibody levels against Lassa virus GPC compared to 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 , approximately 100 times the antibody titer stimulated by GPCv1. Antibody subclasses IgG1 and IgG2a were also significantly higher than those in the GPCv1 group after two injections. In combination with other adjuvants, antibodies against GP1 were also significantly higher than those in the control GPCv1 group.

[0021] In these applications, the prefusion conformational variant of the Lassa virus GPC protein can be used alone or in combination with other small chemical molecules or biomacromolecules to form compositions or conjugates for the preparation of Lassa fever treatments. These drugs can be formulated into common pharmaceutical dosage forms such as injections, sprays, and tablets. The GPC-modified antigen GPCv2 of the present invention significantly increases the level of neutralizing antibodies against Lassa virus in immunized animals compared to GPCv1. The antigen preparation method of the present invention is simple and has promising application prospects.

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

[0023] Seventh, the present invention provides a use of a molecule containing the aforementioned polynucleotide in the preparation of a nucleic acid vaccine. In a specific embodiment of the present invention, the aforementioned use can be provided using conventional techniques in the art based on the aforementioned polynucleotide molecule as shown in SEQ ID NO: 3 or 5. For example, a Lassa fever DNA vaccine can be constructed by using a eukaryotic expression vector such as pcDNA3.1 as a backbone, adding a Kozak sequence before the ATG start codon of the aforementioned polynucleotide gene, and then immunizing the host to achieve immune protection through intramuscular administration, electroporation, or a combination of both.

[0024] Finally, the present invention provides the use of the aforementioned mRNA in the preparation of a Lassa fever mRNA vaccine. For example, based on the mRNA sequence transcribed from the DNA molecule represented by SEQ ID NO: 3 or 5, the 5' UTR and 3' UTR are selected, the mRNA secondary structure is optimized, the Poly A tail is optimized, the mRNA codons are optimized, and modified nucleotides are selected to obtain a stably expressed optimized sequence. The mRNA vaccine can then be delivered to the host using a high-efficiency delivery system such as liposomes to exert its immune protective 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 amino acid sequence position 328; (2) replacement of the S1P restriction site with a flexible G4S linker; and (3) addition of a T4 phage trimer sequence at 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 significantly improved. Mice produced neutralizing antibodies after two immunizations with GPCv2, and the neutralizing antibodies lasted until 120 days 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 between GPCv1 and GPCv2 variants;

[0027] Figure 2 Schematic diagram of the three-dimensional structure of GPCv1 and GPCv2 variants;

[0028] Figure 3.SDS-PAGE profiles of GPCv1 and GPCv2 variants;

[0029] Figure 4 HPLC analysis of GPCv1 and GPCv2 variants;

[0030] Figure 5 ELISA analysis of the binding activity of neutralizing antibodies to GPCv1 and GPCv2 proteins;

[0031] Figure 6 SPR analysis of the binding activity of neutralizing antibodies to GPCv1 protein;

[0032] Figure 7 SPR analysis of the binding activity of neutralizing antibodies to GPCv2 protein;

[0033] Figure 8 ELISA was used to detect the titer of GPC-specific binding antibodies in the serum of immunized mice at different time points;

[0034] Figure 9 Analysis of serum antibody subclasses in immunized mice;

[0035] Figure 10 . Analysis of receptor-binding subunit GP1-specific antibodies in immunized mice;

[0036] Figure 11 .Analysis of neutralizing activity of serum antibodies in immunized mice;

[0037] Figure 12 Fluorescence analysis of the protective effect of GPCv2 on pseudovirus-challenged mice;

[0038] Figure 13 .Statistical graph of fluorescent protein Fluc values ​​in mice after Lassa pseudovirus challenge;

[0039] Figure 14 .ELISA was used to detect the specific antibody levels induced by GPCv2 and different adjuvants;

[0040] Figure 15 .Cellular immune analysis of GPCv2. DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to 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.

[0042] Example 1. Design, Construction, and Expression of GPC Protein Trimer Prefusion Conformation Variants

[0043] In order to design a pre-fusion GPC mutant antigen that is soluble and structurally stable, the present invention intercepted the extracellular region sequence of wild-type GPC (Josiah Strain) (SEQ ID NO.1) and introduced mutations based on the crystal structure of the pre-fusion conformation of GPC (PDB: 7PUY; 5VK2). (1) To improve stability and prevent the dissociation of GP1 and GP2, the S1P site (site 1 protease) "RRLL" was replaced with a flexible linker of GGGGSGGGGSGGGGS. (2) To stabilize the soluble GPC extracellular region in the pre-fusion conformation, we introduced a proline mutation at A328 in the loose region of GP2 amino acid sequence 326-333, which is unstable in conformation, to spatially hinder the refolding process within GP2. This GPC mutant was named GPCv1 (SEQ ID NO.2).

[0044] On the basis of 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, so that the GPC monomer formed a natural trimer structure, and named this GPC mutant GPCv2 (SEQ ID NO.4) (see Figure 1 and Figure 2 ).

[0045] GPC variant cDNAs were codon-optimized and synthesized using pcDNA 3.1-based eukaryotic expression vectors (optimized codons for GPCv1 are shown in SEQ ID NO. 3, and optimized codons for GPCv2 are shown in 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, 5% CO2, and 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 as follows: 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).

[0046] Example 2. Structural Analysis of GPCv2

[0047] After affinity purification with Strep-Tag and streptomycin, the product was analyzed using high-performance liquid chromatography (HPLC). The running buffer was PBS, and the analytical column used was a G5000PWXL (TSK, Japan). After degassing and rinsing the instrument, the column was connected and PBS was run at a flow rate of 1 mL / min. After the baseline stabilized, the sample information and run time were set in the software. The sample was added to the HPLC sample bottle, capped, and placed in the corresponding position on the machine's turntable. The program was started to run the experiment. HPLC analysis showed that both GPCv1 and GPCv2 exist in both monomeric and trimeric protein conformations, but GPCv2 has a higher trimeric content ( Figure 4 ).

[0048] Example 3. ELISA and SPR analysis of neutralizing antibody epitopes displayed by GPCv2

[0049] To verify whether the designed GPC mutants maintain the pre-fusion trimeric conformation, we verified the antigenicity of GPCv2 by enzyme-linked immunosorbent assay and surface plasmon resonance binding assay.

[0050] Following the reported grouping of neutralizing antibodies 37.7H, 37.2D, 25.1C, 12.1F, and 8.9F (Natcommunication 7:1 1544 (2016)), we selected one or two neutralizing antibodies from each of the GPC-A, GPC-B, GPC-C, and GP1-A groups and first assessed their binding to GPCv2 using ELISA. The specific procedure was as follows: 1 µg / mL of Lassa virus GPCv2 protein was coated in carbonate-bicarbonate buffer (pH 9.6) onto a 96-well high-binding microplate (Corning, USA) and incubated overnight at 4°C. The plates were then blocked in PBS containing 2% BSA at 37°C for 1 hour and washed with PBST (PBS + 0.1% Tween-20). Mouse serum serially diluted in dilution buffer (PBS containing 0.2% BSA) was added to the plate and incubated at room temperature for 1 hour. HRP-conjugated goat anti-mouse IgG (Abcam, UK) was diluted 10,000-fold and added to the plate. The plate was then incubated for 1 hour and washed with PBST. Color was developed for 5 minutes in the dark at room temperature using 100 µL of TMB substrate solution (Solaibao Technology Co., Ltd., Beijing, China). The reaction was terminated by adding 50 µL of stop solution (Solaibao Technology Co., Ltd., Beijing, China), and emission was measured at 450 nm (SPECTRA MAX 190). The endpoint titer was defined as the reciprocal of the highest serum dilution that produced an absorbance ≥ 2.1 times that of the negative control serum. ELISA results showed that all mAbs in each group, except for the neutralizing antibody 8.9F in group GPC-C, stably bound to GPCv2. Neutralizing antibody 37.2D in group GPC-B bound to the region between two monomers on GPC, representing a conformational epitope. We observed that GPCv2 had significantly improved binding activity to 37.2D (approximately 10-fold higher affinity) compared to monomeric GPCv1, suggesting that it may better represent the neutralizing antibody epitope of the GPC-B group ( Figure 5 ).

[0051] Secondly, monoclonal antibody binding analysis was performed on a Biacore 3000 (GE Healthcare, UK). A Lassa virus neutralizing antibody was used at 0.5 μg / ml on a Cytiva sensor chip Protein A. The Lassa virus neutralizing antibody was first captured at concentrations between 50 (0) and 500 (Rmax) "Response" for interaction analysis. The buffer used was HEPES-EP buffer (pH 7.5). After antibody conjugation, Lassa virus GPC variants were injected at different molar concentrations ranging from 100 nM to 0.78125 nM. After the experiment was completed, kinetic parameters and affinities were determined using a 1:1 antigen-antibody affinity curve fit. The SPR results also showed that the affinity of GPCv2 for 37.2D was significantly improved (the affinity of GPCv1 was 5.06 nM and that of GPCv1 was 0.379 nM, an increase of approximately 10 times). This result indicates that GPCv2 has better restored the bottom side of the pre-fusion conformation trimer, and the GPC-B family neutralizing antibody epitope ( Figure 6-Figure 7 The binding kinetic parameters of the six antibodies to GPCv1 and GPCv2 are shown in Table 1.

[0052] Table 1. Binding kinetic parameters of 6 antibodies to GPCv2

[0053]

[0054] Example 4. Analysis of Antibody Binding Activity in Sera from Immunized Mice

[0055] To assess 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 Lassa virus GPC-specific and Lassa virus-neutralizing antibody titers.

[0056] 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 both reached their peak values, and there was no significant difference between the two groups ( Figure 8 Serum antibody subclass analysis showed that the antibody titers of IgG1 and IG2a were comparable ( Figure 9 ).

[0057] In addition, we also found that on the 28th day after the initial immunization, mice in the GPCv2 group produced higher levels of receptor-binding subunit GP1-specific antibodies ( Figure 10 ) (GPCv1 average antibody titer 452, GPCv2 average antibody titer 77840), indicating that GPCv2 effectively induced the production of antibodies targeting GP1.

[0058] Example 5. Neutralizing activity analysis of serum antibodies in immunized mice

[0059] We constructed a lentivirus-based Lassa virus pseudovirus and analyzed the neutralizing antibody titers of immune sera using this pseudovirus neutralization assay.

[0060] Lassa virus pseudoviruses carrying full-length GPC were generated within 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). Pseudovirus-containing supernatants were harvested at 48 and 72 hours and replenished with fresh medium. Lassa virus pseudoviruses were filtered through a 0.45 μm filter, aliquoted, and stored at −80°C. Serial dilutions of heat-inactivated serum and titrated pseudoviruses were incubated at 37°C for 60 minutes and plated together with the 293T cell suspension in a 96-well microplate. Luciferase activity was measured using a Luciferase Assay System (Promega, Madison, WI, USA). The Reed–Muench method was used to calculate the neutralization titer of the half-maximal inhibitory concentration (IC50) of each mouse serum sample. No neutralizing antibody response was detected in any of the experimental groups after the initial immunization or the first booster dose. However, after the second booster dose, neutralizing antibody responses were detected in all six mice in the GPCv2 group, with IC50 values ​​ranging from 20 to 80. This neutralizing antibody response persisted until 120 days, while no significant neutralizing antibody response was detected in the PBS group or the GPCv1 group. Figure 11 ).

[0061] Example 6. Evaluation of the protective ability of immune GPCv2 against Lassa virus infection

[0062] Mice were challenged with Lassa virus pseudovirus. Through interaction between the GPC on the pseudovirus surface and viral receptors on the cell surface, the pseudovirus entered cells and expressed the Fluc reporter gene. Mice (n=4) were immunized with 20 μg of protein, 20 μg of CpG, and 100 μg of aluminum adjuvant on days 0, 14, and 28. Control animals (n=4) received three immunizations with the same volume of PBS. Two weeks after the third vaccination, mice were injected intraperitoneally with the pseudovirus.

[0063] The expression of the Fluc gene carried by the pseudovirus can be detected from the first to the seventh day after infection ( Figure 12 The experimental results showed that on the 7th day after 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 ( Figure 12 ), the numerical statistical analysis of fluorescent protein is shown in Figure 13.

[0064] Example 7. Analysis of Antibody Levels after Combination of GPCv2 Antigen with Different Adjuvants

[0065] The results of GPC-specific antibody detection on days 14 and 28 after GPCv2 antigen was combined with different adjuvants are as follows: Figure 14 As shown. On day 14, the Al + CpG adjuvant produced the highest antibody level, reaching 4.73, followed by Addvax (3.70), R848 (3.41), and AS01 (3.27). The PBS group had the lowest antibody level, at 2.02. On day 28, the Al + CpG1826 adjuvant maintained the highest antibody level, at 6.30. Addvax and AS01 produced similar antibody levels, at 6.09 and 5.20, respectively. R848 produced an antibody level of 4.70, while the PBS group still had the lowest antibody level, at 2.10. Statistical analysis showed that on day 14, the Al + CpG group had a significant difference in antibody levels compared with the other groups (p < 0.05). On day 28, the Al + CpG group still had a significant difference compared with the other groups (p < 0.05), but 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.

[0066] Example 8. Analysis of cellular immunity in mice after immunization with GPCv2

[0067] As shown in Figure 15, the ELISpot plate images of different treatment groups (PBS, GPCv1, and GPCv2) show 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 the number of spots per 2×10 5 The number of spot-forming cells (SFCs) per spleen cell was expressed. The PBS group, serving as the control group, showed relatively few spots. The number of GPCv1 and GPCv2 spots was significantly higher than that in the PBS group. This suggests that GPCv2 has potential advantages in stimulating cellular immunity and warrants further study as an immunogen.

[0068] Statistical Analysis in Examples of the Present Invention: All assays were performed at least three times independently, and data are presented as mean ± standard error. Statistical analyses were performed in GraphPad Prism (version 9.0). Statistical significance between groups was assessed using a standard one-way analysis of variance with either Dunnett's 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 conformational variant of the Lassa virus GPC protein, characterized in that: The mutation positions of the pre-fusion conformational variant of the Lassa virus GPC protein based on the wild-type Lassa virus GPC protein as 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. The amino acid sequence of the pre-fusion conformational variant of the Lassa virus GPC protein is shown in SEQ ID NO:

4.

2. A DNA molecule encoding the prefusion conformational variant of the Lassa virus GPC protein according to claim 1, characterized in that: The sequence of the DNA molecule is shown in SEQ ID NO:

5.

3. An mRNA molecule obtained by transcribing the DNA molecule according to claim 2. A vector comprising the DNA molecule according to claim 2.

5. The carrier according to claim 4, characterized in that The vector is a lentiviral vector. A host cell containing the vector according to claim 5.

7. Use of the prefusion conformational variant of the Lassa virus GPC protein according to claim 1 in the preparation of a drug for the treatment or prevention of Lassa fever.

8. Use of the prefusion conformational variant of the Lassa virus GPC protein according to claim 1 in the preparation of a vaccine for preventing Lassa fever.

9. Use of the DNA according to claim 2 in the preparation of a Lassa fever DNA vaccine.

10. Use of the mRNA according to claim 3 in the preparation of a Lassa fever mRNA vaccine.

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