Nipah virus infectious clone based on alphavirus replicon and application of Nipah virus infectious clone in preparation of attenuated vaccine

Abstract

The invention belongs to the technical field of biology, and particularly discloses a Nipah virus infectious clone based on an alphavirus replicon and application of the Nipah virus infectious clone in preparation of attenuated vaccines. The infectious clone is obtained by further screening after an envelope glycoprotein for coding the Nipah virus is inserted on the basis of an alphavirus replicon vector. The virus-like vesicles rescued by utilizing the infectious clone disclosed by the invention can be continuously and autonomously replicated and amplified, are high in virion titer, stably express antigen protein, and have good safety, immunogenicity and immune protection efficacy, so that the virus-like vesicles can effectively resist challenge infection of wild type viruses, and can be used for preparing safe and effective attenuated vaccine strains.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a Nipah virus infectious clone based on an alphavirus replicon and its application in the preparation of an attenuated vaccine. Background Art

[0002] Nipah virus (NiV) is a highly pathogenic zoonotic virus belonging to the genus Henipavirus of the family Paramyxoviridae. It has diverse transmission routes, including contact with infected animals or consumption of contaminated food, and may also spread between humans. Preventing and controlling the spread of Nipah virus is crucial for protecting human health.

[0003] Vaccines are one of the most effective means of preventing virus infection and transmission. Currently, there is no approved vaccine for Nipah virus on the market, and most vaccines are still in the research and development stage, involving various strategies such as inactivated vaccines, subunit vaccines, attenuated vaccines, nucleic acid vaccines, and viral vector vaccines. Among them, live attenuated vaccines can induce strong humoral and cellular immunity and are an ideal vaccine type. However, traditional live attenuated vaccines are mainly obtained by continuous passage of the virus to attenuate its virulence, and there is a risk of reversion through in vivo replication. In recent years, recombinant viral vector vaccines formed by chimeric expression of viral envelope antigen proteins using vesicular stomatitis virus, adenovirus, etc. have provided new strategies for the development of novel attenuated vaccines. Although these recombinant vector vaccines have higher safety, they contain multiple self-structural proteins, and related research shows that there are still potential safety hazards. Therefore, it is urgent to continue to develop safe and effective novel vector vaccine technologies for the prevention and control of such viruses.

[0004] To address the above problems, the present invention prepared an infectious clone of a recombinant vector vaccine that expresses only the Nipah virus envelope protein using an alphavirus replicon as a vector. The genome of the recombinant vector vaccine only contains the non-structural proteins of the alphavirus and the envelope protein of the Nipah virus, and there is no possibility of assembling into pathogenic alphavirus or Nipah virus under natural conditions, which fully reduces the safety hazards of the recombinant virus vaccine. Summary of the Invention

[0005] The object of the present invention is to provide a Nipah virus infectious clone based on an alphavirus replicon, and the nucleotide sequence of the infectious clone is shown as SEQ ID NO.2.

[0006] An object of the present invention is to provide virus-like vesicles rescued from the above infectious clone.

[0007] The last object of the present invention is to provide the application of the above infectious clone or virus-like vesicles in the preparation of a Nipah virus vaccine. To achieve the above object, the present invention takes the following technical measures:

[0008] Obtaining a Nipah virus infectious clone based on an alphavirus replicon:

[0009] The applicant tandemly constructed the Nipah virus Malaysia strain NiV-G and NiV-F gene fragments into the TC-83 replicon. After passage screening, an infectious clone was obtained that could rescue virus particles with immunogenicity, stable yield, and high virus titer. The polynucleotide sequence of the infectious clone is shown in SEQ ID NO.2.

[0010] The protection scope of the present invention also includes:

[0011] RNA corresponding to the above-mentioned infectious clone DNA.

[0012] Virus-like vesicles rescued from the above-mentioned infectious clone or RNA.

[0013] A recombinant cell containing the above RNA and / or virus-like vesicles.

[0014] The above-mentioned cell, preferably, is a Vero cell or a BHK-21 cell.

[0015] A pharmaceutical composition comprising the above-mentioned infectious clone, RNA, virus-like vesicles, and / or recombinant cell.

[0016] Use of the above-mentioned infectious clone, RNA, virus-like vesicles, recombinant cell, and / or pharmaceutical composition in the preparation of a drug for treating or preventing Nipah virus infection.

[0017] Use of the above-mentioned infectious clone, RNA, virus-like vesicles, recombinant cell, and / or pharmaceutical composition in the preparation of a Nipah virus vaccine.

[0018] For the above-mentioned use, preferably, the vaccine is a live attenuated vaccine.

[0019] Compared with the prior art, the present invention has the following advantages and effects:

[0020] 1. The present invention constructs an infectious clone of Nipah virus based on an alphavirus replicon for the first time through reverse genetics technology and successfully rescues immunogenic virus-like vesicles.

[0021] 2. The infectious clone provided by the present invention can be cultured and amplified in Vero to obtain high-titer virus. The acquisition method is simple, which provides convenience for the subsequent production of vaccines and saves costs.

[0022] 3. The virus particles rescued from the infectious clone provided by the present invention can be continuously amplified during continuous passage, the antigen gene stably exists, and the antigen protein is stably expressed, having good genetic stability.

[0023] 4. The virus particles rescued by the infectious clone provided by the present invention will not cause diseases even when hamsters are infected at high doses, and are proven to be sufficiently attenuated in hamsters, so they have high safety.

[0024] 5. The virus particles rescued by the infectious clone provided by the present invention can be used as an immunizing agent to directly immunize hamsters and can induce a high level of specific antibody response. After challenge with a high lethal dose of WT NiV, it can provide complete immunoprotection for hamsters.

[0025] Using the infectious clone provided by the present invention, a safe and effective live attenuated vaccine strain can be prepared, which has good application prospects and important strategic significance for coping with the potential threat of Nipah virus in China and the world. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the construction of the Nipah virus infectious clone VEEV-NiV-G-F and indirect immunofluorescence detection of virus-like vesicles rescued by the infectious clone;

[0027] Among them, A: Schematic diagram of the VEEV-NiV-G-F clone expressing NiV-G and NiV-F with the Venezuelan equine encephalitis virus vaccine strain TC-83 replicon as the vector; B: Indirect immunofluorescence images of BHK-21 cells transfected with in vitro transcribed RNA of VEEV-NiV-G-F at different time points; C: Indirect immunofluorescence images of BHK-21 cells infected with Nipah virus virus-like vesicles VEEV-NiV-G-F VLVs at different time points.

[0028] Figure 2 Plaque morphology of VEEV-NiV-G-F VLVs and growth curve of NiV VLVs;

[0029] Among them, A: Plaque morphology of VEEV-NiV-G-F VLVs on Vero cells; B: Growth curve of NiV VLVs on Vero.

[0030] Figure 3 Detection of the infectivity and antigen expression stability of NiV VLVs;

[0031] Among them, A: Indirect immunofluorescence detection of the expression of NiV-G protein after NiV VLVs of P1, P5, and P10 generations infect Vero cells;

[0032] B: RT-PCR comparison of the sizes of NiV-G and NiV-F protein genes of NiV VLVs of P1, P5, and P10 generations in cells;

[0033] C: Comparison of plaque morphology of NiV VLVs from P1, P5, and P10 generations on Vero cells.

[0034] Figure 4 Pathogenicity detection of NiV VLVs in rodents;

[0035] A: Weight changes of golden hamsters after intraperitoneal injection of NiV VLVs;

[0036] B: Survival rate of golden hamsters after intraperitoneal injection of NiV VLVs;

[0037] C: Viremia of golden hamsters after intraperitoneal injection of NiV VLVs.

[0038] Figure 5 Detection of serum antibody titers in animals after intraperitoneal immunization with NiV VLVs;

[0039] A: ELISA antibody titer detection in golden hamsters immunized with NiV VLVs;

[0040] B: Neutralizing antibody titer detection in golden hamsters immunized with NiV VLVs.

[0041] Figure 6 Detection results of the protection rate provided by NiV VLVs after intraperitoneal immunization of golden hamsters;

[0042] A: Weight changes of golden hamsters immunized with NiV VLVs after challenge with NiV-WT;

[0043] B: Survival rate of golden hamsters immunized with NiV VLVs after challenge with NiV-WT;

[0044] C: Viral loads in the lungs, spleens, and brains of golden hamsters immunized with NiV VLVs after challenge with NiV-WT. Specific implementation manners

[0045] The experimental methods such as PCR, enzyme digestion, ligation, transformation, RNA extraction, RT-PCR, etc. involved in this part, unless otherwise specified, are all conventional methods in this field. The reagents or materials described in the present invention, unless otherwise specified, are all publicly available or sourced from commercial channels. The following are only several specific embodiments of the present invention. Obviously, the present invention is not limited to the following embodiments and there can be many variations. Therefore, any modifications or improvements made by those skilled in the art based on the disclosed content of the present invention shall fall within the scope of protection required by the present invention.

[0046] Example 1:

[0047] Construction of an infectious clone of Nipah virus virus-like vesicles and virus rescue:

[0048] In the process of constructing the infectious clone of this embodiment, we conducted multiple sequence explorations and optimizations to ensure that the clones finally obtained can rescue virus-like vesicles with infectivity and immunogenicity. Specifically, this embodiment first attempts to insert the glycoprotein G and fusion protein F of Nipah virus into the TC-83 replicon separately to construct VEEV-NiV-G and VEEV-NiV-F clones. However, no significant increase in cytopathic or immunofluorescence-positive cells was observed after transfection of cells, indicating that the expression of NiV-G or -F protein alone is not enough to form infectious virus-like vesicles. On this basis, this embodiment further expresses NiV-G, NiV-F and matrix protein NiV-M (known to promote Nipah virus assembly) in pairs through the TC-83 subgenomic promoter, and constructs VEEV-NiV-GF, VEEV-NiV-GM and VEEV-NiV-FM clones respectively. The experimental results showed that only the VEEV-NiV-GF clone, a combination that simultaneously expresses NiV-G and NiV-F, could successfully rescue Nipah virus-like vesicles that efficiently replicate and amplify, while other combinations failed to achieve this goal.

[0049] 1. Construction of virus-like vesicle VEEV-NiV-GF infectious clone: ​​According to the gene sequence of Nipah virus Malaysia strain (GenBank accession no.AY029767.1), the primers required for the amplification of NiV-G and NiV-F to construct infectious clones were synthesized (primers are shown in Table 1). The templates pET33b-Nipah-G and pET33b-Nipah-F plasmids used for amplification of NiV-G and NiV-F gene fragments were provided by the laboratories of Researcher Guan Wuxiang and Researcher Xiao Gengfu of the Wuhan Institute of Virology, Chinese Academy of Sciences, respectively. The plasmids were constructed by inserting the G or F protein gene in the Malaysia strain of Nipah virus into the pET33b plasmid through the two restriction sites of NdeI and XhoI; the Venezuelan equine encephalitis virus vaccine strain TC-83 replicon plasmid used as a vector was constructed by the Flavivirus Infection and Prevention Discipline Group of the Wuhan Institute of Virology. The genomic sequence of the TC-83 replicon (CN110643632A) is the vaccine strain sequence of TC-83 with the structural protein (Capsid and the entire E protein) deleted (GenBank accessionno.L01443.1), and was constructed into the pACYC177 plasmid vector through the two restriction sites of NotI and XbaI to obtain the vector plasmid of pACYC177-VEEV Replicon (TC-83).

[0050] Amplification of NiV-G and NiV-F gene fragments: Using pET33b-Nipah-G and pET33b-Nipah-F as templates, the primers used are shown in Table 1. PCR amplification was performed with Primestar Max enzyme (purchased from Takara). The PCR reaction system was as follows: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 15 s, treatment at 72°C for 10 min, for 35 cycles.

[0051] The PCR products were recovered. The recovered NiV-G fragment was double-digested with AscI / PacI and ligated with the same enzymes to the Venezuelan equine encephalitis virus vaccine strain TC-83 replicon vector pACYC177-VEEV Replicon (TC-83), and then transformed into Escherichia coli competent HB101. The plasmid was identified as correct by DNA sequencing and named VEEV-NiV-G. On this basis, the recovered NiV-F fragment was double-digested with PacI / NotI and ligated with the same enzymes to VEEV-NiV-G and then transformed into Escherichia coli competent HB101. The plasmid was identified as correct by DNA sequencing and named the infectious clone VEEV-NiV-G-F (shown in SEQ ID NO.1). The schematic diagram of clone construction is as Figure 1 shown in A in

[0052] 2. Linearization of plasmid and phenol-chloroform extraction: Digest 10 μg of the infectious clone plasmid VEEV-NiV-G-F with FastDigest NotI at 37 °C for 30 min. After verifying complete digestion by 1% agarose gel electrophoresis, add 100 μL of Tris-saturated phenol (purchased from Beijing Solarbio Science & Technology Co., Ltd.) to the digestion product, mix well by shaking, centrifuge at 17,000 g for 5 min, aspirate the supernatant into a new centrifuge tube, and add 100 μL of sterile water to the original centrifuge tube, mix well by shaking, and centrifuge at 17,000 g for 5 min; aspirate the supernatant and combine it with the supernatant obtained in the previous step (total volume is about 200 μL), add 200 μL of chloroform (purchased from Sinopharm Chemical Reagent Co., Ltd.), mix well, and centrifuge at 17,000 g for 5 min; aspirate the supernatant into an RNAase-free EP tube (about 150 - 200 μL), add one-tenth volume of sodium acetate, pH 5.2, 3 mol / L (purchased from Sinopharm Chemical Reagent Co., Ltd.) and 2.5 volumes of absolute ethanol (purchased from Sinopharm Chemical Reagent Co., Ltd.), mix well, place at -20 °C overnight, centrifuge at 17,000 g for 5 min; discard the supernatant, add 1 mL of 70% ethanol for washing, centrifuge at 17,000 g for 5 min, and discard the supernatant; air dry at room temperature for 15 min, and finally add 10 μL of RNAase-free water to dissolve. Measure the concentration of DNA using Thermo Scientific NanoDrop 2000, and detect the quality of DNA using 1% agarose gel electrophoresis. Store at -20 °C for later use.

[0053] 3. In vitro transcription of RNA: Use 1 μg of the above phenol-chloroform extracted linearized product as a template, and use the in vitro transcription kit T7 mMESSAGE mMACHINE kit (purchased from Thermo Fisher Scientific, USA) to prepare the RNA of recombinant VEEV-NiV-G-F according to the kit instructions. Measure the concentration of RNA using Thermo Scientific NanoDrop 2000, and detect the quality of RNA using 1% freshly prepared agarose gel electrophoresis. Store at -80 °C for later use.

[0054] 4. Transfect the RNA of VEEV-NiV-G-F obtained by in vitro transcription into BHK-21 cells using the liposome transfection method, and use BHK-21 cells without transfected RNA as a control: One day before transfection, inoculate 2.5×10 5Three 10 mm×10 mm coverslips were placed in each 35 mm cell culture dish with BHK-21 cells, so that the cells reached about 80% on the day of transfection. When transfecting, first discard the medium in the culture dish, wash twice with 1 mL of Opti-MEM and discard, then add 1 mL of Opti-MEM to make the cells in an infiltrated state. Add 1 mL of Opti-MEM to a 1.5 mL RNase-free EP tube, add 4 μL of DMRIE-C, and then add 1 μg of the in vitro transcribed RNA of VEEV-NiV-G-F (the control group does not add RNA), mix well by inverting up and down. Discard the Opti-MEM in the culture dish, and add the mixed solution to the dish (the action should be gentle, do not blow and beat the cells). After culturing in a 37 °C carbon dioxide incubator for 4-6 h, discard the culture, and then add 2 mL of DMEM medium containing 2% FBS to each. Observe the cell status of the experimental group and the control group under a microscope, and fix the cells on one slide with 5% acetone fixative (purchased from Sinopharm Chemical Reagent Co., Ltd.) at 24, 48, and 72 h after transfection respectively. Fix at room temperature for 15 min, wash three times with PBS, store at 4 °C, and collect the cell culture supernatant at 96 h after transfection (at this time, obvious cytopathic effects have occurred) to obtain virus-like vesicles of VEEV-NiV-G-F, named VEEV-NiV-G-F VLVs, and store at -80 °C.

[0055] 5. Indirect immunofluorescence (IFA) was used to detect the expression of envelope glycoproteins of VEEV-NiV-G-F VLVs: Incubate the cell slides stored at 4 °C in the above step 4 at room temperature with the primary antibody, and the antibody is a murine NiV-G antibody (diluted 1:500), which was kindly provided by Researcher Deng Fei of Wuhan Institute of Virology, Chinese Academy of Sciences. Incubate the primary antibody at room temperature for 1 h, wash three times with PBS, and then incubate the secondary antibody at room temperature in the dark. The antibody is a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody (diluted 1:125). After incubating the secondary antibody for 40 min, wash three times with PBS, take out the glass slides, make marks, and place a small drop of 95% glycerol at each marked place. Place the coverslip with the cell side down on the glycerol drop and observe under a fluorescence microscope at a magnification of 100× ( Figure 1 in B).

[0056] The results showed that positive cells could be observed 24 h after transfection of BHK-21 with VEEV-NiV-G-F RNA, and with the extension of the transfection culture time, the positive rate increased significantly, showing an obvious amplification phenomenon. The above results preliminarily indicated that VEEV-NiV-G-F could produce virus and rescue infectious virus-like vesicles.

[0057] 6. IFA detection of the infectivity amplification ability of the rescued VEEV-NiV-G-F VLVs: To further verify the virus production phenomenon of VEEV-NiV-G-F and determine its infectivity, the cell culture supernatant collected 72 h after transfection of RNA in step 4 above was used to infect BHK-21 cells, and IFA detection was performed at 24, 48, and 72 h ( Figure 1 as shown in C in [reference]), and the method was the same as before. The results showed that obvious positive cell clusters could be observed 24 h after VEEV-NiV-G-F VLVs infected BHK-21 cells, and the positive rate increased significantly with the extension of time, which fully indicated that expressing the glycoprotein of Nipah virus with alphavirus replicon could produce infectious virus particles.

[0058] Table 1. Primers used for the construction of the VEEV-NiV-G-F infectious clone

[0059]

[0060] Example 2:

[0061] Plaque morphology identification of VEEV-NiV-G-F VLVs provided by the present invention and obtaining a stable and high-yield virus particle infectious clone:

[0062] The rescued VEEV-NiV-G-F VLVs in Example 1 were subjected to plaque assay to identify the plaque morphology: 1×10 5 Vero cells were seeded in each well of a 24-well cell culture plate. When the cell confluence reached 90%, the medium in the wells was discarded, and 100 μL of the virus collected in step 4 of Example 1 diluted 10-fold with DMEM medium containing 2% FBS was added. Adsorb at 37°C for 1 h, and shake well every 15 min. After adsorption, the virus solution in each well was aspirated and discarded, and a covering material containing DMEM medium with 2% FBS and 1% methylcellulose was added. Incubate in an incubator at 37°C and 5% CO 2 for 3 days. After plaque formation, stain with a staining solution containing 1% crystal violet and 3.7% formaldehyde. After treating at room temperature for 3 h, discard the staining solution in the wells, rinse the bottom of the wells with running water, dry, and then count and convert it into virus titer, and observe the plaque morphology at the same time ( Figure 2 as shown in A in [reference]). It can be seen from the figure that VEEV-NiV-G-F VLVs can produce clear plaque morphology on Vero.

[0063] However, the titer of VEEV-NiV-G-F VLVs 72 h after transfection and rescue was only 1.2×10 2PFU / mL, which is insufficient to meet the requirements of mass production. Therefore, we passaged and domesticated and screened VEEV-NiV-G-F VLVs on Vero cells. After passage to the 30th generation, the virus titer increased significantly, reaching 1×10 5 PFU / mL, and it was named NiV VLVs.

[0064] Through genome sequencing, multiple adaptive mutation sites (nsP1-A67G / nsP1-T521P / nsP2-A264T / nsP2-A792T / NiV-G-V64A / NiV-G-T117A / NiV-F-P418H / NiV-F-D530G) were determined, and an infectious clone capable of efficiently rescuing NiV VLVs was obtained, and its nucleotide sequence is shown in SEQ ID NO.2.

[0065] On this basis, the growth curve of NiV VLVs was measured. 2.5×10 5 Vero cells were inoculated in a 35 mm cell culture dish. Under the culture conditions of 37 °C and 5% CO 2 When the confluence reached 60%, 500 μL of diluted NiV VLVs was added to the small dish according to a multiplicity of infection (MOI) of 0.001. After adsorption in an incubator at 37 °C and 5% CO 2 for 2 h, the virus solution was discarded, and 2 mL of DMEM medium containing 2% FBS was added to each well. The cells were cultured at 37 °C and 5% CO 2 Under the culture conditions, 400 μL of virus supernatant was collected every 24 h after infection, and 400 μL of DMEM medium containing 2% FBS was supplemented as virus-like at different time points and stored at -80 °C. According to the above plaque assay method, the virus titers collected at different time points were measured, and a growth curve was plotted ( Figure 2 B in). The results showed that when NiV VLVs were inoculated on Vero at an MOI of 0.001, the titer reached the highest at 72 h, and the highest titer was 2×10 5 PFU / mL.

[0066] Example 3:

[0067] The NiV VLVs provided by the present invention can be continuously infected and stably express NiV-G:

[0068] 1. Infect Vero cells with the NiV VLVs obtained by subculture screening in Example 2 (as P0) at a multiplicity of infection (MOI) of 0.001. After 3 days, when obvious cytopathic effect (CPE) appears, collect the cell supernatant (P1 generation). Propagate three strains of the virus in parallel for each generation, and repeat this step 10 times. After infecting cells with the viruses of P1, P5, and P10 generations again, detect the protein expression using specific antibodies against NiV-G and NiV-F proteins by immunofluorescence assay (IFA) (the method is the same as in Example 1) ( Figure 3 in A). The IFA results show that NiV-G and NiV-F protein expressions can be detected in the NiV VLVs of P1, P5, and P10 generations; Extract the total cellular RNA from the cells collected in P1, P5, and P10 generations according to the instructions of the Trizol kit, and perform RT-PCR ( Figure 3 in B) using the aforementioned primers for amplifying the NiV-G and NiV-F protein genes, and perform electrophoresis analysis on it. The results show that the sizes of the target bands obtained by RT-PCR amplification of the cell samples of P1, P5, and P10 generations are the same, indicating that after 10 generations of passage of NiV VLVs, the sizes of the NiV-G and NiV-F protein genes do not change.

[0069] 2. Take the supernatant virus liquids collected in P1, P5, and P10 generations for plaque morphology comparison (the method is the same as in Example 2). The plaques ( Figure 3 in C) show that there are no obvious differences in the plaque sizes of the NiV VLVs of P1, P5, and P10 generations, and they are all relatively uniform.

[0070] Example 4:

[0071] Biosafety of the Nipah virus virus-like vesicles NiV VLVs provided by the present invention:

[0072] For the pathogenicity assessment of the virus-like vesicles NiV VLVs: Divide 4- to 6-week-old golden hamsters into two groups (5 per group), and infect the hamsters by intraperitoneal injection with 1×10 6 PFU of NiV VLVs and an equal volume of PBS control. Monitor the body weight changes ( Figure 4 in A), survival rate ( Figure 4 in B), and viremia ( Figure 4 in C) within the first 3 days after infection of the hamsters within 14 days after infection. The results are as Figure 4 shown. After intraperitoneal infection with NiV VLVs, the hamsters are in good health, all survive, their body weights do not decrease significantly, and no viremia is detected. The above results show that NiV VLVs have no obvious pathogenicity to golden hamsters and meet the safety target requirements of the present invention.

[0073] Example 5:

[0074] Application of Nipah virus virus-like vesicles NiV VLVs provided by the present invention in the preparation of live attenuated vaccines:

[0075] This example examines the humoral immunity induced by virus-like vesicles NiV VLVs. Since specific antibodies are the main "weapons" for preventing Nipah virus and Lassa virus infections, artificial active immunization (vaccine immunization) is the main way to induce specific antibodies. Therefore, specific antibodies are one of the indicators for evaluating the efficacy of Nipah virus and Lassa virus vaccines.

[0076] For the evaluation of the humoral immunity induced by the vaccine strain NiV VLVs, female golden hamsters aged 4 - 6 weeks were randomly divided into 3 groups (5 animals / group), and were immunized intraperitoneally with 1×10 5 PFU (abbreviated as 1E5 in the figure), 1×10 6 PFU (abbreviated as 1E6 in the figure) of NiV VLVs twice at an interval of 28 days. Intraperitoneal injection of an equal volume of PBS was used as a control. On the 14th and 28th days after the second immunization, mouse sera were collected, and the specific antibodies against Nipah virus ( Figure 5 A in the figure) and the neutralizing antibody levels ( Figure 5 B in the figure) were detected by enzyme-linked immunosorbent assay ELISA and plaque reduction neutralization test PRNT. The results showed that after the second immunization with the vaccine strain NiV VLVs, dose-dependent NiV-specific IgG antibodies and neutralizing antibodies could be induced in hamsters: the IgG antibody titers could reach 1:800 (1E5 group) and 1:3,200 (1E6 group) respectively on the 14th - 28th days, and the neutralizing antibody titers could reach 1:40 - 80 (1E5 group) and 1:80 - 160 (1E6 group) respectively on the 14th - 28th days. The above results indicate that NiV VLVs have good immunogenicity and meet the immunogenicity target requirements of the present invention.

[0077] Example 6:

[0078] Application of Nipah virus virus-like vesicles NiV VLVs provided by the present invention in the preparation of live attenuated vaccines:

[0079] This example examines the protective effect against NiV-WT challenge after immunizing golden hamsters with the vaccine strain NiV VLVs. Female golden hamsters aged 4 - 6 weeks were randomly divided into 3 groups (5 animals / group), and were immunized intraperitoneally with 1×10 5 PFU (abbreviated as 1E5 in the figure), 1×10 6 PFU (abbreviated as 1E6 in the figure) of NiV VLVs twice at an interval of 28 days. Intraperitoneal injection of an equal volume of PBS was used as a control. On the 28th day after the second immunization, in a biosafety level 4 laboratory, with 1×10 4 TCID 50Hamsters were challenged by intraperitoneal injection with NiV-WT. The changes in body weight of hamsters within 14 days after challenge ( Figure 6 A in Figure 6 ), survival rate ( Figure 6 B in Figure 6 ) and viral loads in the lungs, spleens and brain tissues on the 5th day after challenge ( Figure 6 C in Figure 6 ) were monitored. The results are shown in Figure 6 . As shown, all hamsters in the two-dose immunized groups with NiV VLVs survived (100%), showed no significant change in body weight, and no live virus was detected in the lungs, spleens and brain tissues. After challenge with PBS, all hamsters died within 7 days after challenge, and high-titer virus was detected in the lungs, spleens and brain tissues. The above results indicate that NiV VLVs has good protective effect against challenge as a vaccine candidate strain.

Claims

1. A Nipah virus infectious clone based on an alphavirus replicon, wherein the polynucleotide sequence of the infectious clone is shown in SEQ ID NO.

2.

2. The RNA corresponding to the infectious clone DNA according to claim 1.

3. The virus-like vesicle rescued by the infectious clone of claim 1 or the DNA of claim 2.

4. A recombinant cell, comprising the RNA of claim 2 and / or the virus-like vesicle of claim 3.

5. The recombinant cell according to claim 4, wherein the cell is a Vero cell or a BHK-21 cell.

6. A pharmaceutical composition, comprising the infectious clone of claim 1, the RNA of claim 2, the virus-like vesicle of claim 3 and / or the recombinant cell of claim 4.

7. Use of the infectious clone according to claim 1, the RNA according to claim 2, the virus-like vesicle according to claim 3 and / or the recombinant cell according to claim 4 in the preparation of a medicament for treating or preventing Nipah virus infection.

8. Use of the infectious clone according to claim 1, the RNA according to claim 2, the virus-like vesicle according to claim 3 and / or the recombinant cell according to claim 4 in the preparation of a Nipah virus vaccine.

9. The use according to claim 8, wherein the vaccine is a live attenuated vaccine.

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