Nipah virus and Hendea virus double-antigen vaccine based on chimpanzee adenovirus vector and application of Nipah virus and Hendea virus double-antigen vaccine

Abstract

The invention relates to a Nipah virus and Hendea virus double-antigen vaccine based on a chimpanzee adenovirus vector and application of the Nipah virus and Hendea virus double-antigen vaccine. The double-antigen vaccine is obtained by directionally cloning a human CMV (cytomegalovirus) promoter, codon optimization sequences for coding NiV fusion glycoprotein (F) (NiV F) and HeV attachment glycoprotein (G) (HeV G) and an expression cassette of SV40 to an E1 deletion site of a chimpanzee adenovirus vector, constructing recombinant plasmids, and packaging and processing the recombinant plasmids. According to the present invention, the replication-deficient chimpanzee adenovirus vector is utilized to express the NiV F protein and the HeV G protein, the vaccine aiming at the two viruses is developed, the effect of preventing two diseases through one injection is achieved, and compared with the single-antigen vaccine in the prior art, the double-antigen vaccine of the present invention has great advantages in practicality and economy.

Description

Technical Field

[0001] This application belongs to the field of vaccine technology, specifically relating to a Nipah virus and Hendra virus dual antigen vaccine based on a chimpanzee adenovirus vector and its application. Background Technology

[0002] Nipah virus (NiV) and Hendra virus (HeV) belong to the genus Hennipahvirus in the family Paramyxoviridae. Their natural host is the fruit bat (Pteropodidae) in the genus Pteropodidae. 1，2 Both viruses have the ability to cross the species barrier, causing serious zoonotic diseases with human mortality rates ranging from 40% to 100%. 3 Due to their high pathogenicity, high mortality rate, and lack of approved medical responses, these two viruses are classified as Biosafety Level 4 (BSL-4) pathogens and are listed as priority emerging pathogens by the World Health Organization (WHO). 4 This classification reflects their epidemic potential through animal-to-human (spillover) and human-to-human transmission mechanisms. Model studies indicate that nearly 50% of the global population lives in geographically vulnerable areas. Currently, treatment strategies for Nipah virus and Hendra virus infections are limited to supportive care, while prevention for Hendra virus relies primarily on immunization of horses with the Equivac HeV (HEV-SG) veterinary vaccine, approved in Australia in 2012. The limited implementation of this vaccine underscores the risk of outbreaks caused by Hennipah virus. 5-7 .

[0003] Both Nipah virus (NiV) and Henipa virus (HeV) are single-stranded, negative-sense RNA viruses with a genome RNA length of approximately 18.2 kilobase pairs. Their genomes encode six structural proteins—nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), attachment glycoprotein (G), and large polymerase protein (L)—as well as three non-structural proteins (C, V, W). 4,8 Among them, the membrane attachment glycoprotein (G) and fusion glycoprotein (F), which mediate viral entry through synergistic receptor binding and membrane fusion mechanisms, are the main targets of neutralizing antibody intervention. 9 These surface-exposed glycoproteins exhibit evolutionary conservation among Hennipa virus species, and their structural accessibility and functional indispensability make them a key focus for preventative vaccine development and therapeutic antibody discovery. 10-12 .

[0004] Adenoviruses were first isolated in adenoidectomy in 1953. Due to their optimal balance between immunogenicity and tolerable reactivity, this DNA virus has been a primary vector platform for gene therapy and vaccine development since the 1990s. Adenovirus vector systems elicit a dual protective immune response of humoral and cellular immunity by producing antigen-specific antibodies and activating cytotoxic T lymphocytes. 13 To circumvent pre-existing neutralizing antibodies against common human serotypes, such as human adenovirus type 5 [AdHu5], vaccine developers have turned to adenovirus vectors derived from chimpanzees, including AdC68. These vectors have low seroprevalence in global population studies while maintaining durable immunostimulatory capabilities, making them particularly advantageous in large-scale vaccination programs in developing countries. 13，14 .

[0005] References

[0006] 1.B.Vanmechelen, S.Meurs, M.Horemans, A.Loosen, T.Joly Maes, L.Laenen, VVergote, FRKoundouno, N.Magassouba, MKKonde, ISCondé, MWCarroll andP.Maes, Virus evolution 8(2), veac061(2022).

[0007] 2. B. Rockx, R. Winegar and ANFreiberg, Antiviral research 95(2), 135-149(2012).

[0008] 3.S.-L.Zhai, X.Zhou, H.-C.Gou, K.-L.Zhang and C.-L.Li, The Lancet Microbe4(12)(2023).

[0009] 4.BTEaton, CCBroder, D.Middleton and LFWang, Naturereviews.Microbiology 4(1), 23-35(2006).

[0010] 5.R.Gómez Román,N.Tornieporth,N.G Cherian,A.C.Shurtleff,M.L′AzouJackson,D.Yeskey,A.Hacker,E.Mungai and T.T.Le,The Lancet.Infectious diseases22(1),e13-e27(2022).

[0011] 6.S.Kummer and D.C.Kranz,PLoS neglected tropical diseases 16(2),e0010157(2022).

[0012] 7.H.Li,J.V.Kim and B.S.Pickering,Frontiers in microbiology 14,1167085(2023).

[0013] 8.J.Quarleri,V.Galvan and M.V.Delpino,GeroScience 44(5),2447-2459(2022).

[0014] 9.R.J.Loomis,G.B.E.Stewart-Jones,Y.Tsybovsky,R.T.Caringal,K.M.Morabito,J.S.McLellan,A.L Chamberlain,S.T.Nugent,G B.Hutchinson,LA.Kueltzo,J.R.Mascola and B.S.Graham,Frontiers in immunology 11,842(2020).

[0015] 1O.S.Yang and S.Kar,Structural chemistry,1-19(2023).

[0016] 11.G.Mishra,V.Prajapat and D.Nayak,Immunology 171(2),155-169(2024).

[0017] 12.N.Thakur and D.Bailey,Microbes and infection 21(7),278-286(2019).

[0018] 13.K.McCoy, N.Tatsis, B.Korioth-Schmitz, MOLasaro, SEHensley, S.-W.Lin, Y Li, W.Giles-Davis, A.Cun, D.Zhou, Z.Xiang, NLLetvin and HCJErtl, Journal of virology 81(12), 6594-6604(2007).

[0019] 14. D. Zhou, T.-L. Wu, KL Emmer, R. Kurupati, S. Tuyishime, Y Li, W. Giles-Davis, X. Zhou, Z. Xiang, Q. Liu, SJ Ratcliffe and HCJ Ertl, Molecular Therapy 21(3), 696-706(2013). Summary of the Invention

[0020] The technical objective of this invention is to provide a Nipah virus and Hendra virus dual antigen vaccine based on a chimpanzee adenovirus vector.

[0021] Another technical objective of this invention is to provide a method for preparing the above-mentioned Nipah virus and Hendra virus dual antigen vaccine based on a chimpanzee adenovirus vector.

[0022] Another technical objective of this invention is to provide the relevant applications of the above-mentioned Nipah virus and Hendra virus dual antigen vaccine based on chimpanzee adenovirus vector in drug and reagent kit development.

[0023] On the one hand, the present invention provides a Nipah virus and Hendra virus dual antigen vaccine based on a chimpanzee adenovirus vector. The dual antigen vaccine is obtained by directionally cloning the human CMV promoter, the codon optimization sequence encoding the NiV fusion glycoprotein (F) (NiV F) and the HeV attachment glycoprotein (G) (HeV G) and the SV40 expression cassette into the E1 deletion site of a chimpanzee adenovirus vector, constructing a recombinant plasmid, and then packaging and processing it to obtain an immunogenic Nipah virus and Hendra virus dual antigen vaccine.

[0024] In a specific embodiment, in the dual-antigen vaccine, NiV F and HeV G are linked together by a P2A self-splitting peptide linker to form a NiV F-P2A-HeV G fragment.

[0025] In a specific embodiment, the amino acid sequence of the NiV F-P2A-HeV G fragment is shown in SEQ ID No. 17.

[0026] In a specific embodiment, the coding sequence of the NiV F-P2A-HeV G fragment is shown in SEQ ID No. 2.

[0027] In a specific embodiment, the chimpanzee adenovirus vector is a replication-defective AdC68 vector.

[0028] In a specific embodiment, the chimpanzee adenovirus vector (e.g., a replication-defective AdC68 vector) also lacks the E3 region.

[0029] In a specific embodiment, the sequence of the recombinant plasmid is shown in SEQ ID No. 1.

[0030] In another aspect, the present invention provides a recombinant plasmid comprising the nucleotide sequence shown in SEQ ID No. 2.

[0031] In a specific embodiment, the nucleotide sequence of the recombinant plasmid is shown in SEQ ID No. 1.

[0032] On the other hand, the present invention provides a method for preparing the above-mentioned Nipah virus and Hendra virus dual antigen vaccine based on a chimpanzee adenovirus vector, the method comprising the following steps:

[0033] 1) The human CMV promoter, the codon optimization sequences encoding the NiV fusion glycoprotein (F) and the HeV attachment glycoprotein (G), and the SV40 expression cassette were directionally cloned into the E1 deletion site of the chimpanzee adenovirus vector to construct a recombinant plasmid.

[0034] 2) After linearizing the recombinant plasmid with enzyme digestion, it was transfected into adenovirus packaging cells and then cultured.

[0035] In a specific embodiment, the adenovirus packaging cells are HEK293 cells.

[0036] In another aspect, the present invention provides the use of the above-mentioned dual-antigen vaccine or the above-mentioned recombinant plasmid in the preparation of medicaments or kits for the prevention or treatment of diseases associated with Nipah virus and Hendra virus infection.

[0037] Beneficial effects

[0038] The chimpanzee adenovirus vector used in this application is a non-replicating viral vector vaccine that can only replicate and proliferate in HEK293 cells with stable E1 gene expression, and cannot replicate and proliferate in normal human bodies, thus ensuring vaccine safety. One of the main advantages of this type of vaccine vector is that it avoids the possibility of pre-existing antibodies in the human body. Although adenoviruses have a broad host range, they have strict host specificity, and adenoviruses that infect chimpanzees generally do not infect humans, thus ensuring that the human population does not have pre-existing antibodies against chimpanzee adenoviruses, thereby reducing the possibility of decreased vaccine efficacy. Simultaneously, the deletion of the E3 region in the chimpanzee adenovirus vector increases the capacity for carrying foreign genes.

[0039] In summary, this application utilizes a replication-defective chimpanzee adenovirus vector to express NiV F and HeV G proteins, developing a vaccine targeting these two strains to achieve a "one-shot protection against two diseases" effect. Compared to existing single-antigen vaccines, this dual-antigen vaccine offers significant advantages in practicality and cost-effectiveness. Research results show that a single AdC68-HNV vaccination regimen induced strong and sustained specific IgG antibodies, neutralizing antibodies, and cellular immune responses in mice. A single intranasal AdC68-HNV vaccination regimen similarly induced strong humoral and cellular immune responses in Syrian hamsters, generating effective mucosal immunity, while providing complete protection against NiV-M, NiV-B, and HeV challenges in vaccinated hamsters. Notably, in the current model, animals were administered a high dose of the virus (1000 LD). 50 This infectious dose may be higher than the dose at which humans are naturally infected. Attached Figure Description

[0040] Figure 1Construction and characterization of AdC68-HNV. (A) Schematic diagram of the transgenic cassette. Codon-optimized Nipah virus (NiV) fusion protein (F) and Hendra virus (HeV) glycoprotein (G) sequences were inserted into the E1 deletion region, under the transcriptional control of the cytomegalovirus (CMV) promoter (CMV p), and carrying the simian virus 40 polyadenylation signal (SV40pA). AdC68-HNV encodes the Nipah virus fusion protein and Hendra virus glycoprotein; AdC68-empty did not have transgene insertion. (B) Cytopathic effect (CPE) observed in HEK293 cells 24 hours post-infection. Scale bar is 100 μm. (C) Western blot analysis of the expression of Nipah virus (NiV) fusion protein (F) and Hendra virus (HeV) glycoprotein (G) in HEK293 cells infected with AdC68-HNV. HEK293 cells infected with empty AdC68 vector were used as a negative control. Samples were from the same experiment, and the blots were processed simultaneously. (D) Immunofluorescence analysis of NiV F and HeV G protein expression after AdC68-HNV infection of Vero cells. In 24-well plates, each well of Vero cells was infected with 5 × 10⁶ cells. 9 VP AdC68-HNV or AdC68-empty vector. After 24 hours, cells were labeled with rabbit monoclonal antibodies against NiV F or HeV G proteins, followed by labeling with goat anti-rabbit IgG antibody in combination with Alexa Fluor 488. Cells were observed under a fluorescence microscope. Scale bar: 275 μm. (E) Negative-stained electron microscopy image of purified virus particles. The intact adenovirus capsid (approximately 90 nm in diameter) showed structural integrity in all constructs. Scale bar: 100 nm.

[0041] Figure 2 BALB / c mouse immunization regimen and vaccine-induced immune response. (A) BALB / c mouse immunization flowchart. Mice in each group (n=9 / group) were immunized by intramuscular injection or intranasal drop on day 0 with 1×10 10VP doses of AdC68-HNV or AdC68 empty vector control vaccine were administered. Serum and spleen tissues were collected at specified time points to assess humoral and cellular immune responses. NiV F(B) and HeV G(C) specific IgG antibodies in the serum of immunized mice were detected by ELISA; the dashed line indicates the detection limit. NiV(D) and HeV(E) pseudovirus neutralizing antibodies in mouse serum were detected by a pseudovirus neutralization assay. NiV F(F) and HeV G(G) specific T-cell immune responses were assessed using an enzyme-linked immunospot (ELISpot) assay, measuring the number of interferon-γ (IFN-γ)-secreting cells per million spleen cells after peptide pool stimulation; SFC represents spot-forming cells. Single spots represent independent biological replicates. Data are presented as mean (Mean ± SD) including standard deviation. * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.

[0042] Figure 3 Immunogenicity of AdC68-HNV vaccine in Syrian hamsters. (A) Flowchart of vaccine immunization and challenge in Syrian hamsters. Using 1×10 10 Syrian hamsters were immunized intranasally with VP's AdC68-HNV or AdC68 empty vector vaccine (n=6 / group). Serum, spleen, and bronchoalveolar lavage fluid (BALF) were collected at designated time points to assess humoral and cellular immune responses. Three weeks after the last immunization, 1000 LD was administered. 50 NiV-M (8.55×10 3 TCID 50 NiV-B (22.33×10) 3 TCID 50 ) and HeV (2×10 5 TCID 50 Hamsters were attacked. NiV F and HeV G specific IgG levels in serum (B) and BALF (E) cells were detected by ELISA. Neutralizing antibodies against NiV and HeV pseudoviruses in serum (C) and BALF (F) cells were detected in HEK293T cells. Neutralizing antibodies against NiV-M, NiV-B, and HeV evoviruses in serum (D) and BALF (G) cells were detected in Vero E6 cells. NiV F and HeV G specific T-cell immune responses in spleen cells (H and J) and BALF cells (I and K) were assessed using enzyme-linked immunospot (ELISpot) assays. The number of cells secreting interferon-γ (IFN-γ) per million cells after peptide pool stimulation was measured, with SFC representing spot-forming cells. Single spots represent independent biological replicates. Data are presented as mean (Mean ± SD) including standard deviation. * indicates p < 0.05; **** indicates p < 0.0001; ns, no significant difference.

[0043] Figure 4 The protective efficacy of the AdC68-HNV vaccine against Syrian hamsters challenged with NiV-M, NiV-B, and HeV. Survival rate (A) and weight change (B) of vaccinated Syrian hamsters were monitored daily for 21 days after NiV-M, NiV-B, and HeV challenge. Four hamsters in each group infected with NiV-M, NiV-B, and HeV were euthanized on days 4, 5, and 3 post-challenge, and lung, brain, and spleen tissues were collected for detection of infectious viral particles (C) and viral RNA (D).

[0044] Figure 5 The efficacy of the AdC68-HNV vaccine against Syrian hamsters challenged with NiV-M, NiV-B, and HeV. Hematoxylin and eosin (H&E) staining of lung, spleen, and brain tissues. Scale bar: 50 μm. Detailed Implementation

[0045] The technical solutions of this application are described in detail below through specific embodiments to enable those skilled in the art to better understand the technical content of this application. However, the provision of these embodiments is not intended to limit the scope of this application.

[0046] Materials and Methods

[0047] Cells and Culture

[0048] The HEK293T (ATCC, ACS-4500), HEK293 (ATCC, CRL-1573), Vero (ATCC, CCL-81), and Vero E6 (ATCC, CRL-1586) cells used in this study were purchased from the Cell Bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS, Gibco), 100 U / mL penicillin, and 100 μg / mL streptomycin (Symploc). All cells were cultured at 37°C in a cell culture incubator containing 5% CO2.

[0049] HEK293 suspension cells were purchased from Shanghai Youyi Biotechnology Co., Ltd. HEK293 suspension cells were placed in HEK293 initial medium (CD05, Youyi Biotechnology) and cultured in a shaker incubator at 37℃, 8% CO2, 120 rpm, and 50 mm amplitude.

[0050] Virus

[0051] Nipah virus Malaysian strain (NiV-M; GenBank: AF212302.2), Nipah virus Bangladeshi strain (NiV-B; GenBank: AY988601.1), and Hendra virus (HeV; GenBank: AF017149.3) were replicated and amplified in fused Vero E6 cell monolayers. All virus strains were provided by the National Virus Resource Bank of Wuhan Institute of Virology, Chinese Academy of Sciences.

[0052] laboratory animals

[0053] The 4-6 week old SPF-grade female Balb / C and 5-6 week old SPF-grade female Syrian hamsters used in the experiment were purchased from Beijing Vital River.

[0054] Data Analysis

[0055] Statistical analysis was performed using GraphPad Prism 10.0.0 software. Two-tailed unpaired Student's t-tests were used to compare differences between the two experimental groups. One-way ANOVA and Tukey's multiple comparison test were used to compare more than two experimental groups. The log-rank test was used for survival statistics. A p-value < 0.05 was considered significant; a p-value > 0.05 was considered not significant. In the figures, * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001; **** represents p < 0.0001; ns, no significant difference.

[0056] Example 1: Production of AdC68-NiVF+HeVG vaccine

[0057] Experimental procedure: Using PrimeSTAR TMMax DNA polymerase (2×Premix, catalog number: R045A, Takara Bio) amplified the coding sequences of Nipah virus fusion glycoprotein (NiV-F) (nucleotide encoding sequence SEQ ID No. 9) and Hendra virus attachment glycoprotein (HeV-G) (nucleotide encoding sequence SEQ ID No. 10). A P2A self-splitting peptide linker was introduced between the two sequences via overlap extension PCR to obtain the NiV F-P2A-HeVG fusion fragment (nucleotide sequence SEQ ID No. 2, amino acid sequence SEQ ID No. 17). The fragment was directionally cloned into the pS2 vector (SEQ ID No. 11) using NotI / KpnI restriction endonuclease (NEB, USA) to construct the recombinant plasmid pShuttle-NiV F+HeV G. A DNA fragment containing the CMV promoter, NiVF+HeVG gene, and SV40 poly-A signal was excised from the pShuttle-NiVF+HeVG plasmid using I-CeuI / PI-SceI restriction endonuclease (NEB, USA). This expression cassette was then inserted into the E1 deletion site of the replication-defective adenovirus backbone vector pAdC68 (E1 region deleted, kindly provided by Professor Zhou Dongming of Tianjin Medical University) to construct the recombinant adenovirus plasmid pAdC68-NiVF+HeVG. Figure 1 (See Figure A, SEQ ID No. 1). The pAdC68-NiV F+HeV G and pAdC68-ΔE 1 plasmids linearized with PacI were transfected into HEK293 cells using Lipofectamine 2000 (Thermo Fisher Scientific) to obtain recombinant adenoviruses AdC68-HNV and AdC68, respectively, as empty vector controls for subsequent experiments.

[0058] Viral particles were amplified by continuous passage in HEK293 cells, purified by ultracentrifugation using a cesium chloride density gradient, and then purified using NanoDrop. TM Determination of A by a 2000 spectrophotometer 260 The values ​​were quantified. After the purified virus was verified to be correct by restriction enzyme digestion, it was aliquoted and stored in an ultra-low temperature freezer at -80°C.

[0059] Example 2: Observation of cytopathic effects and Western blot experiment

[0060] HEK293 cells were seeded in 6-well plates and infected with recombinant adenovirus AdC68-HNV and the empty vector control AdC68. Cell morphological changes were observed and images were recorded using an Olympus IX73 inverted microscope. After the CPE phenomenon stabilized, cells were collected, lysis buffer was added, and then mixed with protein loading buffer (Solarbio, catalog number: P1040). The mixture was heated in a 100°C metal bath for 10 minutes. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (Epizyme, catalog number: PG112) and then transferred to nitrocellulose (NC) membranes. Block the membrane with TBST buffer containing 5% skim milk powder for 1 hour, then add primary antibodies (Anti-HeV G, PVV07901, 1:1000; Anti-NiV F, PVV08101, 1:1000; Anti-GAPDH, CL594-60004, 1:10000) and incubate at room temperature for 1 hour. After washing, add HRP-labeled secondary antibodies (horseradish peroxidase-labeled goat anti-rabbit IgG (H+L), A0208, 1:1000; horseradish peroxidase-labeled goat anti-mouse IgG (H+L), A0216, 1:1000) and incubate at room temperature for 1 hour. After washing, add Tanon... TM The high-Sig ECL chemiluminescent substrate (catalog number: 180-5001) was used for color development, and the images were taken using the Tianneng gel imaging system.

[0061] like Figure 1 As shown in Figure B, AdC68-HNV and AdC68 were successfully rescued and multiplied in HEK293 cells, exhibiting typical adenovirus aggregation and cell shedding cytopathic effects. Figure 1 The results from the C-cell assay show that AdC68-HNV can efficiently express NiV F and HeV G proteins in infected cells. These results demonstrate that this study successfully constructed a replication-deficient chimpanzee adenovirus vector dual-antigen vaccine, achieving efficient expression of NiV F and HeV G glycoproteins. This vaccine platform provides a crucial technological foundation for subsequent systematic evaluation of the immunogenicity of NiV and HeV dual-antigen vaccines.

[0062] Example 3: Immunofluorescence experiment

[0063] Vero cells were seeded in 24-well plates, 1 x 10⁻⁶ 5 Cells / well. On the second day, when cell confluence reached 90%, the cell culture medium was replaced with 500 μL of DMEM containing 2% FBS, and then 5 × 10⁶ cells were added directly to each well. 9The adenovirus vector vaccine for VP was mixed by shaking and then placed in a cell culture plate at 37°C in a cell culture incubator containing 5% CO2. After 24 hours of culture, the cells were washed twice with sterile PBS, fixed with a 1:1 methanol-acetone mixture at room temperature, and permeabilized for 20 min. After washing three times with PBS, the cells were blocked with PBS containing 5% BSA (g / v) at room temperature for 1 hour. After washing with PBS again, the cells were incubated with the primary antibody (Anti-HeV G, PVV07901, 1:1000; Anti-NiV F, PVV08101, 1:1000) diluted in 1% BSA-PBS at room temperature for 2 hours. After washing three times with PBS, the cells were incubated with the secondary antibody (488Goat anti rabbit, A11034, 1:2000) diluted in 1% BSA-PBS at room temperature in the dark for 1 hour. After washing three times with PBS, stain the nuclei with Hochest 34580 (0.2-2 μg / mL in PBS) at a 1:10000 ratio and incubate at room temperature for 10 min. Finally, after washing with PBS, stain the nuclei with EVOS. TM The M7000 imaging system (Invitrogen) allows observation and image acquisition in 100x field of view.

[0064] like Figure 1 The results from the study show that AdC68-HNV can effectively express NiV F and HeV G proteins in infected cells.

[0065] Example 4: Negative staining of virus particles by transmission electron microscopy (TEM)

[0066] Hydrophilication of a 300-mesh ordinary carbon support film (Beijing Zhongjing Keyi Technology, BZ110135b) was performed using a glow discharge system (PELCO easiGlow91000, TEDPELLA, USA). The copper mesh was held with the carbon film side facing upwards, and 1 / 3 to 1 / 2 of the mesh was held with reverse tweezers (Dumont #5; F6521; Merck) to facilitate the formation of a dye gradient. A 10 μL sample was dropped onto the mesh and allowed to stand for 60 seconds. Filter paper was used to absorb the solution perpendicularly along the mesh edge at an angle close to 90 degrees. Immediately, 10 μL of 2% uranium acetate staining solution was added and allowed to stand for 60 seconds. Filter paper was used to absorb the solution perpendicularly along the mesh edge at an angle close to 90 degrees, and the remaining solution was aspirated with tweezers. The mesh was dried and stored in a suitable container (mesh box or petri dish). The mesh was examined and photographed using a field emission cryo-electron microscope (FEI F20, USA).

[0067] like Figure 1 The results showed that all recombinant viruses maintained the typical adenovirus morphology (90-100nm, icosahedral capsid) and showed no structural difference from the empty vector control.

[0068] animal experiments

[0069] Female BALB / c mice aged 4–6 weeks were vaccinated with 1×10⁻⁶ mice via intranasal and intramuscular injection. 10 VPAdC68-HNV vaccine was administered concurrently with the same dose of AdC68 empty vector vaccine as a control group. Serum, spleen, lung, and bronchoalveolar lavage fluid were collected at specified time points for immunological analysis. Figure 2 (A)

[0070] For 5-6 week old female Syrian hamsters, administer intranasal inoculation with 50 μL of 1×10 10 VP AdC68-HNV or AdC68 empty vector. Serum, spleen, and bronchoalveolar lavage fluid were collected at specified time points and subjected to immunological analysis. Figure 3 (Middle A). Three weeks after the last vaccination, the hamsters were transferred to the ABSL-4 laboratory and in 500 μL DMEM with 1000 LD 50 (1,000 median lethal dose [LD50]) 50 NiV-M (8.55×10) 3 TCID 50 NiV-B (22.33×10) 3 TCID 50 ) and HeV (2×10 5 TCID 50 Hamsters were challenged with viral suspensions of NiV-M, NiV-B, and HeV. Four hamsters in each group infected with NiV-M, NiV-B, and HeV were euthanized on days 4, 5, and 3 post-challenge. Lung, brain, and spleen tissues were harvested for live virus detection, quantitative PCR (qRT-PCR), and histopathological evaluation (H&E staining). The remaining six hamsters in each group were monitored daily for weight changes, clinical symptoms, and survival status until day 21 post-challenge.

[0071] The results showed that all hamsters in the AdC68 empty control group died, while all hamsters in the AdC68-HNV vaccine group survived. Figure 4 (A), and no signs of illness or weight loss were observed throughout the experiment. Figure 4 (B)

[0072] Example 5: Enzyme-linked immunosorbent assay (ELISA)

[0073] High-binding 96-well EIA / RIA plates (Corning, Model 3590) were coated with 100 ng / well of recombinant NiV F or HeVG protein and incubated overnight at 4°C. After washing with PBS, the plates were blocked at 37°C for 90 minutes with 100 μL / well blocking buffer (PBS containing 5% w / v nonfat dry milk) to remove non-specific binding sites. Immune mouse or hamster serum was tested and diluted 2-fold in blocking buffer into 96-well deep-well plates (Biosharp, BS-16-DW). 100 μL of solution from each dilution was transferred to the antigen-coated wells along with the negative control (containing only blocking buffer). After incubation at 37°C for 2 hours, the plates were washed four times with PBST (PBS containing 0.05% Tween-20). HRP-linked secondary antibodies (goat anti-mouse IgG, ab6789, 1:30,000; goat anti-mouse IgA, ab97235, 1:10,000; goat anti-Syrian hamster IgG, ab6892, 1:10,000; Abekam Biotechnology, UK) were diluted in blocking buffer and added to 96-well plates, followed by incubation at 37°C for 60 minutes. After five washes with 300 μL / well of PBST, 100 μL / well of TMB reagent (NCM Biotechnology, M30500) was added, and the plates were incubated at room temperature for 10 minutes. The reaction was terminated with 50 μL / well of ELISA stop solution (Solbro Biotechnology, C1058). Within 15 minutes of stopping the reaction, the optical density (OD) was measured at 450 nm (main measurement wavelength) and 630 nm (reference wavelength) using a multi-plate reader (BioTek Synergy H1). The formula for calculating the specific signal is: ΔOD = OD450nm - OD630nm. The cutoff value is defined as: [the average value of the negative control (ΔOD)] × 2.1. When the ΔOD value of the sample is greater than or equal to the cutoff value, the sample is considered positive.

[0074] The response of IgG in serum samples from immunized mice to purified NiV F and HeV G proteins was assessed using an ELISA method, such as... Figure 2 China B and Figure 2 The results showed that the AdC68-HNV vaccine induced high levels of NiVF and HeVG-specific IgG in mice via both immunization methods, with antibody levels gradually increasing over time and then stabilizing. However, no specific antibodies against NiVF and HeVG proteins were detected in the serum of mice immunized with the AdC68 empty vector. The levels of NiVF-specific IgG induced by intranasal and intramuscular injection were comparable in mice, while intranasal immunization induced higher levels of HeVG-specific IgG in mice compared to intramuscular injection.

[0075] Analysis of immunized hamster serum and BALF samples showed that ( Figure 3 In the AdC68 empty vector control group, no specific IgG antibodies were induced. However, the AdC68-HNV primary immunization group induced high titers of NiV F-specific IgG (GMT 1:1600-1:25,600) and HeV G-specific IgG (GMT 1:12,400-1:204,800). The AdC68-HNV booster immunization group induced high titers of NiV F-specific IgG (GMT 1:12,800-1:25,600) and HeV G-specific IgG (GMT 1:51,200-1:204,800). High levels of NiV F-specific IgG (GMT 1:800-1:1,600) and HeV G-specific IgG (GMT 1:1,600-1:3,200) were also detected in BALF samples. Figure 3 (E).

[0076] Example 6: Enzyme-linked immunospot assay (ELISpot)

[0077] Following euthanasia via cervical dislocation, spleens were aseptically removed from BALB / c mice and Syrian hamsters and mechanically separated using a 70-micron cell filter (Corning, 352350) to prepare single-cell suspensions. Bronchoalveolar lavage fluid (BALF) was collected from Syrian hamsters and centrifuged to obtain cells. Red blood cells were lysed for 4 minutes at room temperature using erythrocyte lysis buffer (Solepro, R1010), followed by termination of lysis with RPMI 1640 (Gibco, C11875500BT) containing 10% fetal bovine serum. Pre-coated IFN-γ ELISpot plates (Mabtech, 3321-4APT-2 for mice; 3102-4APW-2 for hamsters) were equilibrated by washing four times consecutively with sterile PBS (200 μL per well). Blocking was performed by incubating cells with RPMI 1640 (200 μL per well) containing 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco, 15140122) at room temperature for at least 30 minutes. The peptide pool (18 amino acid lengths, with 10 amino acid overlaps; GenScript) was diluted to a final concentration of 5 μg / mL and added to the assay medium. Cells were then introduced at a density of 5 × 10⁶ cells per well. 5Cells were seeded at a density of 50 μL and co-cultured with 50 μL of peptide solution. The positive control group was treated with 10 μL of 500 ng / mL phorbol 12-myristate 13-acetate (PMA) and 10 μg / mL iomycin (2030421; Dakowei, Shenzhen, China). The culture plates were incubated at 37°C and 5% CO2 for 20–24 hours. After incubation, the plates were washed five times with PBS. The biotinylation detection antibody (Mabtech) was diluted to 1 μg / mL with PBS containing 0.5% fetal bovine serum, and 100 μL was added to each well. The plates were incubated at 25°C for 2 hours. Subsequently, streptavidin-alkaline phosphatase (Mabtech) (1:1000 diluted in PBS containing 0.5% fetal bovine serum, 100 μL per well) was added, and the plates were incubated at room temperature for 60 minutes. Sterilize the BCIP / NBT-plus alkaline phosphatase substrate by filtration through a 0.45 μm Millipore filter, adding 100 μL to each well. Incubate in the dark at room temperature until distinct spots appear. Stop the colorimetric reaction by rinsing with water. Analyze the air-dried culture plates using an AIDEliSpotReaderClassic (AID, Germany) and count the spots, expressing them as spot-forming units (SFU).

[0078] To determine whether AdC68-HNV elicits a cell-mediated immune (CMI) response in mice, interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assays were performed using lymphocytes isolated from mouse spleens. Peptides from NiV F that have been screened for effective stimulation and peptides covering the full length of the HeV G protein were synthesized. Compared to the AdC68 empty vector group, both intranasal and intramuscular injections of AdC68-HNV induced significant CMI responses in mice. Figure 2 China F and Figure 2 (G).

[0079] To clarify the T-cell response elicited by vaccine-induced mucosal immunity in hamsters, spleen cells and BALF cells were isolated 10 days after booster immunization for ELISPOT assay.

[0080] The results showed that the number of spleen cell antigen-specific spots in the AdC68-HNV intranasal immunization group was low (NiV-F peptide: 23-129 SFU / 10). 6 Cells, HeV-G peptide: 52-179 SFU / 10 6 cell)( Figure 3 H and Figure 3 In the middle I), the number of spots in BALF cells was significantly increased (NiV-F peptide: 22.31-1298.97 SFU / 10). 6 Cellular, HeV-G peptide: 70.25-2050.79 SFU / 10 6(cells), compared to the empty vector group in BALF cells, the spots (NiV-F peptide: 19.61-68.60 SFU / 10) 6 Cellular HUV-G peptide: 94.12-235.98 SFU / 10 6 Cellular) significantly improved ( Figure 3 China J and Figure 3 The results (K) indicate that nasal immunization can preferentially activate local T-cell immunity in the respiratory tract.

[0081] Example 7: Neutralizing antibody titer determination

[0082] Neutralization test based on NiV and HeV pseudoviruses

[0083] Nipah virus / Hendra virus pseudoviruses were generated in 293T cells using a three-plasmid co-transfection system via Lipofectamine 3000 (Thermo Fisher Scientific, L3000150). The transfection mixture contained either pNL4-3.Luc.RE (SEQ ID No. 12), pcDNA3.1(+)-Nipah-F-25 (SEQ ID No. 13), or pcDNA3.1(+)-Nipah-G-33 (SEQ ID No. 15), or pNL4-3.Luc.RE (SEQ ID No. 12), pcDNA3.1(+)-Hendra-F-25 (SEQ ID No. 14), or pcDNA3.1(+)-Hendra-G-33 (SEQ ID No. 16), in a mass ratio of 12:1:2. Transfected cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 for 48 hours. The supernatant containing pseudovirus was clarified by centrifugation at 1000×g for 5 minutes, aliquoted, and stored at -80°C. Heat-inactivated (56°C, 30 minutes) test-immunized mouse or hamster serum was serially diluted 1:20 (v / v) in serum-free DMEM. Equal volumes (50 μL) of pseudovirus stock solution and diluted serum were co-incubated in 96-well plates (Nest, 701001) at 37°C for 60 minutes. 3 × 10⁶ cells were seeded into each well of the mixture. 4 293T cells were cultured for 48 hours (37°C, 5% CO2), then the supernatant was discarded and cultured using Bright-Globe technology. TM Luciferase activity was quantified using a luciferase detection system (Promega, E2620). The neutralization endpoint was defined as a ≥50% reduction in relative optical units (RLU) compared to the virus-only control group.

[0084] The serum neutralizing antibody response induced by the vaccine in mice was characterized using a pseudovirus neutralization assay. Results showed that the AdC68-HNV vaccine produced neutralizing antibodies against NiV and HeV pseudoviruses in mice via both immunization routes, and that intranasal administration induced higher levels of neutralizing antibodies in mice compared to intramuscular injection. Figure 2 China D and Figure 2 (E).

[0085] Analysis of serum and BALF samples from immunized hamsters showed ( Figure 3 In the AdC68 empty vector control group, neutralizing antibodies could not be induced, while the AdC68-HNV primary immunization group could induce significant neutralizing antibody activity against the pseudovirus (NiV neutralizing titer IC50). 50 4,882-13,112, HeV neutralization titer IC50 50 468.5-3,290), the AdC68-HNV booster immunization group can induce significant neutralizing antibody activity against the pseudovirus (NiV neutralizing titer IC50). 50 4,419-17,887, HeV neutralization titer IC50 50 418.5-4,761). The neutralizing activities against NiV and HeV pseudotoxicities in BALF were IC50, 418.5-4,761 ... 50 89.8-312.7 (NiV) and IC 50 1.93-21.74 (HeV) ( Figure 3 (F).

[0086] NiV and HeV live virus neutralization test

[0087] Hamster serum samples (n=6) were inactivated by heating in a water bath at 56°C for 30 minutes, and then serially diluted 3-fold in serum-free DMEM, starting at a 1:20 (volume ratio). Each diluted sample of immunized hamster serum was then mixed with an equal volume (1:1) of 100 TCID50. 50 Nipah virus-Malaysian strain (NiV-M), Nipah virus-Bangladesh strain (NiV-B), or Hendra virus (HeV) were mixed. The neutralization complex was incubated in a humidified CO2 incubator at 37°C for 60 min. The virus-serum mixture (200 μL per well) was transferred to a 96-well plate onto a monolayer of 90% confluent Vero E6 cells and adsorbed at 37°C with 5% CO2 for 60 min. After adsorption, the inoculum was replaced with DMEM containing 2% fetal bovine serum. Cytopathic effect (CPE) was assessed at 120 h post-infection (hpi). The inhibitory dose (ID50) was calculated using the Reed-Muench method. 50 ), defined as the reciprocal of the highest serum dilution that completely inhibits CPE in at least 2 out of 4 replicate wells.

[0088] Analysis of serum and BALF samples from immunized hamsters showed ( Figure 3 In the AdC68 empty vector control group, no neutralizing antibodies were induced, while the AdC68-HNV primary immunization group induced significant neutralizing antibody activity against the true virus (NiV M neutralizing titer IC50). 50 20-34.64, NiV B neutralization titer IC50 50 20-46.56, HeV neutralizing titer IC50 50 (41.60-103.92), the AdC68-HNV booster immunization group can induce significant neutralizing antibody activity against the true virus (NiV M neutralizing titer IC50). 50 86.53-271.76, NiV B neutralization titer IC50 50 86.53-311.77, HeV neutralization titer IC50 50 259.60-311.77). The neutralizing activities against NiV and HeV true toxins in BALF were IC50, IC50, and IC50, respectively. 50 5.66-13.63 (NiV M), IC 50 10.08-35.92 (NiV B) and IC 50 16-32(HeV)( Figure 3 (G).

[0089] Example 8: Virus Titration Test

[0090] 24 hours before infection, Vero E6 cells were injected at a rate of 5 × 10⁻⁶. 4 Cells were seeded per well in 96-well microplates (Corning, 3599) that had undergone tissue culture treatment. Virus inoculation was performed when cells reached 90% confluence. 100 μL of a 5-fold diluted clear tissue homogenate was inoculated into each well, and a parallel negative control was inoculated with an equal volume of sterile PBS.

[0091] After inoculation, the plates were incubated at 37°C and 5% CO2 for 60 minutes to allow virus attachment. After adsorption, the inoculum was discarded, and the cells were washed twice with PBS. 100 μL of maintenance medium (DMEM containing 2% FBS and 1% PS) was added. Cytopathic effect (CPE) was assessed daily; characteristic manifestations of CPE included syncytial formation, cytoplasmic vacuolization, and cell detachment. At 120 hours post-infection, the 50% tissue culture infection dose (TCID) was determined using the Reed-Muench statistical method. 50 / mL).

[0092] like Figure 4 The results showed that no infectious live virus particles were detected in the lungs, spleen, and brain tissue of hamsters in the AdC68-HNV vaccine group, while no infectious live virus particles were detected in the lungs of animals in the empty vector control group (NiV M: 5.62-85.6 TCID).50 / mg; NiV B: 0-5.09TCID 50 / mg; HeV: 59.5-217 TCID 50 (mg) and spleen (NiV M: 4.28-78.1 TCID) 50 / mg; NiV B: 0-2.78TCID 50 / mg; HeV: 12.5-133 TCID 50 High levels of live virus were detected in both tissues ( / mg), but no live virus was isolated from brain tissue.

[0093] Example 9: Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

[0094] Lung, spleen, and brain tissues were collected from Syrian hamsters after euthanasia for viral load detection. The tissues were homogenized in 1 mL DMEM, and 100 μL of the homogenate supernatant was added to 300 μL of TRIzol. TM The virus was inactivated using LS reagent, and high-purity viral nucleic acid was rapidly extracted using the VAMNE Virus DNA / RNA Extraction Kit 3.0. Viral load was detected using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) on a CFX96 Real-time System (Bio Rad) with the HiScriptII One Step qRT-PCR Probe Kit.

[0095] The primers and probes used for detecting HeV are: forward primer (5'-AAGAGATTATTTGCCCAGATGG3-3'SEQ ID No. 3), reverse primer (5'-CTGCCACCCTGCTGATCTC-3'SEQ ID No. 4), and probe (5'-FAM-TCTCTAATGGACATCAATCCTTGGCTCA-MGB-3'SEQ ID No. 5), in a 25 μL reaction system (10 9 -10 4 A standard curve with 6 points was constructed in the sample, and samples with Cq values ​​> 35 were considered negative.

[0096] The primers and probes used for NiV detection were: forward primer (5'-AACATCAGCAGGAAGGCAAGA-3'SEQ ID No. 6), reverse primer (5'-GCCACTCTGTTCTATAGGTTCTTC-3'SEQ ID No. 7), and probe (5'-FAM-TTGCTGCAGGAGGTGTGCTC-BHQ1-3'SEQ ID No. 8), in a 20 μL reaction system (10 9 -10 1 A standard curve with 9 points was constructed in the sample, and samples with Cq values ​​> 35 were considered negative.

[0097] like Figure 4 As shown in Figure D, the viral RNA load in the lung, spleen, and brain tissues of hamsters in the AdC68-HNV vaccine group was below the detection limit; while the viral RNA load in the lungs of the empty vector control group reached 1.38 × 10⁻⁶. 7 -7.64×10 7 Copies / mg (NiV M), 5.04 × 10⁻⁶ 6 -1.09×10 7 Copies / mg (NiV B) and 5.55×10 8 -1.29×10 9 The viral RNA load in the spleen reached 1.07 × 10⁻⁶ copies / mg (HeV). 6 -74.20×10 6 Copies / mg (NiV M), 5.79 × 10 4 -2.96×10 5 Copies / mg (NiV B) and 8.86×10 7 -4.14×10 8 Copy / mg (HeV). Notably, although no live virus was isolated from the brain tissue of the empty vector control group, viral RNA (NiV M: 8.19 × 10⁻⁶) could be detected. 3 -1.90×10 5 Copies / mg; NiV B: 1.03×10 1 -7.12×10 3 Copies / mg; HeV: 4.78 × 10⁻⁶ 6 -8.11×10 6 The result (copy / mg) suggests the possible passive transfer of non-replicating viral particles.

[0098] Example 10: Histology and Histopathology

[0099] Hamster lung and spleen tissues were fixed in 10% neutral buffered formalin for at least 7 days, with the fixative changed twice before transferring the samples out of the ABSL-4 facility. The samples were then embedded in paraffin, sectioned at a thickness of 4 μm, and stained with hematoxylin and eosin (H&E). Images were acquired using a Pannoracic MIDI system (3DHISTECH Ltd., HUN).

[0100] The results are as follows Figure 5 As shown in the figures, the lung tissue of hamsters immunized with AdC68 empty vector exhibited thickened alveolar septa, alveolar wall and alveolar hemorrhage, widespread infiltration of lymphocytes and mononuclear macrophages, significant inflammatory response in blood vessels and pleura, edema fluid, and fibrous alveolar exudate. In contrast, no obvious histopathological abnormalities were observed in the lung tissue of the AdC68-HNV inoculated group. In the control group, spleen lymphocytes showed single-cell necrosis or focal necrosis, and a significant reduction in white pulp lymphocytes. However, the white pulp lymphocytes in the vaccine group were not reduced or only slightly reduced. Brain tissue sections from both groups showed diffuse glial cell activation.

[0101] In summary, this application developed a Nipah virus and Hendra virus dual-antigen vaccine, AdC68-HNV, based on a chimpanzee adenovirus vector. This vaccine demonstrated high immunogenicity in Syrian hamsters after a single intranasal immunization, simultaneously preventing infection with both NiV and HeV. The vaccine shows great promise in controlling NiV and HeV infection and transmission during outbreaks. After validating its broad-spectrum immunoprotective effect in susceptible animals, this vaccine could be developed for the prevention and treatment of Nipah virus and Hendra virus infections in humans and animals.

Claims

1. A Nipah virus and Hendra virus dual-antigen vaccine based on a chimpanzee adenovirus vector, wherein the dual-antigen vaccine is obtained by directionally cloning the human CMV promoter, the codon optimized sequences encoding the NiV fusion glycoprotein (F) (NiV F) and the HeV attachment glycoprotein (G) (HeV G), and the SV40 expression cassette into the E1 deletion site of a chimpanzee adenovirus vector, constructing a recombinant plasmid, and then packaging and processing it to obtain an immunogenic Nipah virus and Hendra virus dual-antigen vaccine.

2. The dual-antigen vaccine according to claim 1, wherein, In the dual-antigen vaccine, NiV F and HeV G are linked by a P2A self-splitting peptide linker to form a NiV F-P2A-HeV G fragment.

3. The dual-antigen vaccine according to claim 2, wherein, The amino acid sequence of the NiV F-P2A-HeV G fragment is shown in SEQ ID No.

17.

4. The dual-antigen vaccine according to claim 1, wherein, The chimpanzee adenovirus vector is a replication-defective AdC68 vector.

5. The dual-antigen vaccine according to claim 1, wherein, The chimpanzee adenovirus vector also lacks the E3 region.

6. The dual-antigen vaccine according to claim 1, wherein, The sequence of the recombinant plasmid is shown in SEQ ID No.

1.

7. A recombinant plasmid comprising the nucleotide sequence shown in SEQ ID No. 2, preferably, the nucleotide sequence of the recombinant plasmid is shown in SEQ ID No.

1.

8. A method for preparing a Nipah virus and Hendra virus dual-antigen vaccine based on a chimpanzee adenovirus vector as described in any one of claims 1-6, the method comprising the following steps: 1) The human CMV promoter, the codon optimization sequences encoding the NiV fusion glycoprotein (F) and the HeV attachment glycoprotein (G), and the SV40 expression cassette were directionally cloned into the E1 deletion site of the chimpanzee adenovirus vector to construct a recombinant plasmid. 2) After linearizing the recombinant plasmid with enzyme digestion, it was transfected into adenovirus packaging cells and then cultured.

9. The method according to claim 8, wherein, The adenovirus packaging cells were HEK293 cells.

10. The use of the dual-antigen vaccine as described in any one of claims 1-6 or the recombinant plasmid as described in claim 7 in the preparation of a medicament or kit for the prevention or treatment of diseases associated with Nipah virus and Hendra virus infection.

Images