A Nipah virus disease vaccine using a human replication-defective adenovirus as a vector

Abstract

This invention provides a polynucleotide encoding the extracellular domain of the Nipah virus G protein, a recombinant human adenovirus type 5 containing the polynucleotide, and the application of the recombinant human adenovirus type 5 in the preparation of a vaccine to prevent Nipah virus disease. The vaccine provided by this invention has good immunogenicity and can effectively and rapidly stimulate humoral and cellular immune responses in BALB / c mice.

Description

Technical Field

[0001] This invention discloses an adenovirus vector vaccine, belonging to the field of nucleic acid and pharmaceutical technology. Background Technology

[0002] Nipah virus (NiV) is a single-stranded, negative-sense RNA virus that is zoonotic. Hendra virus, belonging to the same genus as Nipah virus in the family Paramyxoviridae, is a biosafety level 4 (BSL-4) pathogen. The natural host of Nipah virus is the fruit bat (Pteropodidae), which has a very wide geographical distribution.

[0003] Nipah virus can cause respiratory and neurological diseases in humans, and is highly pathogenic and has a high mortality rate. Nipah virus has been proven to be transmissible between humans, and therefore has the potential to cause a pandemic.

[0004] Nipah virus disease was listed by the WHO as one of the 10 potential high-risk infectious diseases of concern in 2018, and to date, no approved human vaccine or treatment has been released. Nipah virus enters host cells by binding to its attachment (G) and fusion (F) glycoproteins. Its G glycoprotein recognizes and binds to the host cell surface receptors ephrin-B2 (EFNB) and ephrin-B3 (EFNB3), triggering a conformational cascade reaction of the F protein, leading to membrane fusion. Therefore, immunization strategies against Nipah virus mainly involve its G and F proteins. Currently, various vaccines are under development, including viral vector vaccines, protein subunit vaccines, virus-like particles, and mRNA vaccines, but none have yet been approved for marketing. The purpose of this invention is to provide a vaccine for the prevention of Nipah virus disease. Summary of the Invention

[0005] To achieve the above objectives, this invention first provides a polynucleotide molecule encoding the extracellular domain of the Nipah virus G protein, the sequence of which is shown in SEQ ID NO.1. The sequence of the polynucleotide molecule was obtained through artificial optimization based on the wild-type sequence of the Nipah virus G protein extracellular domain (Genbank: AY988601.1). Compared to the wild-type sequence, the codon fitness index (CAI) was 0.72 before optimization and reached 0.99 after optimization. Simultaneously, a secretory expression signal peptide was added to improve its expression level in mammalian cells.

[0006] Secondly, the present invention provides a recombinant human adenovirus type 5 containing the above-mentioned polynucleotides, wherein the recombinant human adenovirus type 5 expresses the extracellular domain of the Nipah virus G protein encoded by the polynucleotide molecule.

[0007] In a preferred embodiment, the recombinant human adenovirus type 5 is a recombinant human adenovirus type 5 replication-defective adenovirus.

[0008] In a more preferred embodiment, the recombinant human adenovirus type 5 is a human adenovirus type 5 replication-defective with E1 and E3 deletions. In this invention, a recombinant human adenovirus type 5 vector is obtained by packaging polynucleotide molecules with sequences as shown in SEQ ID NO. 1 using an E1 and E3 deletion replication-defective human adenovirus type 5 as a vector and HEK293 cells with integrated adenovirus E1 gene as a packaging cell line.

[0009] Third, the present invention provides the application of the above-mentioned recombinant human adenovirus type 5 in the preparation of a vaccine for the prevention of Nipah virus disease.

[0010] In a preferred embodiment, the recombinant human adenovirus type 5 is prepared as an injection, nasal drops, spray, or inhaler.

[0011] Fourth, this invention provides a shuttle plasmid vector containing the aforementioned polynucleotides. The shuttle plasmid vector can be co-transformed with a corresponding backbone plasmid into host cells and packaged as a recombinant adenovirus. In one specific embodiment of this invention, the shuttle plasmid vector is the pShuttle-G plasmid (purchased from ABM Biotechnology (China) Co., Ltd.).

[0012] Finally, the present invention provides a method for preparing the above-mentioned recombinant human adenovirus type 5, the method comprising the following steps:

[0013] (1) Construct a shuttle plasmid vector containing the above-mentioned sequence encoding the Nipah virus as shown in SEQ ID NO.1;

[0014] (2) The shuttle vector described in step (1) is co-transformed with the backbone plasmid into the host cell to package a recombinant replication-defective adenovirus;

[0015] (3) Cultivate the host cells described in step (2);

[0016] (4) Extract human replication-deficient recombinant virus expressing Nipah virus G protein from the host cell described in step (3).

[0017] In a preferred embodiment, the host cell in step (2) is a HEK293 cell.

[0018] In another preferred embodiment, the backbone plasmid in step (2) is a vector containing a human adenovirus type 5 genome with a combined deletion of the E1 / E3 region. In a specific embodiment of the present invention, the backbone plasmid is a pAdeno vector (purchased from ABM Biotechnology (China) Co., Ltd.).

[0019] This invention provides a Nipah virus disease vaccine, Ad5-NiV, using a human replication-defective adenovirus as a vector. The vaccine uses a replication-defective human adenovirus type 5 (A5) with combined E1 and E3 deletions as a vector, and HEK293 cells with integrated adenovirus E1 gene as the packaging cell line. The protective antigen gene it carries encodes the extracellular domain of an optimized Nipah virus G protein. Experimental results show that the Ad5-NiV vaccine has good immunogenicity and can effectively and rapidly elicit humoral and cellular immune responses in BALB / c mice. Specifically, a single Ad5-NiV immunization can elicit a neutralizing antibody response against Malaysian and Bangladeshi Nipah viruses; after a booster immunization, the neutralizing antibody titer further increases, exceeding 10. 3 The results showed significantly better efficacy than the recombinant protein vaccine immunization group. After a single immunization of mice, the IFN-γ concentration reached 30,000 pg / ml, and the TNF concentration reached 1,000 pg / ml. These data indicate that a single immunization with Ad5-NiV can elicit a strong specific cellular immune response against Nipah virus. The vaccine provided by this invention is rapid and simple to prepare, and can be mass-produced in a short period of time to respond to sudden outbreaks, demonstrating industrial applicability. Attached Figure Description

[0020] Figure 1 Schematic diagram of recombinant adenovirus packaging plasmid construction;

[0021] Figure 2 Schematic diagram of the Ad5-NiV protective antigen sequence and its cytopathic effect after infection;

[0022] Figure 3 Nipah virus G protein-specific antibody titers in mouse serum 14 days after the initial Ad5-NiV immunization and 21 days after the booster immunization;

[0023] Figure 4 . Neutralizing antibody titer against Bengal Nipah virus 14 days after the initial Ad5-NiV immunization;

[0024] Figure 5 . Neutralizing antibody titer against Bengal Nipah virus 21 days after Ad5-NiV booster immunization;

[0025] Figure 6 . Neutralizing antibody titer against Malaysian Nipah virus 14 days after the initial Ad5-NiV immunization;

[0026] Figure 7 . Neutralizing antibody titer against Malaysian Nipah virus 21 days after Ad5-NiV booster immunization;

[0027] Figure 8 Concentration of IFN-γ cytokine secreted by mouse spleen cells after Ad5-NiV immunization;

[0028] Figure 9 Concentration of TNF cytokine secreted by spleen cells in mice after Ad5-NiV immunization;

[0029] Figure 10 The concentration of IL-10 secreted by spleen cells in mice after Ad5-NiV immunization;

[0030] Figure 11 The concentration of IL-6 secreted by spleen cells in mice after Ad5-NiV immunization;

[0031] Figure 12 The concentration of IL-2 secreted by spleen cells in mice after Ad5-NiV immunization;

[0032] Figure 13 Concentration of IL-17A secreted by spleen cells in mice after Ad5-NiV immunization. Detailed Implementation

[0033] 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 a result of the description. 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.

[0034] Example 1: Preparation of a Nipah virus disease vaccine using human replication-defective adenovirus as a vector

[0035] 1. Design and construction of recombinant adenovirus packaging plasmids

[0036] 1.1 Optimization and Synthesis of Extracellular Region of Nipah Virus G Protein

[0037] The gene sequence of the Bengal-type Nipah G protein (Gene ID: 920955) was obtained from the Genbank database. The original extracellular region sequence, as shown in SEQ ID NO.2, was selected, and the secretory expression tPA signal peptide was added before the coding region. Since the frequency of synonymous codon usage varies among different species, the expression level of the foreign gene is significantly increased when the frequency of synonymous codon usage matches that of the expressing host. Codon optimization was performed according to the Human Cell Genetic Codon Table (https: / / www.ncbi.nlm.nih.gov / Taxonomy / Utils / wprintgc.cgi#SG1), replacing codons in the wild-type sequence with commonly used human codons.

[0038] This invention uses the Codon Adaptation Index (CAI) to evaluate the expression adaptability of optimized codons. The CAI measures the similarity between the codon preference of a gene and that of the high-expression genes, using a set of genes with high expression levels as a reference. If a gene uses all the codons used by the high-expression genes, its CAI value is 1. This index is currently widely used to predict gene expression levels. The calculation method can be found in the reference (Sharp PM, Li WH. The codon Adaptation Index--a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 1987 Feb 11;15(3):1281-95. doi: 10.1093 / nar / 15.3.1281. PMID: 3547335; PMCID: PMC340524.). The codon fitness index used in this invention is only an indicator of the expressibility of codons after optimization and does not provide technical guidance for the artificial optimization process. The optimization of the codons described in this invention requires comprehensive consideration of multiple indicators such as codon preference, GC content, and hairpin structure formation before final manual determination. In this invention, the codon CAI before optimization was 0.72, and after optimization, it reached 0.99, thereby improving its expression level in mammalian cells. A kozak sequence (CGCCACC) was added before the coding region, and a TGA stop codon was added after the coding region to form the packaging sequence as shown in SEQ ID NO.1. The packaging sequence was constructed into the eukaryotic expression vector PCDNA3.1 using BamHI (GGATCC) and EcoRI (GAATTC) restriction sites (gene expression and plasmid construction were completed by General Biosystems (Anhui) Co., Ltd.).

[0039] 1.2 Construction of recombinant adenovirus packaging plasmid

[0040] The shuttle plasmid pShuttle-G carrying the G protein gene (vector from ABM Bioscience (China) Co., Ltd.) was digested using PI-SceI / I-CeuI restriction endonuclease. The DNA was incubated at 37°C for 3 hours, and then purified using the phenol-chloroform method. The reagent ratio in the extraction system was phenol:chloroform:isoamyl alcohol (25:24:1).

[0041] The expression cassette carried by the pShuttle vector was subcloned into the pAdeno vector (vector sourced from ABM Bioscience (China) Co., Ltd.) containing the backbone plasmid of human adenovirus type 5 genome with E1 / E3 region deletion. The G protein expression cassette was ligated into the pAdeno vector using standard methods with PI-SceI / I-CeuI restriction endonucleases. The ligation product was digested with Swal enzyme to digest the pAdeno vector without the target gene ligated. The reaction product was purified using phenol-chloroform extraction. The purified ligation product was transformed into DH5α competent cells, cultured overnight, and then colony PCR was performed for identification. Plasmids were extracted from positive clones and sequenced for verification. The correctly sequenced recombinant plasmid was named pAdeno-G (…). Figure 1 ).

[0042] 2. Packaging, preparation and identification of recombinant adenovirus

[0043] 2.1 Packaging of recombinant adenovirus

[0044] HEK293T cells were seeded in 6-well plates and cultured at 37°C with 5% CO2 for 12-24 hours. When the cells reached 70% confluence, they were transfected using DNA fectin transfection reagent, with 5-7 μg of pAdeno-G plasmid per well. After 3-5 days, the cells were digested with trypsin and transferred to T75 cell culture flasks for further culture. Cytopathic effects were monitored periodically. When 90% of the cells showed cytopathic effects and detached from the bottom of the culture flask, the culture medium was collected, centrifuged at 1500 g for 5 min at 23°C, and the pellet was resuspended in 500 μL PBS. Three freeze-thaw cycles were performed to lyse the cells, and the obtained viral solution was stored at -80°C. In this invention, the obtained recombinant adenovirus is defined as "Ad5-NiV".

[0045] 2.2 Amplification and Identification of Recombinant Adenovirus

[0046] HEK293T cells were seeded in 10cm culture dishes and cultured for 12-24 hours. When the cell confluence reached 60-70%, the cells were infected with 200μL of adenovirus stock solution. Cytopathic effects were observed daily. When 95% of the cells showed cytopathic effects and detached from the culture dish, the culture medium was collected, and three freeze-thaw cycles were performed. Cell debris was removed by centrifugation at 3000 rpm for 10 min at room temperature. The recombinant adenovirus was purified using double cesium chloride density gradient centrifugation, and the purified virus solution was stored in DMEM medium containing 2.5% glycerol. Bacterial and fungal contamination was detected using direct culture, and mycoplasma contamination was detected using PCR. The adenovirus titer was determined using an endpoint dilution assay. The results showed that 293T cells infected with recombinant adenovirus developed grape-like cytopathic effects (CPE). Figure 2It is free from fungal, bacterial, and mycoplasma contamination and can be used as a toxin for large-scale production.

[0047] Example 2. Immunological evaluation of Nipah virus disease vaccine in mouse models

[0048] 1. Animal immunization

[0049] Vaccine immunization experiments were conducted using 6-8 week old female BALB / c mice free of specific pathogens. Six mice were used in each group, and each mouse was immunized with 1×10⁻⁶ mmol / L. 7 The control group was immunized with 10 μg of recombinant Nipah virus G protein (GeneID: 920955) or recombinant Hendra virus G protein (GeneID: NC_001906.3) and 200 μg of aluminum adjuvant. The blank group was immunized with PBS. All mice were administered 100 μL of the drug via intramuscular injection.

[0050] 2. Specific antibody detection

[0051] Purified Nipah virus and Hendra virus soluble G proteins (Gene ID: 1446471) were coated onto 96-well microplates at a concentration of 1 μg / mL using coating buffer (50 mM carbonate buffer, pH 9.6) and incubated overnight at 4°C. After discarding the supernatant, 100 μL of blocking buffer (PBS containing 2% BSA) was added to all wells, and the plates were blocked at 37°C for 1 hour. After discarding the supernatant, the plates were washed three times with PBST (PBS containing 0.1% Tween 20), and then diluent (PBS containing 0.2% BSA) and serum were added. The serum was serially diluted, and the plates were incubated at 37°C for 1 hour. After discarding the supernatant, wash the plate three times with PBST (PBS containing 0.1% Tween 20). Add 100 μL / well of HRP-bound goat anti-mouse IgG (Abcam, UK) diluted 1:50000 and incubate at 37°C for 45 minutes. After discarding the supernatant, wash the plate three times with PBST (PBS containing 0.1% Tween 20). Add 100 μL / well of TMB single-component substrate solution and incubate in the dark for 5 minutes. Then add 50 μL / well of ELISA stop solution and measure the absorbance at 450 nm - 630 nm using a microplate reader.

[0052] Nipah virus G protein-specific IgG antibodies produced in mice were detected 14 days after the initial Ad5-NiV immunization and 21 days after the booster immunization. Figure 3The results showed that 14 days after the first immunization, the mean log10 titer of anti-Nipah virus G protein-specific antibodies induced by the Ad5-NiV group was 4.6, 4.0 in the Nipah virus G protein plus aluminum adjuvant group, 3.2 in the Hendra virus G protein plus aluminum adjuvant group, and undetectable in the PBS group. Figure 3 ).

[0053] Buffet immunization stimulated a stronger Nipah virus G protein-specific antibody response, with antibody titers generally increasing in vaccinated mice. The average log10 titer of anti-Bangladesh Nipah virus G protein-specific antibodies induced by Ad5-NiV was 5.5, compared to 5.1 in the Nipah virus G protein plus aluminum adjuvant group and 4.0 in the Hendra virus G protein plus aluminum adjuvant group. No antibody was detected in the PBS group. This indicates that Ad5-NiV immunization effectively elicits a Nipah virus G protein-specific antibody response and is superior to recombinant protein vaccines using Nipah or Hendra virus G proteins as antigens.

[0054] 3. Nipah virus neutralizing antibody detection

[0055] The neutralizing antibody response to Ad5-NiV immune stimulation was detected using a liquid chromatography-array method, a mainstream approach for detecting Nipah virus neutralizing antibodies. Isolated mouse serum was placed in a black, opaque 96-well plate. Magnetic microspheres coated with the G protein of both Malaysian and Bangladeshi Nipah virus types were added to the culture plate, 1500 microspheres of each type per well. Diluted biotin-conjugated ephrin-B2 receptor was added and the plate was incubated at 800 rpm for 60 minutes with shaking. Streptavidin-r-phyreythrin (SAPE) was then added and the plate was incubated at 800 rpm for 30 minutes with shaking. The supernatant was aspirated using a magnetic plate separator and washed three times with PBS (containing 1% BSA). Finally, the binding was measured using a Luminex MAGPIX liquid chromatography-array analyzer.

[0056] The results showed that a single Ad5-NiV immunization could elicit a neutralizing antibody response against Nipah virus. In the detection of neutralizing antibodies against Bengal Nipah virus, 14 days after the initial immunization, the average log10 neutralizing antibody titer induced by the Ad5-NiV group was 2.7, 2.5 in the Nipah virus G protein with aluminum adjuvant group, 1.7 in the Hendra virus G protein with aluminum adjuvant group, and undetectable in the PBS group. Figure 4 The booster immunization stimulated a stronger neutralizing antibody response against Bengal Nipah virus, with a general increase in neutralizing antibody titers in vaccinated mice. Twenty-one days after the booster immunization, the average log10 neutralizing antibody titer against Bengal Nipah virus induced by the Ad5-NiV group was 3.4, 3.7 in the Nipah virus G protein with aluminum adjuvant group, 2.5 in the Hendra virus G protein with aluminum adjuvant group, and undetectable in the PBS group. Figure 5 ).

[0057] In the detection of neutralizing antibodies against the Malaysian Nipah virus, 14 days after primary immunization, the mean log10 neutralizing antibody titer induced by the Ad5-NiV group was 2.7, 2.4 in the Nipah virus G protein with aluminum adjuvant group, 1.8 in the Hendra virus G protein with aluminum adjuvant group, and undetectable in the PBS group. Figure 6 ) Boosted immunization stimulated a stronger neutralizing antibody response against the Malaysian Nipah virus, with a general increase in neutralizing antibody titers in vaccinated mice. 21 days after boosted immunization, the average log10 neutralizing antibody titer against the Malaysian Nipah virus was 3.4 in the Ad5-NiV group, 3.6 in the Nipah virus G protein with aluminum adjuvant group, and 2.4 in the Hendra virus G protein with aluminum adjuvant group. It was undetectable in the PBS group. Figure 7 ).

[0058] The results above demonstrate that Ad5-NiV can rapidly and efficiently elicit neutralizing antibody responses against Malaysian and Bangladeshi Nipah virus in mouse models, preliminarily validating its effectiveness as a candidate vaccine.

[0059] 4. Detection of cellular immune responses

[0060] 4.1 Peptide Library Synthesis

[0061] The Nipah virus G protein (Gene ID: 920955) has a full length of 602 amino acids. Peptide library synthesis was performed using the extracellular region of the G protein, specifically amino acids 71-602. Peptides were selected in 15-amino acid segments with a step size of 4, and each segment had 11 overlaps. A total of 130 peptides were synthesized and desalted (by Jier Biochemical Co., Ltd.) to construct a T-cell epitope peptide library. T-cell epitope peptides can specifically stimulate immune cells, requiring no internalization or processing by APCs, and can be directly presented to T cells. Each T-cell epitope peptide corresponds to a MHC molecule, representing a specific type of cellular immunity. Multiple T-cell epitope peptides are mixed to form a T-cell epitope peptide library or peptide pool. Stimulation using the peptide library can effectively induce specific T cells to secrete cytokines, thereby detecting the level of specific cellular immunity evoked by candidate vaccines.

[0062] 4.2 CBA Flow Cytometry Cytokine Detection Technology

[0063] Vaccine immunization experiments were conducted using 6-8 week old female BALB / c mice free of specific pathogens. Six mice were used in each group, and each mouse was immunized with 1×10⁻⁶ mmol / L. 7The control group was immunized with 10 μg of recombinant Nipah virus G protein (Gene ID: 920955) or recombinant Hendra virus G protein (Gene ID: NC_001906.3) and 200 μg of aluminum adjuvant. The blank group was immunized with PBS. All mice were administered 100 μL of the drug via intramuscular injection.

[0064] Spleens were removed and ground under aseptic conditions from mice euthanized with recombinant adenovirus and recombinant protein vaccines. The mouse spleen cells were diluted to 4 × 10⁶ cells / mL using 1640 medium containing 10% FBS. 6 / mL, and 50 μL of cell suspension was added to a 96-well cell culture plate. Then, the T-cell epitope peptide library or Nipah virus G protein dilution was added for stimulation, with a peptide library concentration of 1 μg / mL per peptide and a protein concentration of 20 μg / mL. The 96-well plate was incubated at 37°C for 3 days. After stimulation, the plate was centrifuged at 500g for 5 minutes and the supernatant was collected. Cytokine assays were performed using a mouse Th1 / Th2 / Th17 CBA kit (BD Pharmingen, USA). The reagent used to capture the magnetic beads was added to a mixture of 50 μL of sample and PE detection antibody. The reaction was carried out in the dark at room temperature for 2 hours. All unbound antibodies were removed by adding 1 mL of wash buffer before centrifugation. The captured beads were then resuspended in 300 μL of wash buffer and analyzed using FACS (BD). Six standard curves were obtained for each experiment using four-parameter linear fitting, with serially diluted standards ranging from 0 to 5000 pg / mL.

[0065] The results showed that spleen cells of Ad5-NiV-immunized mice stimulated by the peptide pool secreted large amounts of cytokines ( Figures 8-13 The levels of IFN-γ, TNF, IL-2, IL-6, IL-10, and IL-17A secreted by the Ad5-NiV group were 40777.3, 1282.2, 231.8, 928.1, 247.0, and 287.3 pg / mL, respectively. The levels of IFN-γ, TNF, IL-2, IL-6, and IL-10 secreted by the Nipah virus G protein with aluminum adjuvant group were 16.3, 238.7, 38.0, 41.5, and 18.0 pg / mL, respectively, with IL-17A not detected. The TNF concentration secreted by the Hendra virus G protein with aluminum adjuvant group was 45.2, while IFN-γ, IL-2, IL-6, IL-10, and IL-17A were not detected. This indicates that a single immunization with Ad5-NiV can elicit a strong specific cellular immune response against Nipah virus, significantly superior to that of the recombinant protein vaccine.

Claims

1. The application of a recombinant human adenovirus type 5 in the preparation of a vaccine for the prevention of Nipah virus disease, characterized in that, The recombinant human adenovirus type 5 expresses the extracellular domain of the Nipah virus G protein encoded by the polynucleotide molecule shown in SEQ ID NO. 1; The recombinant human adenovirus type 5 is a human type 5 replication-defective adenovirus with combined deletions of E1 and E3.

2. The application according to claim 1, characterized in that, The recombinant human adenovirus type 5 was prepared as an injection, nasal drops, spray, or inhaler.

3. A recombinant human adenovirus type 5 containing the polynucleotide of claim 1.

4. The method for preparing recombinant human adenovirus type 5 according to claim 3, the method comprising the following steps: (1) Construct a shuttle plasmid vector containing the polynucleotides described in claim 1; (2) The shuttle vector described in step (1) is co-transfected with the backbone plasmid into the host cell to package a recombinant replication-defective adenovirus; (3) Cultivate the host cells described in step (2); (4) Extract human replication-deficient recombinant virus expressing Nipah virus G protein from the host cell described in step (3).

5. The method according to claim 4, characterized in that, The host cell in step (2) is HEK293 cell.

6. The method according to claim 4, characterized in that, The backbone plasmid in step (2) is a vector containing the genome of human adenovirus type 5 with a combined deletion of the E1 / E3 region.

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