Recombinant nucleic acid construct, recombinant vaccinia virus, vaccine composition and application thereof

Abstract

The invention discloses a recombinant nucleic acid construct, a recombinant vaccinia virus, a vaccine composition and application thereof, and relates to the recombinant nucleic acid construct for preventing human adenovirus infection, the recombinant vaccinia virus prepared from the recombinant nucleic acid construct, the vaccine composition containing the virus, and preparation and application of the recombinant nucleic acid construct and the recombinant vaccinia virus. The technical problems that an existing multivalent vaccine is not uniform in immune effect and tedious in construction process are solved. The invention discloses a recombinant nucleic acid construct which comprises a vaccinia virus homologous recombination arm and an expression cassette unit located between the vaccinia virus homologous recombination arm. The expression cassette unit comprises human type-55, type-3 and type-7 adenovirus HEXON protein coding genes, the type-55 gene is reverse, and the type-3 and type-7 genes are forward. The recombinant vaccinia virus constructed by the method can realize stable co-expression of multivalent antigens, and is used for preparing multivalent vaccines for preventing infection of human type 3, type 7 and type 55 adenoviruses. The method is suitable for the fields of vaccine research and development, infectious disease prevention and control, viral vector technology platforms, recombinant protein or multivalent antigen expression technology and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a recombinant nucleic acid construct for preventing human adenovirus infection, a recombinant vaccinia virus prepared from the construct, a vaccine composition containing the virus, and the preparation and application thereof. Background Technology

[0002] Human adenoviruses are common respiratory pathogens that can cause severe acute respiratory illnesses, including pneumonia and bronchitis, in children and immunocompromised individuals. HAdV-3 (Ad3) and HAdV-7 (Ad7) are the most prevalent types causing sporadic infections and outbreaks in children, while HAdV-55 (Ad55) infection is associated with localized outbreaks and more severe pneumonia outcomes. Currently, there are no specific antiviral drugs targeting these adenovirus types, nor are there any safe and effective adenovirus vaccines available for public use. Therefore, developing a vaccine that can simultaneously prevent multiple prevalent adenoviruses is of significant public health importance.

[0003] Vaccine virus (VTT), as a classic viral vector, possesses advantages such as large genome capacity, broad host range, and the ability to induce strong cellular and humoral immune responses. The Tian Tan strain of Vaccine Virus (VTT) is a vaccine strain unique to my country with a long history of proven safety. By using homologous recombination technology to insert exogenous antigen genes into non-essential regions of the VTT genome (such as the TJ2R fragment), recombinant viruses expressing exogenous antigens can be constructed.

[0004] However, the development of multivalent adenovirus vaccines still faces challenges: First, simple mixed immunization with multiple antigens may lead to antigen competition, resulting in a weakened immune response against certain types; second, stable and efficient co-expression of multiple exogenous genes in a vector is required; and third, the traditional recombinant virus screening process is cumbersome and time-consuming. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant nucleic acid construct, a recombinant vaccinia virus prepared therefrom, and a vaccine composition containing the virus, so as to solve the technical problems of uneven immunization effect and cumbersome construction process of multivalent vaccines in the prior art, thereby achieving safe, efficient and stable combined prevention against human adenovirus types 3, 7 and 55.

[0006] Solution A recombinant nucleic acid construct comprising homologous recombination arms of vaccinia virus and expression cassette units located between the homologous recombination arms; The expression cassette unit contains expression cassettes for the HEXON protein-coding genes of human adenovirus types 55, 3, and 7, wherein the expression cassette for the HEXON protein-coding genes of human adenovirus type 55 is reversed, and the expression cassettes for the HEXON protein-coding genes of human adenovirus types 3 and 7 are forward-oriented.

[0007] In a further preferred embodiment, the recombinant nucleic acid construct is the shuttle plasmid pTJ2R-AD, and the sequence of the left recombinant arm of the homologous recombinant arm is shown in SEQ ID NO: 1, and the sequence of the right recombinant arm is shown in SEQ ID NO: 2.

[0008] In a further preferred embodiment, the sequence of the expression box unit is shown in SEQ ID NO:3.

[0009] A recombinant vaccinia virus, wherein the genome of the recombinant vaccinia virus integrates an expression cassette unit carried by the recombinant nucleic acid construct.

[0010] A vaccine composition comprising the recombinant vaccinia virus and a pharmaceutically acceptable vector or adjuvant.

[0011] A method for preparing recombinant vaccinia virus includes the following steps: Step 1: Co-transfect mammalian cells with the recombinant nucleic acid construct and the Tian Tan strain of vaccinia virus; Step 2: Use the exogenous screening markers to screen and purify recombinant viral plaques.

[0012] A further preferred approach is to use a dual-label screening method with EGFP and BrdU.

[0013] The recombinant nucleic acid construct is used in the preparation of drugs for the prevention of human adenovirus type 3, 7 and / or 55 infection.

[0014] The recombinant vaccinia virus is used in the preparation of drugs for the prevention of human adenovirus type 3, 7 and / or 55 infection.

[0015] The use of the vaccine composition in the preparation of a medicament for the prevention of human adenovirus type 3, 7 and / or 55 infection.

[0016] The beneficial effects of this invention compared to the prior art are as follows: The recombinant nucleic acid constructs and the multivalent vaccines prepared therefrom, by constructing the HEXON protein-encoding genes of human adenovirus types 55, 3 and 7 in a specific order in the same expression cassette unit, achieve a balanced, efficient and antigen-free synergistic immune protection against the three prevalent adenovirus types, ensuring the core efficacy of the multivalent vaccine.

[0017] The recombinant nucleic acid construct described in this application achieves convenient purification and identification of exogenous proteins by fusing his, flag, and HA tag coding sequences to the HEXON gene and separating adjacent expression cassettes with meaningless nucleic acid sequences. At the same time, it effectively avoids sequence loss or rearrangement caused by homologous recombination when multiple genes are tandemly linked, thereby improving the genetic stability of the recombinant virus.

[0018] The recombinant nucleic acid construct and virus preparation method described in this application, by introducing a screening system including the Cre / Loxp system and the EGFP and BrdU dual-marker genes, achieves rapid, visualized, and efficient screening and purification of recombinant vaccinia virus, significantly improving the construction efficiency of the vaccine platform.

[0019] The vaccine composition described in this application utilizes the Tian Tan strain of vaccinia virus (VTT), whose safety has been validated over a long period, as a vector. An antigen expression cassette is integrated into the TJ2R non-essential region of its genome, while virulence-related genes can be deleted. This enables the construction of a multivalent adenovirus vaccine vector platform with strong immunogenicity, high safety, and suitability for large-scale preparation.

[0020] The recombinant nucleic acid constructs, recombinant vaccinia virus, vaccine compositions, and their applications described in this invention are applicable to fields such as vaccine research and development and infectious disease prevention and control, viral vector technology platforms, and recombinant protein or multivalent antigen expression technologies. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the construction of the shuttle plasmid pTJ2R-AD as described in Embodiment 1; Figure 2 It is the Hind III enzyme digestion diagram described in Implementation Method 1; Figure 3 It is the virus screening chart described in Implementation Method 2; Figure 4 The genetic stability detection diagrams described in Implementation Method 3 are as follows: (a) Electrophoresis results of Ad3 HEXON gene PCR amplification products; (b) Electrophoresis results of Ad7 HEXON gene PCR amplification products; (c) Electrophoresis results of Ad55 HEXON gene PCR amplification products; (d) Electrophoresis results of TJ site PCR amplification products; and (e) Electrophoresis results of EGFP selection marker gene PCR amplification products. Figure 5 The following are diagrams illustrating protein expression as described in Embodiment 4: (a) Ad3 HEXON protein; (b) Ad7 HEXON protein; (c) Ad55 HEXON protein; Figure 6 This is a graph showing the change in mouse body weight as described in Implementation Method 5; Figure 7This is a diagram showing the T lymphocyte activation results as described in Implementation Method Six; Figure 8 The following is a statistical chart of the number of activated T lymphocytes as described in Implementation Method Six: (a) the proportion of CD4+ helper T cells in total T cells; (b) the proportion of CD8+ cytotoxic T cells in total T cells; (c) the secretion of IFN-γ. (d) The proportion of CD4+ T cells secreting IL-4; (e) The proportion of CD4+ T cells secreting IFN-γ. (f) The proportion of CD8+ T cells secreting IL-4; Figure 9 The specific antibody level detection graphs described in Implementation Method 7 are: (a) dynamic changes in specific antibody levels against Ad3; (b) dynamic changes in specific antibody levels against Ad7; and (c) dynamic changes in specific antibody levels against Ad55. Figure 10 The neutralizing antibody level detection graphs described in Embodiment 8 are: (a) neutralizing antibody titer against Ad3; (b) neutralizing antibody titer against Ad7; and (c) neutralizing antibody titer against Ad55. Detailed Implementation

[0022] Various embodiments of the present invention will now be clearly and completely described with reference to the accompanying drawings. The embodiments described with reference to the drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0023] Implementation Method 1: Design and Synthesis of Shuttle Plasmid pTJ2R-AD Homologous recombination arms flanking the TJ2R nucleic acid fragment were designed and synthesized based on the complete genome sequence of the Tian Tan strain of vaccinia virus (VTT, GenBank: AF095689.1). The left recombinant arm sequence is shown in SEQ ID NO: 1, which is 500 bases from position 79784 to position 80283 of the VTT full genome sequence; the right recombinant arm sequence is shown in SEQ ID NO: 2, which is 1015 bases from position 80841 to position 81855 of the VTT full genome sequence. Subsequently, four expression cassettes were inserted between the two homologous recombination arms. The sequences of the expression cassettes are shown in SEQ ID NO: 3. Each expression cassette contains the vaccinia virus-specific early and late strong promoter RE / L and the termination signal T5nT sequence. Each expression cassette is separated by a meaningless sequence. The first expression cassette contains the HEXON gene of human adenovirus type 55 with a his tag sequence, the second expression cassette contains the HEXON gene of human adenovirus type 3 with a flag tag sequence, the third expression cassette contains the HEXON gene of human adenovirus type 7 with a HA tag sequence, and the fourth expression cassette contains the exogenous selection marker EGFP gene. The first expression cassette is in reverse expression mode, and the other three expression cassettes are in forward expression mode. The construction diagram is shown below. Figure 1 As shown; After plasmid synthesis, it is digested with enzymes and sequenced for identification, such as Figure 2 As shown, this embodiment successfully constructed a shuttle plasmid containing four expression cassettes, and obtained an 8600bp band by Hind III digestion, which was then successfully sequenced.

[0024] Implementation Method 2: Construction of Recombinant Virus In this embodiment, recombinant vaccinia virus rVTT-AD is constructed by co-transfection of the shuttle plasmid pTJ2R-AD with VTT. The specific process is as follows: BHK-21 cells (a mammalian cell line highly sensitive to vaccinia virus and commonly used for its replication and recombination) were passaged into 6-well plates. After 12-24 hours, when the cells grew to about 80% of the bottom wall area of ​​the cell plate, the culture medium in the wells was aspirated, 600 μL of DMEM culture medium was added, and 0.1 MOI of VTT was seeded. The cells were gently shaken to mix, and then placed in an incubator for 2 hours. Add 500 μL of Opti-MEM culture medium to sterile EP tubes (Eppendorf tubes). Add 10 μg of large-scale plasmid pTJ2R-AD to one EP tube and 10 μL of liposomes to another EP tube. Mix well and let stand for 5 min. Add the liquid containing plasmids to the EP tube containing liposomes, mix gently and let stand for 20 min. Discard the liquid in the 6-well plate, add the plasmid and liposome mixture to the wells, and incubate at 37℃ and 5% CO2 for 6 h. Then discard the liquid in the wells, add 2% FBS DMEM culture medium, and incubate for 48 h. After 48 hours, the cell slurry in the wells was aspirated, and green fluorescent plaques were observed under a fluorescence microscope, indicating successful transfection. The cells were cultured for another 72 hours, after which the supernatant and adherent cells were collected. The cells were then subjected to three freeze-thaw cycles and centrifuged at 500g for 5 minutes. The supernatant was collected and stored at -80°C for later use. The virus solution was then re-infected with BHK-21 cells, and the presence of fluorescent plaques indicated successful homologous recombination during the 48-72 hour period. Plaques with high fluorescence intensity were picked under a fluorescence microscope and dissolved in 400 μL of DMEM culture medium. The virus solution was then freeze-thawed three times and inoculated into BHK-21 cells again using the same method. After 48-72 hours, more fluorescent plaques were picked, and this process was repeated 10 times. Finally, a vaccinia virus rVTT-AD expressing only green fluorescent protein (RFP) was obtained through screening, i.e., recombinant vaccinia virus. The virus screening process is as follows: Figure 3 As shown.

[0025] Implementation Method 3: Genetic Stability Identification This embodiment confirms the stable existence of the expression cassette unit and that the original TJ2R nucleic acid fragment has not undergone reversion mutation, and evaluates the genetic stability of the deletion-type vaccinia virus rVTT-AD: The recombinant vaccinia virus rVTT-AD constructed in Implementation Method 2 was passaged in BHK-21 cells for 20 generations. The viral genomes of the 5th, 10th, 15th and 20th generations were extracted and used as templates. The VTT genome was used as a positive control for PCR identification. The 5 pairs of primers required for PCR are shown in Table 1. Table 1

[0026] PCR system: 4 μL genome template, 12.5 μL 2×PCR Master Mix, 0.5 μL upstream primer, 0.5 μL downstream primer, 7.5 μL ddH2O; PCR reaction conditions: 95℃ for 5 min, 94℃ for 30 s, 56℃ for 30 s, 72℃ for 30 s, for a total of 30 cycles; extension at 72℃ for 10 min; Finally, electrophoretic identification was performed. BHK-21 cells were infected with the recombinant vaccinia virus rVTT-AD, and the genome of the diseased cells was extracted and identified by RT-PCR. The results are as follows: Figure 4 As shown, the constructed vaccinia virus exhibits good genetic stability.

[0027] Implementation Method 4: Identification of Protein Expression Levels BHK-21 cells were passaged into 6-well plates. After 12-24 hours, when the cells had grown to about 80% of the bottom wall area of ​​the cell plate, the culture medium in the wells was aspirated, 600 μL of DMEM culture medium was added, and 0.1 MOI of VTT and rVTT-AD were seeded. The cells were gently shaken to mix, and then placed in an incubator for 2 hours. 48 hours later, cells were collected and proteins were extracted using a total protein extraction kit (Kang Wei Century, Beijing). Proteins were quantified using the BCA kit instructions (Beyotime, Shanghai). 30 μg of protein from each sample was separated using 12.5% ​​(Kangwei Century, Beijing) SDS-PGA and then transferred to an NC membrane; Then, the membrane was sealed with TBST containing 5% skim milk powder for 2 hours, and the primary antibody (1:1000) for each tag was added overnight at 4°C. After washing with TBST, the membrane was incubated with secondary antibody (1:1000) for 40 minutes, and then washed with TBST. Finally, protein expression levels were detected using an ECT colorimetric kit (Thermo, USA) and a gel imaging system. Total protein was extracted from diseased cells and analyzed by Western blotting. The results are as follows: Figure 5 As shown, the results indicate that the recombinant vaccinia virus rVTT-AD successfully transcribed and expressed an antigenic protein of 55 kDa.

[0028] Implementation Method 5: Mouse Immunization Experiment Dilute the virus samples (rVTT-AD and VTT) with 0.1 mL of PBS to a dilution of 1 × 10⁻⁶. 6 PFU / 0.1 mL, with a PBS control group included. At week 0, the first immunization was performed, with blood collected from the tail vein before the first immunization and from the tail vein once a week after the first immunization. Three weeks after immunization, blood was drawn from the orbital artery once, and a booster immunization was given at the same dose as at week 0. Two weeks after the booster immunization, blood was drawn from the orbital artery. The mice were grouped as shown in Table 2.

[0029] Table 2

[0030] Body weight changes in BALB / c mice 5 weeks after immunization are as follows Figure 6As shown, the weight of mice in the PBS control group maintained a steady increase; the rVTT-AD group showed no significant change compared to the control group; while the weight growth rate of mice in the VTT group was lower than that of the other two groups. This indicates that the knockout of the TJ2R gene reduced the toxicity of VTT, and the expression of recombinant vaccinia virus-associated adenovirus protein did not have a serious impact on the weight of BALB / c mice.

[0031] Implementation Method Six: Splenic Lymphocyte Isolation and Flow Cytometry Detection The spleen lymphocyte isolation and flow cytometry detection includes the following steps: (1) The mouse was euthanized by neck dislocation, the skin on the back of the mouse was disinfected with alcohol swabs, the left back of the mouse was cut open with scissors to expose the spleen, the spleen was removed and washed with 2ml of Hank's solution; (2) Gently grind the spleen with a sterile glass slide, then filter the cell suspension through a 200-mesh nylon mesh into a 15ml centrifuge tube, centrifuge at 1000rpm for 2min, slowly discard the supernatant, and then add 1ml of 1640 and mix well. (3) Slowly add the lymphocyte suspension to a 15ml centrifuge tube containing 4ml of mouse lymphocyte separation solution and centrifuge at 2000rpm for 10min.

[0032] (4) After centrifugation, the liquid can be observed to be divided into four layers. The second layer of milky white ring-shaped lymphocytes is aspirated, and the aspirated cells are transferred to another 15ml centrifuge tube. 4ml of 10% FBS 1640 culture medium is added, and then centrifuged at 1500rpm for 5min. (5) Slowly discard the supernatant and add 500µL of 10% FBS 1640 culture medium to resuspend the cells; (6) Cell counting. Dilute the cells to a final concentration of 2 × 10⁶ cells / ml; (7) The extracted mouse spleen cells were stained with APC-CD3, FITC-CD4, and PE-CD8 and then analyzed by flow cytometry. The changes in CD3+CD4+ and CD3+CD8+ in each group of mice were counted. Subsequently, the cells were stained with PerCP-IL-4 and PerCP-IFN-γ and then analyzed by flow cytometry. The changes in CD3+CD4+ / IL-4 / IFN-γ and CD3+CD8+ / IL-4 / IFN-γ in each group of mice were counted. like Figure 7 and Figure 8As shown, analysis of T cell subsets revealed that the number of CD3+CD4+ T cells and CD3+CD8+ T cells in the rVTT-AD group and VTT group were significantly higher than those in the PBS group (P<0.05), with the rVTT-AD-H group showing a significantly higher number than the VTT group (P<0.05). Immunization with the rVTT-AD vaccine significantly activated T lymphocytes and induced cytokine secretion. The levels of CD3+CD4+ / IL-4 / IFN-γ and CD3+CD8+ / IL-4 / IFN-γ were significantly higher than those in the VTT and PBS groups (P<0.05), indicating that immunization with the rVTT-AD vaccine can induce both humoral and cellular immunity.

[0033] Implementation Method Seven: Detection of Specific Antibody Levels The detection of the specific antibody level includes the following steps: (1) Coating antigen: Dilute Ad3, Ad7, and Ad55 to 2×10 6 PFU / ml, inactivated by water bath at 60℃ for 30 min, diluted 10-fold with coating buffer, and then 100µL / well was added to the ELISA plate and coated overnight at 4℃; (2) Discard the coating solution, wash the plate 3 times, and pat dry; (3) Add 200µL of blocking solution to each well and incubate at 37℃ for 2h; (4) Wash the plate 5 times and pat it dry; (5) Add 100µL of serum sample diluted 10 times to each well and incubate at 37°C for 2 hours; (6) Wash the board 5 times and pat it dry; (7) Add 100 µL of HRP-labeled antibody to each well and incubate at 37°C for 2 h; (8) Wash the board 5 times and pat it dry; (9) Add 100µL of 1×TMB solution to each well and incubate at room temperature for 15 min; (10) Add 50µL of stop solution to each well; (11) Detect absorbance at 450 nm; Specific antibody test results as follows Figure 9 As shown, starting from week 1 after the initial immunization, the level of anti-adenovirus specific antibodies in the rVTT-AD group was significantly higher than that in the VTT and PBS groups. After the booster immunization, the difference in specific antibody levels between the rVTT-AD group and the VTT and PBS groups became even more significant (P<0.001), indicating that rVTT-AD can stimulate the body to produce an anti-adenovirus specific immune response.

[0034] Implementation Method 8: Neutralizing Antibody Detection BHK-21 cells were administered at a rate of 1×10⁻⁶. 4The virus was inoculated into 96-well plates and cultured for 24 hours. The original culture medium was discarded and the virus mixture was inoculated into a 3-week and 5-week mouse serum collected at 56°C for 30 minutes. The mouse serum-virus mixture was prepared as follows: Mouse serum was serially diluted with DMEM culture medium at 2-fold intervals, with dilution gradients ranging from 2-1 to 2-10, and each dilution was replicated three times; 100 PFU of Ad3, Ad7, and Ad55 virus solutions were thoroughly mixed with the diluted mouse serum, and DMEM culture medium was added to a final volume of 100 μL. The mixture was then incubated at 37°C for 1 h to allow the virus and serum to fully react. A blank control group, a virus control group, and a serum toxicity control group were set up; After infecting cells at 37℃ for 2 h, discard the mixture, add 100 μL of 2% DMEM culture medium per well, and culture for another 72~96 h. Record the number of wells where plaques appear. The IC50 of the experiment was analyzed using the Reed-Muench method, and the titer was defined as the highest serum dilution that could inhibit 50% of the ingested viral load compared to the control. like Figure 10 As shown, neutralizing antibody detection in the third and fifth weeks revealed significant neutralizing activity in the serum of the rVTT-AD group (P<0.01), while no neutralizing activity was found in the serum of the VTT and PBS groups.

Claims

1. A recombinant nucleic acid construct, characterized in that, The recombinant nucleic acid construct includes homologous recombination arms of vaccinia virus and expression cassette units located between the homologous recombination arms; The expression cassette unit contains expression cassettes for the HEXON protein-coding genes of human adenovirus types 55, 3, and 7, wherein the expression cassette for the HEXON protein-coding genes of human adenovirus type 55 is reversed, and the expression cassettes for the HEXON protein-coding genes of human adenovirus types 3 and 7 are forward-oriented.

2. The recombinant nucleic acid construct according to claim 1, characterized in that, The recombinant nucleic acid construct is the shuttle plasmid pTJ2R-AD. The sequence of the left recombinant arm of the homologous recombinant arm is shown in SEQ ID NO: 1, and the sequence of the right recombinant arm is shown in SEQ ID NO:

2.

3. The recombinant nucleic acid construct according to claim 1, characterized in that, The sequence of the expression box unit is shown in SEQ ID NO:

3.

4. A recombinant vaccinia virus, characterized in that, The recombinant vaccinia virus genome integrates the expression cassette unit carried by the recombinant nucleic acid construct according to any one of claims 1-3.

5. A vaccine composition, characterized in that, The vaccine composition comprises the recombinant vaccinia virus of claim 4 and a pharmaceutically acceptable vector or adjuvant.

6. The use of the recombinant nucleic acid construct according to any one of claims 1-3 in the preparation of a medicament for preventing human adenovirus type 3, 7 and / or 55 infection.

7. The use of the recombinant vaccinia virus according to claim 4 in the preparation of a medicament for the prevention of human adenovirus type 3, 7 and / or 55 infection.

8. The use of the vaccine composition according to claim 5 in the preparation of a medicament for the prevention of human adenovirus type 3, 7 and / or 55 infection.

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