SAMRNA VACCINE AND PREPARATION METHOD THEREFOR

Abstract

Disclosed is an SamRNA vaccine, including a recombinant viral vector which includes: i) a viral gene replication complex including nucleotide sequences encoding viral gene replication-related proteins nsP1, nsP2, nsP3, and nsP4; and ii) a nucleotide sequence encoding at least one antigen. According to the SamRNA vaccine of the present invention, in addition to that a promoter of a modified adenoviral vector itself can transcribe an antigen gene to form mRNA, the viral gene replication-related proteins nsP1-4 use RNA as a template to synthesize a large amount of mRNAs, and the immune effect of a target antigen is greatly improved.

Claims

1. An SamRNA vaccine, comprising a recombinant viral vector which comprises: i) a viral gene replication complex comprising nucleotide sequences encoding viral gene replication-related proteins nsP1, nsP2, nsP3, and nsP4; and ii) a nucleotide sequence encoding at least one antigen.

2. The SamRNA vaccine according to claim 1, wherein the recombinant viral vector is a recombinant adenovirus, a chimpanzee adenovirus, a recombinant vesicular stomatitis virus, a recombinant poxvirus, a recombinant dengue virus, a recombinant Kunjin virus, a recombinant sendai virus, or a recombinant canine distemper virus.

3. The SamRNA vaccine according to claim 1, wherein the antigen causes an immune response against bacteria, viruses, fungi or parasites.

4. The SamRNA vaccine according to claim 3, wherein the antigen is a human herpes zoster virus gE protein, a rotavirus VP4 or VP7, an HPV-L1 protein, or an Ebola virus gP protein.

5. The SamRNA vaccine according to claim 1, wherein the antigen is a tumor-specific antigen, and is selected from NY-ESO-1, SSX2, SCP1, RAGE, BAGE, GAGE, MAGE family polypeptides, p53, p21/Ras, CDK4, MUM1, caspase-8, CIA0205, HLA-A2-R1701, (3-catenin, TCR, BCR-abl, triosephosphate isomerase, KIA0205, CDC-27, LDLR-FUT, Galectin 4, Galectin 9, protease 3, WT 1, carbonic anhydrase, aldolase A, PRAME, HER-2/neu, mammaglobin, alpha-fetal protein, KSA, gastrin, telomerase catalytic protein, MUC-1, G-250, p53, carcino-embryonic antigens, melanoma-melanocyte differentiation antigens, PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, p15, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EB viral antigens, EBNA, human papillomavirus antigens, hepatitis B and C viral antigens, human T-lymphotropic viral antigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791Tgp72, β-HCG, BCA225, BTAA, CA 125, CA 15-3(CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733(EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TAAL6, TAG72, TLP, and TPS.

6. The SamRNA vaccine according to claim 1, wherein the recombinant viral vector is obtained by co-transfecting a modified adenoviral skeleton plasmid and a shuttle plasmid containing a nucleotide sequence encoding at least one antigen, wherein the modified adenoviral skeleton plasmid comprises a viral gene replication complex which is gene sequences encoding viral gene replication-related proteins nsP1, nsP2, nsP3 and nsP4.

7. The SamRNA vaccine according to claim 6, wherein the adenoviral skeleton plasmid is selected from pAdEasy-1, pAdEasy-2, pBHG11, pBHG-fiber5 or pBHG-fiber35.

8. The SamRNA vaccine according to claim 1, wherein the recombinant viral vector further comprises: iii) a promoter for transcribing an antigen gene.

9. The SamRNA vaccine according to claim 1, wherein the recombinant viral vector further comprises: iv) a nucleotide sequence encoding at least one adjuvant.

10. The SamRNA vaccine according to claim 8, wherein the adjuvant is selected from C3b, GM-CSF, IL-17, IFN, IL-15, IL-21, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-α, INF-γ and CpG.

11. A method for preparing the SamRNA vaccine according to claim 1, comprising the steps of: constructing a modified adenoviral skeleton plasmid; cloning an antigen gene fragment; constructing a shuttle plasmid; co-transfecting and packaging the shuttle plasmid and the adenoviral skeleton plasmid, wherein the modified adenoviral skeleton plasmid comprises a viral gene replication complex which is gene sequences encoding viral gene replication-related proteins nsP1, nsP2, nsP3, and nsP4.

12. (canceled)

13. (canceled)

14. A modified adenoviral skeleton plasmid, comprising a viral gene replication complex which is gene sequences encoding viral gene replication-related proteins nsP1, nsP2, nsP3 and nsP4, wherein the adenoviral skeleton plasmid is selected from pAdEasy-1, pAdEasy-2, pBHG11, pBHG-fiber5, or pBHG-fiber35.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1 shows the identification results of inserting a viral gene replication complex protein gene into a skeleton plasmid;

[0051] FIG. 2 shows the identification results of inserting a promoter sequence into the skeleton plasmid;

[0052] FIG. 3 shows a modified skeleton plasmid;

[0053] FIG. 4 shows a flow diagram of constructing Ad-SamRNA-gE;

[0054] FIG. 5 shows the double enzyme digestion verification of Ad-SamRNA-gE;

[0055] FIG. 6 shows a chromatogram of Ad-SamRNA-gE purified by CL-4B;

[0056] FIG. 7 shows the purity analysis of Ad-SamRNA-gE;

[0057] FIG. 8 shows the immunogenicity of different vectors, wherein the left side is the immunogenicity of primary immunization, and the right side is the immunogenicity of secondary immunization;

[0058] FIG. 9 shows the expression quantities of gE genes of different vectors; and

[0059] FIG. 10 shows that the molecular adjuvant enhances the immunogenicity of Ad-SamRNA-gE, wherein the left side is the immunogenicity of primary immunization, and the right side is the immunogenicity of secondary immunization.

DETAILED DESCRIPTION

[0060] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are merely a part of embodiments rather than all embodiments of the present invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

Embodiment 1 Insertion of Viral Gene Replication Complex and Promoter Gene

[0061] Fragments into a Skeleton Plasmid

[0062] Synthesis of viral gene replication complex and promoter gene fragments: In a specific implementation, the gene sequences of the viral gene replication complexes nsP1, nsP2, nsP3 and nsP4 were SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, respectively, and the viral gene replication complex gene fragment is synthesized according to the aforementioned sequences, wherein four antigen protein genes were synthesized into a large gene fragment.

[0063] For the promoter, a gene fragment of the promoter was synthesized according to SEQ ID NO.5, and meanwhile, enzymatic cleavage sites were added at both terminals of the gene fragment of the promoter.

[0064] Taking a pAdEasy-1 adenoviral skeleton plasmid as an example, a process of inserting the viral gene replication complex and the promoter into pAdEasy-1 was presented. Firstly, the gene fragment of the viral gene replication complex was inserted into the pAdEasy-1 adenoviral skeleton plasmid, wherein the 4 synthesized long protein gene fragments of the viral gene replication complex contained pad enzymatic cleavage sites on both sides. pAdEasy-1 and the 4 protein genes of the viral gene replication complex were respectively digested with a pac I enzyme. After gel extraction, ligation was conducted by a T4 DNA ligase. Theoretically, there were two ligation manners of the protein genes of the viral gene replication complex (insertion into the skeleton plasmid in an inverted order and insertion into the skeleton plasmid in a sequential order). After the ligation product was transformed into DH5a, monoclones were selected, and the vector in which the genes were inserted into the skeleton plasmid as expected was identified by a PCR method. The results of PCR identification were shown in FIG. 1. The clone No. 1 and the clone No. 2 were both correctly inserted into the protein gene fragment of the viral gene replication complex, and the clone No. 1 was selected to be subjected to an operation of inserting the gene fragment of the promoter.

[0065] The gene of the promoter and the skeleton plasmid into which the gene of the viral gene replication complex has been inserted, were digested with ClaI, and then ligated with a T4 DNA ligase after gel extraction. Theoretically, there were two ligation manners of the protein genes of the promoter (insertion into the skeleton plasmid in an inverted order and insertion into the skeleton plasmid in a sequential order). After the ligation product was transformed into DH5a, monoclones were selected, and the vector in which the genes were inserted into the skeleton plasmid as expected was identified by a PCR method. The results of PCR identification were shown in FIG. 2. In this step of operation, clones No. 3, No. 4 and No. 5 were correctly inserted into the promoter sequence.

[0066] After the aforementioned two steps, the skeleton plasmids pAdEasy-No. 13, No. 4 and No. 5 with addition of the sequences of the viral gene replication complex and the promoter, will be obtained. The positions of the promoter and the viral gene replication complex in the pAdEasy-1 skeleton plasmid were shown in FIG. 3.

[0067] According to the same method, the insertion of sequences of the promoter and the viral gene replication complex could be completed on other skeleton plasmids.

Embodiment 2 Construction of Ad-SamRNA-gE

[0068] Taking the herpes zoster gE protein antigen as an example, the display of a construction process of an Ad (adenovirus)-SamRNA vaccine was conducted.

[0069] A gE antigen gene was synthesized, and meanwhile enzymatic cleavage sites were added on both sides of the gene fragment, the specific enzymatic cleavage sites were determined according to the selected recombination system and shuttle plasmid, and this method was mastered by those skilled in the art. The Adeasy vector system was taken as an example to display the construction process of the Ad-SamRNA vaccine hereafter.

[0070] Firstly, the gE antigen gene was ligated into the shuttle plasmid pS-C to form pS-C-gE, and the ligation method adopted an experimental method commonly used in the industry. The shuttle plasmid pS-C and the gE antigen gene were subjected to double enzyme digestion with KpnI and XhoI at the same time (see Table 1 below for the enzyme digestion reaction system). After the enzyme digestion reaction was completed, the target fragments were recovered with a gel extraction kit (see the instructions for the recovery method). Then the linearized antigen fragment and the plasmid were ligated by a T4 DNA ligase (see Table 2 below for the reaction system).

[0071] The construction methods adopting other adenoviral vector systems were similar to the above.

TABLE-US-00001 TABLE 1 Double-enzyme digestion reaction system (50 μl) Reagent Volume Enzyme A 2 μl Enzyme B 2 μl Buffer 5 μl Shuttle plasmid or gE gene fragment 30 μl  water 11 μl 

[0072] The double enzyme digestion reaction was carried out at 37° C. for at least 4 h or above.

TABLE-US-00002 TABLE 2 Enzyme ligation reaction system (10 μl) Reagent Volume Shuttle plasmid 2 μl gE gene fragment 2 μl Buffer 1 μl T4 DNA ligase 1 μl H2O

[0073] The ligation reaction was carried out at 4° C. overnight.

[0074] The shuttle plasmid pS-C-gE containing an antigen gene, and the adenoviral skeleton plasmid (such as pAdEasy-1) in which the viral gene replication complex and the promoter were inserted, were used for co-transfection, the two plasmids would undergo homologous recombination, and Ad-SamRNA-gE was obtained through separation. The flow diagram of constructing Ad-SamRNA was shown in FIG. 4.

[0075] The constructed Ad-SamRNA-gE was verified by a double enzyme digestion method, and the successfully constructed vector would be used for production and immunogenicity evaluation of the vaccine. The results of double enzyme digestion were as shown in FIG. 5 below: a lane M was a marker, a lane 1 was double enzyme digestion of the adenoviral vector, and a lane 2 was double enzyme digestion of Ad-SamRNA-gE. The results showed that the gE antigen gene was correctly integrated into the adenoviral vector system.

Embodiment 3 Preparation of Ad-SamRNA-gE Vaccine

[0076] Taking the herpes zoster gE protein antigen as an example, the display of a preparation process of an Ad-SamRNA vaccine was conducted.

[0077] An adenoviral vector could massively propagate in 293 cells. HEK293 cells were mostly used for the Admax adenoviral vector system, while AD293 cells were used for Adeasy recombinant adenoviral vector cells.

[0078] 293 cells were infected with Ad-SamRNA-gE at MOI=5-10 for at least 40 h, and then centrifuged at 8,000 g for 10 min to collect cell precipitates. The cell precipitates were dissolved in PB or a lysis buffer (2 mM MgCl.sub.2, 50 mM HEPES, pH 7.5), and then repeatedly frozen and thawed at −80° C. for three times for cell lysis, the cell debris was removed by centrifugation, and the supernatant passed through CL-4B, and subjected to one-step chromatography to obtain a target virus.

[0079] Besides cell debris, various impure proteins in the cell lysis solution were Ad-SamRNA-gE, and the cell debris could be removed by centrifugation. Compared with impure proteins, the molecular weight of Ad-SamRNA-gE was much larger than those of impure proteins, and therefore, a pure virus could be obtained by a one-step process with CL-4B.

[0080] The result of purifying Ad-SamRNA-gE by CL-4B was shown in FIG. 6. The molecular weight of Ad-SamRNA-gE was the largest, and thus was eluted first. The peak 1 in FIG. 6 was Ad-SamRNA-gE.

[0081] The purity of the harvested Ad-SamRNA-gE was analyzed by HPLC. A TSK5000 column was selected for the experiment. The HPLC result was shown in FIG. 7. It could be seen that no impurity peak could be seen for the harvested virus, and thus the purity of the virus solution was very high.

Embodiment 4 Study on Immunogenicity of Ad-SamRNA-gE Vaccine

[0082] Taking the herpes zoster gE protein antigen as an example, the display of the immunogenicity of the Ad-SamRNA vaccine was conducted.

[0083] An experimental group: an Ad-SamRNA-gE group prepared in Embodiment 3;

[0084] A control group 1: a group directly injected with mRNA of the gE protein, which was referred to as mRNA-gE for short;

[0085] A control group 2: gE protein antigen gene vaccine using a common adenovirus as a vector, referred to as Ad-gE for short;

[0086] A negative control group: a normal saline immunization group

[0087] Experimental animals: NIH mice were taken, with 10 in each group, and a weight of 12-14 g.

[0088] Immunization mode: subcutaneous injection, wherein the experimental group, the control group 1 and the control group 2 were injected with drugs at a single injection dose of 1×10.sup.8 IFU. Each mouse was immunized with two injections, with an interval of 4 weeks between the two injections. Primary immunization blood was collected from the orbit before the immunization with the second injection, and the eyeball was enucleated to collect the secondary immunization serum 28 days after the last immunization. The antibody titer in the serum was determined by ELISA, and the results were shown in FIG. 8.

[0089] It could be seen according to the results shown in FIG. 8 that the immunogenicity of Ad-SamRNA-gE was superior to that of Ad-gE and significantly superior to that of mRNA-gE, with significant difference. Therefore, it was proved that the antigen gene vaccine taking the modified adenovirus as the vector, prepared by the present invention, could greatly improve the immunogenicity of the mRNA vaccine.

Embodiment 5 Determination of Expression Quantities of Ad-SamRNA-gE and Ad-gE

[0090] 293 cells were infected with the constructed Ad-SamRNA-gE and Ad-gE at MOI=10, incubated in a 5% CO.sub.2 incubator at 37° C. for 40 h, and then centrifuged to collect a cell supernatant and cell precipitates, and the cell precipitates was lysed with a lysis buffer (2 mM MgCl.sub.2, 50 mM HEPES, pH 7.5); and the expression quantities of the gE protein in the two vectors was analyzed by SDS-PAGE. The results were shown in FIG. 9.

[0091] FIG. 9 demonstrated that compared with the control group Ad-gE, the expression quantity of the gE protein was significantly higher after the cells were infected with the sample to be tested Ad-SamRNA-gE. The result corresponded to the case that the immunogenicity of Ad-SamRNA-gE is much higher than that of Ad-gE in Embodiment 4.

Embodiment 6 Immunogenicity of Molecular Adjuvant Against Ad-SamRNA-gE

[0092] Taking the herpes zoster gE protein antigen as an example, the display of the immunogenicity of the Ad-SamRNA vaccine was conducted.

[0093] Experimental groups: Ad-SamRNA-gE with C3b and Ad-SamRNA-gE without C3b were prepared respectively.

[0094] A negative control group: a normal saline immunization group

[0095] Experimental animals: NIH mice were taken, with 10 in each group, and a weight of 12-14 g.

[0096] Immunization mode: subcutaneous injection, wherein the experimental group, the control group 1 and the control group 2 were injected with drugs at a single injection dose of 1×10.sup.8 IFU. Each mouse was immunized with two injections, with an interval of 4 weeks between the two injections. Primary immunization blood was collected from the orbit before the immunization with the second injection, and the eyeball was enucleated to collect the secondary immunization serum 28 days after the last immunization. The antibody titer in the serum was determined by ELISA, and the results were shown in FIG. 10.

[0097] According to the results in FIG. 10, C3b could enhance the immunogenicity of Ad-SamRNA-gE.

[0098] Finally, it should be noted that the embodiments described above are only illustrative of the technical solutions of the present invention, rather than limiting the present invention; although the present invention is described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skills in the art that modifications still can be made to the technical solutions described in the foregoing embodiments or equivalent replacements can be made to some or all technical features in the foregoing embodiments; and these modifications and replacements would not make the nature of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.