Recombinant Viral Vector, Immunogenic Composition Comprising Same, and Uses

Abstract

A recombinant viral vector, an immunogenic composition comprising the same, and uses. The recombinant viral vector comprises a polynucleotide encoding a cytokine, the cytokine being one or more selected from IL-7, IL-15, IL-21 or GM-CSF. The recombinant viral vector is useful in preparing an antitumor vaccine.

Claims

1. A recombinant viral vector, comprising a polynucleotide encoding a cytokine, wherein the cytokine is one or more selected from IL-7, IL-15, IL-21 and GM-CSF.

2. The recombinant viral vector according to claim 1, wherein the IL-15 is human IL-15; an amino acid sequence of the human IL-15 is shown in SEQ ID NO: 2; and an encoding nucleic acid sequence of the human IL-15 is shown in SEQ ID NO: 1.

3. The recombinant viral vector according to claim 1, wherein the GM-CSF is human GM-CSF; an amino acid sequence of the human GM-CSF is shown in SEQ ID NO: 4; and an encoding nucleic acid sequence of the human GM-CSF is shown in SEQ ID NO: 3.

4. The recombinant viral vector according to claim 1, wherein the IL-7 is human IL-7; an amino acid sequence of the human IL-7 is shown in SEQ ID NO: 6; and an encoding nucleic acid sequence of the human IL-7 is shown in SEQ ID NO: 5.

5. The recombinant viral vector according to claim 1, wherein the IL-21 is human IL-21; an amino acid sequence of the human IL-21 is shown in SEQ ID NO: 8; and an encoding nucleic acid sequence of the human IL-21 is shown in SEQ ID NO: 7.

6. The recombinant viral vector according to claim 1 any one of claims 1 to 5, wherein the cytokine comprises human IL-15, human GM-CSF, human IL-7 and human IL-21; a nucleic acid sequence of a polynucleotide encoding the cytokine is shown in SEQ ID NO: 15; and an amino acid sequence encoded by the polynucleotide is shown in SEQ ID NO: 16.

7. The recombinant viral vector according to claim 1, wherein the viral vector is a vaccinia virus vector, a replicative vaccinia virus vector, vaccinia virus Tiantan strain, 752-1 strain; or a non-replicating vaccinia virus vector, or vaccinia virus modified vaccinia Ankara (MVA).

8. An immunogenic composition, comprising a prophylactically and/or therapeutically effective amount of the recombinant viral vector according to claim 1, and a pharmaceutically acceptable carrier.

9. A tumor vaccine, comprising a prophylactically and/or therapeutically effective amount of the recombinant viral vector according to claim 1, and a pharmaceutically acceptable carrier.

10. A kit comprising the recombinant viral vector according to claim 1, the immunogenic composition according to claim 8, or the tumor vaccine according to claim 9, and instructions for use thereof.

11. Uses of the recombinant viral vector according to claim 1, the immunogenic composition according to claim 8, or the tumor vaccine according to claim 9 in preparing a medicament for treating and/or preventing a tumor; wherein the tumor is a malignant tumor; and the malignant tumor is breast cancer or colon cancer.

12. A method for treating and/or preventing a tumor, comprising administrating a prophylactically and/or therapeutically effective amount of the recombinant viral vector according to claim 1, the immunogenic composition according to claim 8, or the tumor vaccine according to claim 9 to a subject in need thereof; wherein the tumor is a malignant tumor; or the malignant tumor is breast cancer or colon cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, in which:

[0033] FIG. 1 and FIG. 2 are a plasmid map and a double digestion identification map of shuttle plasmid vector pSC65CY with the cytokine encoding sequence, respectively.

[0034] FIG. 3 is the detection results of the activity of human GM-CSF in Example 5.

[0035] FIG. 4 is the detection results of the activity of human IL-21 in Example 6.

BEST MODES FOR CARRYING OUT THE INVENTION

[0036] The present invention will be further described below in conjunction with the examples, and it should be understood that the examples are only used to further illustrate and set forth the present invention, and not intended to limit the present invention.

[0037] Unless otherwise defined, all technical and scientific terms in the present description have the same meanings as commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein can be used in the experiments or practical applications, the materials and methods are described hereinafter. If contradicted mutually, the definitions contained in the present description shall prevail. In addition, the materials, methods and examples are illustrative only and not limiting.

Example 1 Construction of Shuttle Vector pSC65CY

[0038] All the amino acid sequences and nucleic acid sequences thereof for human cytokines were obtained from the NCBI database. The sequences of human cytokine IL-15 (NM_000585.4, the nucleic acid sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2), GM-CSF (NM_000758.3, the nucleic acid sequence is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO. 4), IL-7 (NM_000880.3, the nucleic acid sequence is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6), IL-21 (NM_001207006.2, the nucleic acid sequence is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8) were linked in tandem after the terminator codons were removed. The nucleic acid sequences of the respective cytokines were spaced by P2A nucleic acid sequence which is well known to those of ordinary skill in the art (the nucleic acid sequence is shown in SEQ ID NO: 9, the amino acid sequence is shown in SEQ ID NO: 10, and this endogenous proteinic cleavage site can result in effective cleavage of the fusion protein to form active monomeric cytokines (see Reference Kim JI-1, Lee S-R, Li L-H, Park H-J, Park J-H, et al., (2011) High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice. PLoS ONE 6(4): e18556.), and finally linked in tandem to a nucleic acid sequence for expression of green fluorescent protein EGFP (the nucleic acid sequence is shown in SEQ ID NO: 11, and the amino acid sequence is shown in SEQ ID NO: 12) which is well known to those of ordinary skill in the art for screening by marker. Finally, a quadruplet nucleic acid sequence for cytokine expression CY was formed (the nucleic acid sequence is shown in SEQ ID NO: 13, and the amino acid sequence is shown in SEQ ID NO: 14). This sequence was synthesized by Suzhou GENEWIZ Co., Ltd., and then inserted between restriction sites Xho I and Bam HI in shuttle vector pSC65 (addgene, Cat. No.: 30327) by molecular cloning technology to construct shuttle vector pSC65CY that can express 4 cytokines (the plasmid map is shown in FIG. 1), and identified by sequencing for confirmation and warehoused. Restriction endonuclease EcoR V was used to identify vector pSC65CY (the enzyme digestion system is shown in Table 1), and its enzyme digestion identification map is shown in FIG. 2.

TABLE-US-00001 TABLE 1 Enzyme digestion identification system for plasmid pSC65CY (enzyme digestion at 37° C. for 2 hours) Enzyme digestion system Volume Plasmid pSC65CY 3 μL, about 1 μg EcoRV (Takara, Cat. No.: 1068A) 1 μL Enzyme digestion buffer 1 μL ddH.sub.2O q.s. 10 μL

Example 2 Construction of Recombinant Vaccinia Virus Vector Rvv-CY

[0039] The recombinant vaccinia virus vector was obtained in 143B cells, and the specific method was as follows. On Day 1, 143B cells (ATCC® CRL-8303) were plated in a 6-well cell culture plate (JET, TCP-010-006) at 1×106/well, and incubated overnight at 37° C. in a carbon dioxide cell incubator. On Day 2, vaccinia virus wild strain 752-1 (provided by Beijing Institute of Biological Products Co., Ltd.) was added at 0.05 MOI (i.e., 5×10.sup.4 PFU (plaque-forming units)/well), and then placed and incubated in the carbon dioxide cell incubator at 37° C. for two hours, during which a shuttle vector/transfection reagent complex was prepared, wherein the shuttle vector was pSC65CY obtained in Example 1, and the transfection reagent is Turbofect (Thermo Fisher Scientific, R0531). The dose of the transfection reagent and compounding method can be found in the instructions of the transfection reagent. After the complex system was completed, the 143B cell supernatant was replaced with a DMEM maintenance medium containing 2% fetal bovine serum (FBS) at 2 mL/well, and then the shuttle vector/transfection reagent complex was added. 48 hours after the transfection, the supernatant was removed, the cells were collected and resuspended in 0.5 mL of maintenance medium, freeze-thawed repeatedly three times, and then the lysate of the recombinant cells was added to new 143B cells (containing 50 μg/mL BrdU) and incubated at 37° C. for 1 to 2 days. Cell lesions were observed during this period, and when an appropriate number of viral plaques appeared (less than 20 plaques/well), purification of single plaque was performed.

[0040] Purification of Single Plaque:

[0041] Viral plaques emitting green fluorescence were observed under a fluorescence microscope and labeled.

[0042] The supernatant was removed, and a plurality of well-dispersed green fluorescent plaques were picked up from each well and transferred to Ep tubes containing 0.5 mL of maintenance medium, respectively.

[0043] The Ep tubes containing the virus were shaken for uniform mixing, freeze-thawed repeatedly three times (about 5 minutes in a −80° C. refrigerator, and about 2 minutes at room temperature), finally shaken for uniform mixing, and then stored at −80° C.

[0044] At least six rounds of purification of single plaque were repeated until the purity reached 100%.

Example 3 Amplification Preparation and Titration of Recombinant Vaccinia Virus Vector Rvv-CY

[0045] The recombinant vaccinia virus vector rvv-CY constructed in Example 2 and vaccinia virus wild strain (rvv-WT) were amplified in Vero cells (ATCC® CCL-81) respectively, and the amplification method was as follows:

[0046] On the previous day, Vero monolayer cells at a confluence of 100% were prepared (1×10.sup.7 cells/dish), for a total of 10 dishes.

[0047] The supernatant was removed and replaced with a maintenance medium, the vaccinia virus to be amplified was inoculated into the cells (0.01 PFU/cell) and incubated in an incubator at 37° C. for 2-3 days, and obvious cell lesions were observed.

[0048] The cells were scraped off and collected, and centrifuged at 1,800 g for 5 minutes, and the supernatant was removed.

[0049] The cells were resuspended with 5 mL of maintenance medium, and sonicated on ice with an ultrasonic cell disruptor with 50 watts and 5 sec sonication/5 sec interval, for a total of 15 minutes.

[0050] The resultant was freeze-thawed repeatedly two times (about 5 minutes in a −80° C. refrigerator, and about 2 minutes at room temperature), and finally shaken for uniform mixing.

[0051] Subpackage into 1.5 mL centrifuge tubes was performed in a Biological Safety Level 2 cabinet, 1 mL/tube, and the centrifuge tubes were cryopreserved at −80° C.

[0052] The vaccinia virus prepared by amplification was titrated on Vero cells for determining infectious titer. The specific method was as follows:

[0053] On the previous day, Vero cells at a confluence of 100% were prepared in a 24-well plate, 3×10.sup.5 cells/well.

[0054] The supernatant is removed, and 200 μL of maintenance medium was added to each well to prevent the cells from drying out.

[0055] 100 μL of vaccinia virus to be tested was taken and added with 900 μL of maintenance medium, subject to a tenfold dilution, i.e., a serial dilution by 10.sup.2, 10.sup.2, 10.sup.3, . . . until 10.sup.9 times. Note: considering the dilution was carried out from higher concentrations to lower concentrations, the pipette tip should be replaced before each dilution to a lower concentration.

[0056] The virus was added to the 24-well plate in the order of from higher concentrations to lower concentrations (10.sup.9, 10.sup.8, . . . 10.sup.4), with 400 μL of dilution solution per well and two replicates, and determination was carried out continuously for 6 dilution ratios. After the addition was completed, the 24-well plate was placed and incubated in a cell incubator at 37° C. for 2 days.

[0057] The number of viral plaques was counted under a microscope, and recorded as 20+ if it was more than 20. The average counting of two duplicate wells with a plaque number within 20 (including 20)×2.5 (1000 μL/400 μL)×the dilution ratio of the corresponding well was calculated as the recombinant virus titer (PFU/mL).

[0058] The titration results of the vaccinia virus vector titers are shown in Table 2.

TABLE-US-00002 TABLE 2 Titration of vaccinia virus vector titers Vaccinia virus Titer (PFU/mL) Vaccinia virus wild type rvv-wt 1.5 × 10.sup.8 Recombinant vaccinia virus rvv-CY 1.0 × 10.sup.8

Example 4 Detection of Cytokine Expression

[0059] The supernatants of vero cells infected with vaccinia virus wild-type rvv-wt and recombinant vaccinia virus rvv-CY respectively were obtained from Example 3, and the contents of cytokines in the infected supernatants were detected by ELISA methods. The ELISA kits for the detection of human IL-7 (Cat. No.: SEK11821), human IL-15 (Cat. No.: SEK10360), and human GM-CSF (Cat. No.: SEK10015) were purchased from Beijing Sino Biological. The ELISA kit for the detection of human IL-21 (Cat. No.: 88-8218) was purchased from Thermo Fisher Scientific. Reference was made to the kit instructions for the detection methods. The contents of various cytokines measured in the virus-infected supernatants are shown in Table 3. The results show that there was no cytokine expression in the supernatant infected with vaccinia virus wild-type rvv-wt, and the detection was less than the detection limit (<0.01 ng/mL). The expression of various cytokines could be detected for the prepared recombinant vaccinia virus rvv-CY having the cytokine nucleic acid sequence, wherein the secretion of human GM-CSF was the strongest, reaching a level of 144.6 ng/mL, while the secretions of the remaining cytokines were comparable, ranging from 1-6 ng/mL.

TABLE-US-00003 TABLE 3 Detection of cytokine contents (ng/mL) Human Human Human Human Sample IL-7 IL-15 GM-CSF IL-21 rvv-WT infected supernatant <0.01 <0.01 <0.01 <0.01 rvv-CY infected supernatant 3.7 1.9 144.6 5.7

Example 5 Detection of Cytokine Human GM-CSF Activity

[0060] TF-1 cells (ATCC® CRL-2003) were cultured in a complete RPMI-1640 medium (10% fetal bovine serum (FBS), 1% penicillin-streptomycin (PS), 2 ng/mL human IL-3) to a log phase, and 125 g of culture was centrifuged for 10 minutes to collect the cells; the cells were resuspended in a serum-free RPMI-1640 medium, and centrifuged again to collect the cells. The cells were diluted to 1×10.sup.5 cells/mL with a complete RPMI-1640 medium, mixed uniformly, and added to a 96-well plate at 100 μl per well (1×10.sup.4 cells).

[0061] A human GM-CSF standard (Novoprotein, Cat. No.: CC79) was diluted stepwise by a two-fold gradient, added to a 96-well plate from lower concentrations to higher concentrations respectively, and used as blank controls, with two duplicate wells for each concentration, a total of 12 gradients. The plate was placed and cultured in a 37° C., 5% CO.sub.2 incubator for 96 hours.

[0062] Then, 10 microliters of CCK-8 (MCE, Cat. No.: HY-K0301) reagent was added to each well of the 96-well plate, which was put back in the incubator and continuously cultured for 2-4 hours, and OD450 was measured with a microplate reader.

[0063] The test results are shown in FIG. 3. The human GM-CSF secreted in the supernatant infected with the recombinant vaccinia virus rvv-CY having the cytokine nucleic acid sequence prepared in Example 3 had the activity (the half effective concentration (EC.sub.50) is 4.2 ng/mL), which was comparable to that the human GM-CSF standard (EC.sub.50 is 2.8 ng/mL).

Example 6 Detection of Cytokine Human IL-21 Activity

[0064] Mino cells (ATCC® CRL-3000) were cultured in a complete RPMI-1640 medium (10% fetal bovine serum (FBS), 1% penicillin-streptomycin (PS)) to a log phase, and 125 g of culture was centrifuged to collect the cells, and diluted to 2×10.sup.5/mL by the complete medium.

[0065] A human IL-21 standard (Beijing Sino Biological, Cat. No.: 10584-HNAE) was diluted stepwise in a 96-well plate by a two-fold gradient, and used as blank controls, a total of 12 gradients, 100 microliters per well; after the dilution was completed, 50 microliters of uniformly mixed cells (1×10.sup.4 cells) were added, a total of 150 microliters of liquid per well; and the 96-well plate was placed and cultured in a 37° C., 5% CO.sub.2 incubator for 6-7 days.

[0066] microliters of CCK-8 reagent (MCE, Cat. No.: HY-K0301) was added to each well of the 96-well plate after the culture, the 96-well plate was put back into the incubator for a continued culture of 4-8 hours (the time was determined according to color change), and OD450 was measured with a microplate reader.

[0067] The test results are shown in FIG. 4. The human IL-21 secreted in the supernatant infected with the recombinant vaccinia virus rvv-CY having the cytokine nucleic acid sequence prepared in Example 3 had the activity (EC.sub.50 is 1.1 ng/mL), which was comparable to that of the human IL-21 standard (EC.sub.50 is 7 ng/mL).

[0068] The results of Example 5 and Example 6 show that the prepared recombinant vaccinia virus rvv-CY having the cytokine nucleic acid sequences can correctly express biologically active cytokines.

Example 7 Tumor Treatment Experiment

[0069] 20 female BAL B/c mice aged 6-8 weeks were purchased from the Animal Experiment Center of Soochow University and feeded in the SPF grade animal room of the Animal Experiment Center of Soochow University. On Day 0, all mice were subcutaneously inoculated with tumor cells CT26 (ATCC® CRL-2638) at a dose of 1×10.sup.5 cells/mouse, and then randomly divided into two groups. On Day 1, Day 14 and Day 28 after the tumor cell inoculation, the mice were inoculated with the recombinant virus vector rvv-CY prepared in Example 3 or wild strain rvv-WT of vaccinia virus vector as a control, respectively. Correspondingly, on Day 1, Day 14 and Day 28 after the tumor cell inoculation, the mice of the vaccinia group were inoculated with the vaccinia virus vector prepared in Example 3 at the tibialis anterior muscle of lower leg (see Table 4 for the specific vaccination plan). The tumor growth was observed and measured continuously after the inoculation. The tumor volume was calculated according to the following formula:

Tumor volume (mm.sup.3)=length×width.sup.2/2.

[0070] The mice were sacrificed when the tumor volume exceeded 2000 mm.sub.3.

TABLE-US-00004 TABLE 4 Grouping of experimental animals and vaccination plan Day 1 Day 14 Day 28 Grouping Vaccine Dose Vaccine Dose Vaccine Dose Control rvv-WT 2 × 10.sup.6 LMNB/ 2 × 10.sup.6 LMNB/ 2 × 10.sup.6 group PFU IFA PFU CFA PFU (n = 10) Treatment rvv-CY 2 × 10.sup.6 rvv-CY 2 × 10.sup.6 rvv-CY 2 × 10.sup.6 group PFU PFU PFU (n = 10)

[0071] The overall survival time of mice in the group treated by the vaccinia virus vector vaccine rvv-CY was significantly better than that of the mice in the control group. The results show that the vaccinia virus vector vaccine rvv-CY can improve the survival of mice with expressed tumors.

[0072] Although the present invention has been described to a certain extent, it will be apparent that suitable changes may be made to various conditions without departing from the spirit and scope of the present invention. It may be understood that the present invention is not limited to the embodiments described, but depends on the scope of the claims which include the equivalents for each of the elements described.