Virus for treatment of tumor

Abstract

Provided are an enterovirus D68 (EV-D68) or a modified form thereof, or a nucleic acid molecule comprising a genomic sequence or cDNA sequence of the EV-D68 or a modified form thereof, or a complementary sequence of the genomic sequence or cDNA sequence, or a pharmaceutical composition comprising the EV-D68 or a modified form thereof, or the nucleic acid molecule, and use of the EV-D68 or a modified form thereof, or the nucleic acid molecule in the manufacture of a pharmaceutical composition for treating a tumor.

Claims

1. A method of treating a lymphoma, the method comprising administering by intratumoral injection, to a subject in need thereof, an effective amount of a modified EV-D68, an isolated nucleic acid molecule, or a medicament comprising the modified EV-D68, or the isolated nucleic acid molecule, wherein the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: (1) a genomic sequence or cDNA sequence of the modified EV-D68; and (2) a complementary sequence of the genomic sequence or cDNA sequence; wherein, as compared to a genome of the wild-type EV-D68, a genome of the modified EV-D68 has a substitution of the internal ribosome entry site (IRES) sequence in a 5′ untranslated region (5′UTR) with an exogenous IRES sequence from human rhinovirus 2 (HRV2); and the modified EV-D68 has the genomic sequence as set forth in SEQ ID NO: 13 or has the cDNA sequence as set forth in SEQ ID NO: 8; and wherein the subject is a human.

2. The method of claim 1, wherein: (1) the isolated nucleic acid molecule consists of the genomic sequence of the the modified EV-D68; or (2) the isolated nucleic acid molecule is a vector comprising the cDNA sequence of the modified EV-D68, or the complementary sequence of the cDNA sequence.

3. The method of claim 1, wherein the modified EV-D68, or the isolated nucleic acid molecule is administered in combination with an additional pharmaceutically active agent having antitumor activity.

4. A modified EV-D68, a genome of which has a substitution of the internal ribosome entry site (IRES) sequence in a 5′ untranslated region (5′UTR) with an exogenous IRES sequence from human rhinovirus 2 (HRV2) as compared to a genome of a wild-type EV-D68; and the modified EV-D68 has the genomic sequence as set forth in SEQ ID NO: 13 or has the cDNA sequence as set forth in SEQ ID NO: 8.

5. An isolated nucleic acid molecule, comprising a sequence selected from the group consisting of: (1) the genomic sequence or cDNA sequence of the modified EV-D68 of claim 4; and (2) the complementary sequence of the genomic sequence or cDNA sequence.

6. The isolated nucleic acid molecule of claim 5, wherein: (1) the isolated nucleic acid molecule consists of the genomic sequence of the EV-D68 or the modified EV-D68; or (2) the isolated nucleic acid molecule is a vector comprising the cDNA sequence of the EV-D68 or the modified EV-D68, or the complementary sequence of the cDNA sequence.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows photomicrographs of the in vitro killing tests of the wild-type EV-D68 on human umbilical vein endothelial cell line HUVEC, human esophageal cancer cell line TE-1, human thyroid cancer cell lines SW-579 and TT in Example 2, wherein MOCK represents cells that are not infected with the virus. The results showed that the EV-D68 had a significant oncolytic effect on human tumor cell lines TE-1, SW-579, and TT after 72 hours of infection at a multiplicity of infection (MOI) of 10, but had no effect on HUVEC of human normal cells.

(2) FIG. 2 shows the photos of crystal violet staining of the in vitro killing tests of the wild-type EV-D68 on human liver cancer cell lines HepG2, SMMC7721, BEL7404, BEL7402, and Huh7, human cervical cancer cell lines Hela and Caski, human lung cancer cell lines NCI-H1299 and A549, human foreskin fibroblast cell line human embryonic kidney cell line HEK-293, and differentiated human liver progenitor cell line HepaRG in Example 2, wherein MOCK represents cells that are not infected with the virus. The results showed that the EV-D68 had significant oncolytic effects on human tumor cell lines HepG2, SMMC7721, BEL7404, BEL7402, Huh7, Hela, Caski, NCI-H1299 and A549 after 72 hours of infection at MOIs of 10, 1, and 0.1, but had limited effect on HFF-1, HEK-293 and differentiated HepaRG of human normal cells.

(3) FIG. 3 shows an electrophoresis image of four samples of wild-type EV-D68 virus genomic RNA of the same batch obtained by the in vitro transcription method in Example 2.

(4) FIG. 4 shows the killing effect of the wild-type EV-D68 virus genomic RNA on human cervical cancer tumor cell line Hela in Example 2. The results showed that Hela cells showed obvious CPE after 24 hours of transfection with EV-D68 genomic RNA, and were almost all lysed to death by 48 hours.

(5) FIGS. 5A to 5C show the results of in vivo antitumor experiment of the wild-type EV-D68 in Example 3 on human cervical cancer cell line Hela (A), human glioma cell line U118-MG (B), and human lymphoma cell line Raji (C). The results showed that, in the challenge experimental group, 10.sup.6 TCID50 per tumor mass of EV-D68 were injected intratumorally every third day. After 5 treatments in total, the growth of tumors formed by subcutaneous inoculation of Hela, U118-MG, or Raji cells in SCID mice significantly slowed down and arrested, and the tumors were even lysed and disappeared. In contrast, the tumors of the negative group (CTRL) without treatment of oncolytic virus maintained the normal growth, and their tumor volumes are significantly larger than those in the challenge group.

(6) FIG. 6 shows the results of toxicity detection of EV-D68-WT in BALB/c mice in Example 4. FIG. 6A shows the survival rates and health scores of 1-day-old BALB/c mice after challenge with EV-D68 at different doses (10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, and 10.sup.7 TCID50/mouse) by intraperitoneal injection; FIG. 6B shows the survival rates and health scores of BALB/c mice of different ages (1-day-old, 2-day-old, 3-day-old, 7-day-old and 14-day-old) challenged with a very high dose (10.sup.7 TCID50/mouse) by intraperitoneal injection. The overall toxicity of EV-D68 to BALB/c mice was relatively weak, and only high doses caused the death of 1-day-old to 3-day-old BALB/c mice, but had no effect on 4- or more-day-old BALB/c mice, indicating that EV-D68 had good safety in vivo.

SEQUENCE INFORMATION

(7) Information of a part of sequences involved in the present invention is provided in Table 1 as below.

(8) TABLE-US-00001 TABLE 1 Sequence description SEQ ID NO: Description 1 cDNA sequence of wild type EV-D68 (EV-D68-WT) 2 RNA sequence of the internal ribosome entry site of human rhinovirus 2 (HRV2) 3 RNA sequence of the target sequence of miR-133 4 RNA sequence of the target sequence of miR-206 5 RNA sequence of tandem sequence of miR-133 target sequence and miR-206 target sequence 6 DNA sequence of human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene 7 DNA sequence of anti-human programmed death receptor 1 single chain antibody (Anti-PD-1 scFv) 8 cDNA sequence of the modified form of EV-D68 (EV-D68-HRV2) 9 cDNA sequence of the modified form of EV-D68 (EV-D68-miR133&206T) 10 cDNA sequence of the modified form of EV-D68 (EV-D68-GM-CSF) 11 cDNA sequence of the modified form of EV-D68 (EV-D68-Anti-PD1) 12 Genomic sequence of wild-type EV-D68 (EV-D68-WT) 13 Genomic sequence of the modified form of EV-D68 (EV-D68-HRV2) 14 Genomic sequence of the modified form of EV-D68 (EV-D68-miR133 & 206T) 15 Genomic sequence of the modified form of EV-D68 (EV-D68-GM-CSF) 16 Genomic sequence of the modified form of EV-D68 (EV-D68-Anti-PD1) 17 DNA sequence of miR-133 target sequence 18 DNA sequence of miR-206 target sequence 19 DNA sequence of tandem sequence of miR-133 target sequence and miR-206 target sequence 20 DNA sequence of the internal ribosome entry site sequence of human rhinovirus 2 (HRV2)

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION

(9) The present invention is now be described with reference to the following examples which are intended to illustrate the present invention (rather than to limit the present invention).

(10) Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention were carried out substantially by referring to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; restriction enzymes were used under conditions recommended by the product manufacturer. If the specific conditions were not indicated in the examples, the conventional conditions or the conditions recommended by the manufacturer were used. If the reagents or instruments used were not specified by the manufacturer, they were all conventional products that were commercially available. Those skilled in the art will understand that the examples describe the present invention by way of examples, and are not intended to limit the scope of protection claimed by the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1: Obtainment and Preparation of EV-1368 and its Modified Form

(11) 1.1 Isolation of Enterovirus EV-D68 from Patient Clinical Sample

(12) (1) A throat swab of patient was gained from the Center for Disease Control and Prevention of Xiamen City, China; African green monkey kidney cells (Vero cells; ATCC® Number: CCL-81™) were was kept by the National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, China, and cultured in MEM medium containing 10% fetal bovine serum, as well as glutamine, penicillin and streptomycin.

(13) (2) Sample processing: the throat swab of patient was sufficiently agitated in a sample preservation solution to wash off the virus and virus-containing cells adhering to the swab, and then the sample preservation solution was subjected to a high speed centrifugation at 4000 rpm and 4° C. for 30 min;

(14) (3) Inoculation and observation:

(15) A) The Vero cells were plated in a 24-well plate with 1×10.sup.5 cells/well. The growth medium (MEM medium, containing 10% fetal bovine serum, as well as glutamine, penicillin and streptomycin) was aspirated, and 1 mL of maintenance medium (MEM medium, containing 2% fetal calf serum, as well as glutamine, penicillin and streptomycin) was added in each well. Then except the negative control wells, each well was inoculated with 50 μL of the sample supernatant, and cultured in an incubator at 37° C., 5% CO.sub.2.

(16) B) The cells were observed under a microscope every day for one week, and the occurrence of specific cytopathic effect (CPE) in the inoculated wells was recorded.

(17) C) If the enterovirus-specific cytopathic effect appeared in the cells in the inoculated wells within 7 days, the cells and supernatant were collected and frozen at −80° C.; if no CPE appeared after 7 days, the cells were subjected to blind passage.

(18) D) If CPE appeared within 6 blind passages, the cells and supernatant were collected and frozen at −80° C.; If CPE did not appear after 6 blind passages, the cells were determined as negative.

(19) (4) Isolation and cloning of viruses:

(20) RT-PCR (Piralla et al., J Clin Microbiol 2015, 53 (5): 1725-1726) and enzyme-linked immunospot method based on specific antibody (Yang et al., Clin Vaccine Immunol 2014, 21 (3): 312-320; Hou et al., J Virol Methods 2015, 215-216: 56-60) were used to identify the viruses isolated from the clinical sample, and EV-D68 positive cultures were selected and subjected to at least 3 cloning experiments. The virus clones obtained by the limiting dilution method in each experiment were also identified by RT-PCR and ELISPOT, and the EV-D68 positive clones were selected for the next round of cloning. A single EV-D68 strain with strong growth viability was selected as a candidate oncolytic virus strain.

(21) 1.2 Rescued Strain of Enterovirus EV-D68 and its Modified Form Obtained by Infectious Cloning and Reverse Genetics Technology

(22) This example used wild-type EV-D68 (SEQ ID NO: 1) as an example to show how to obtain EV-D68 and its modified form for the present invention through reverse genetics technology. The specific method was as follows.

(23) (1) Construction of viral infectious clone: the cDNA sequence of wild-type enterovirus EV-D68 (named EV-D68-WT) was shown in SEQ ID NO: 1, and its genomic RNA sequence was SEQ ID NO: 12; or gene insertion or replacement based on the cDNA (SEQ ID NO: 1) of enterovirus EV-D68 was performed, comprising:

(24) Modified form 1: the internal ribosome entry site sequence of wild-type EV-D68 was replaced with the internal ribosome entry site sequence of human rhinovirus 2 (which has a DNA sequence shown in SEQ ID NO: 20) to obtain the cDNA (SEQ ID NO: 8) of the recombinant virus (named as EV-D68-HRV2), which has a genomic RNA sequence shown as SEQ ID NO: 13;

(25) Modified form 2: the tandem sequence (which has a DNA sequence shown in SEQ ID NO: 19) of miR-133 target sequence (which has a DNA sequence shown in SEQ ID NO: 17) and miR-206 target sequence (which has a DNA sequence shown in SEQ ID NO: 18) was inserted between 7293-7294 bp of the 3′ untranslated region of the cDNA (SEQ ID NO: 1) of the wild-type EV-D68, to obtain the cDNA (SEQ ID NO: 9) of the recombinant virus (named EV-D68-miR133&206T), which has a genomic RNA sequence shown as SEQ ID NO: 14;

(26) Modified form 3: the human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene (SEQ ID NO: 6) was inserted between the VP1 gene and 2A gene of the cDNA (SEQ ID NO: 1) of wild-type EV-D68 to obtain the cDNA (SEQ ID NO: 10) of the recombinant virus (named EV-D68-GM-CSF), which has a genomic RNA sequence shown as SEQ ID NO: 15;

(27) Modified form 4: the sequence (SEQ ID NO: 7) encoding the single chain antibody against human programmed death receptor 1 (Anti-PD-1 scFv) was inserted between the VP1 gene and 2A gene of the cDNA (SEQ ID NO: 1) of wild-type EV-D68 to obtain the cDNA (SEQ ID NO: 11) of the recombinant virus (named EV-D68-Anti-PD-1), which has a genomic RNA sequence shown as SEQ ID NO: 16.

(28) Then, the cDNA sequences (SEQ ID NO: 1, 8-11) of the above five oncolytic viruses were sent to the gene synthesis company (Shanghai Biotech Engineering Co., Ltd.) for full gene synthesis, and ligated into the pSVA plasmid (Hou et al. Virus Res 2015, 205: 41-44; Yang et al., Virus Res 2015, 210: 165-168) to obtain the infectious cloning plasmids of enterovirus EV-D68 or modified forms thereof (i.e., EV-D68-WT, EV-D68-HRV2, EV-D68-miR133&206T, EV-D68-GM-CSF and EV-D68-Anti-PD-1).

(29) (2) Plasmid mini-kit and E. coli. DH5α competent cells were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; Hela cells (ATCC® Number: CCL-2™) and human rhabdomyosarcoma cells (RD cells; ATCC® Number: CCL-136™) were kept by National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, China, and were cultured with DMEM and MEM media respectively, in which 10% fetal bovine serum as well as glutamine, penicillin and streptomycin were added; transfection reagents Lipofactamine2000 and Opti-MEM were purchased from Thermo Fisher Scientific Company.

(30) (3) The infectious cloning plasmids containing the cDNA sequences of the above five oncolytic viruses were transformed into E. coli DH5α competent cells, the monoclonal strains were picked out and shaken after the outgrowth of clones, and the plasmids were extracted using the plasmid mini-kit, and then sent to the company (Shanghai Biotech Engineering Co., Ltd.) for sequencing analysis.

(31) (4) The infectious cloning plasmids with correct sequence and the helper plasmid pAR3126 were co-transfected into the cells to rescue virus (Hou et al. Virus Res 2015, 205: 41-44; Yang et al. Virus Res 2015, 210: 165-168). Hela cells were first transfected according to the instructions of the transfection reagent; then observed under a microscope. When CPE appeared in Hela cells, the cells and culture supernatant were harvested, and inoculated with RD cells followed by passaging and culturing, thereby obtaining the candidate strain of oncolytic virus.

Example 2: In Vitro Antitumor Experiment of EV-D68 and Modified Form Thereof

(32) 2.1 Viruses and Cell Lines as Used

(33) (1) Viruses: this example used EV-D68-WT (SEQ ID NO: 12), EV-D68-HRV2 (SEQ ID NO: 13), EV-D68-miR133&206T (SEQ ID NO: 14), EV-D68-GM-CSF (SEQ ID NO: 15) and EV-D68-Anti-PD-1 (SEQ ID NO: 16) as provided in Example 1.

(34) (2) Cell lines: human rhabdomyosarcoma cell RD (ATCC® Number: CCL-136™); human cervical cancer cell lines Hela (ATCC® Number: CCL-2™), SiHa (ATCC® Number: HTB-35™), Caski (ATCC® Number: CRL-1550™) and C-33A (ATCC® Number: HTB-31™); human ovarian cancer cell lines SKOV-3/TR (drug-resistant strain of SKOV-3), SKOV-3 (ATCC® Number: HTB-77™) and Caov3 (ATCC® Number: HTB-75™); human endometrial cancer cell lines Hec-1-A (ATCC® Number: HTB-112™), Hec-1-B (ATCC® Number: HTB-113™) and Ishikawa (ECACC No. 99040201); human lung cancer cell lines SPC-A-1 (CCTCC Deposit Number: GDC050), NCI-H1299 (ATCC® Number: CRL-5803™) NCI-H1417 (ATCC® Number: CRL-5869™), NCI-H1703 (ATCC® Number: CRL-5889™), NCI-H1975 (ATCC® Number: CRL-5908™), A549 (ATCC® Number: CCL-185™), NCI-H661 (ATCC® Number: HTB-183™), EBC-1 (Thermo Fisher Scientific, Catalog #: 11875101), and DMS114 (ATCC® Number: CRL-2066™); human liver cancer cell lines MHCC97H (purchased from the Institute of Liver Cancer, Fudan University), C3A (ATCC® Number: CRL-10741™), Hep3B (ATCC® Number: HB-8064™), HepG2 (ATCC® Number: HB-8065™), SMMC7721 (purchased from the Basic Medical Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, number: 3111C0001CCC000087), BEL7402 (CCTCC Deposit Number: GDC035), BEL7404 (purchased from the Cell Resource Center, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, number: 3131C0001000700064), Huh7 (CCTCC Deposit Number: GDC134), PLC/PRF/5 (ATCC® Number: CRL8024™) and SK-Hep-1 (ATCC® Number: HTB-52™); human kidney cancer cell lines A-498 (ATCC® Number: HTB-44™), 786-0 (ATCC® Number: CRL-1932™) and Caki-1 (ATCC® Number: HTB-46™); human neuroblastoma cell lines SH-SYSY (ATCC® Number: CRL-2266™) and SK-N-BE (2) (ATCC® Number: CRL-2271™); human glioma cell lines U87-MG (ATCC® Number: HTB-14™) and U118-MG (ATCC® Number: HTB-15™); human breast cancer cell lines MCF-7 (ATCC® Number: HTB-22™), BcaP37 (CCTCC Deposit Number: GDC206), BT-474 (ATCC® Number: HTB-20™), MDA-MB-231 (ATCC® Number: CRM-HTB-26™) and MDA-MB-453 (ATCC® Number: HTB-131™); human melanoma cell lines A-375 (ATCC® Number: CRL-1619™), SK-MEL-1 (ATCC® Number: HTB-67™) and MeWo (ATCC® Number: HTB-65™); human prostate cancer cell lines PC-3 (ATCC® Number: CRL-1435™), LNCap (ATCC® Number: CRL1740™) and DU145 (ATCC® Number: HTB-81™); human bladder cancer cell lines J82 (ATCC® Number: HTB-1™) and 5637 (ATCC® Number: HTB-9™); human pancreatic cancer cell lines Capan-2 (ATCC® Number: HTB-80™), HPAF-2 (ATCC® Number: CRL-1997™), and PANC-1 (ATCC® Number: CRL-1469™); human gastric cancer cell lines AGS (ATCC® Number: CRL-1739™), SGC7901 (CCTCC Deposit Number: GDC150), BGC823 (CCTCC Deposit Number: GDC151), and NCI-N87 (ATCC® Number: CRL-5822™); human colorectal cancer cell lines DLD-1 (ATCC® Number: CCL-221™), SW1116 (ATCC® Number: CCL-233™), SW480 (ATCC® Number: CCL-228™), HCT-116 (ATCC® Number: CCL247™) and HT-29 (ATCC® Number: HTB-38™); human esophageal cancer cell line TE-1 (purchased from the Cell Resource Center, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, No. 3131C0001000700089); human thyroid cancer SW-579 (ATCC® Number: HTB-107™) and TT (ATCC® Number: CRL-1803™); human laryngeal cancer Hep-2 (ATCC® Number: CCL-23™); osteosarcoma 143B (ATCC® Number: CRL-8303™) and U2OS (ATCC® Number: HTB-96™); human lymphoma and leukemia cell lines K562 (ATCC® Number: CCL-243™), U937 (ATCC® Number: CRL-1593.2™), THP-1 (ATCC® Number: TIB-202™), Raji (ATCC® Number: CCL-86™), Daudi (ATCC® Number: CCL-213™), Jurkat (ATCC® Number: TIB-152™) and MT-4 (obtained from the National Institutes of Health, USA); human normal cell lines include: human embryo lung fibroblast cell line MRC-5 (ATCC® Number: CCL-171™), human embryonic kidney cell line HEK-293 (ATCC® Number: CRL-1573™) human foreskin fibroblast cell line FIFF-1 (ATCC® Number: SCRC-1041™), human skin keratinocyte cell line HaCat (CCTCC Deposit Number: GDC106), human prostate stromal cell line WPMY-1 (ATCC® Number: CRL-2854™), human umbilical vein endothelial cell line HUVEC (Thermo Fisher Scientific, Catalog #: C01510C), and differentiated human liver progenitor cell line HepaRG (with characteristics of primary hepatocytes; Thermo Fisher Scientific, Catalog #: HPRGC10). The above cells were kept by National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, China. HepaRG cells were cultured in WME medium (added with 1.5% DMSO), AGS and TT were cultured with F-12K medium, SW-579 was cultured with L-15 medium, SH-SY5Y and SK-N-BE (2) were cultured with DMEM:F12 (1:1) medium, RD, C-33A, EBC-1, J82, SK-Hep-1, SK-MEL-1 and DU145 were cultured with MEM medium, K562, U937, THP-1, Raji, Daudi, Jurkat, MT-4, 5637, 786-O, TE-1, Caski, NCI-H1417, NCI-H1703, NCI-H1975, NCI-H661, SGC7901, BGC823, DLD-1, SW1116, Hep-2, and LNCap were cultured with RPMI-1640 medium, other cells were cultured with DMEM medium. All the mediums mentioned above were supplemented with 10% fetal bovine serum, glutamine and penicillin-streptomycin. All the above cells were cultured under the standard conditions of 37° C. and 5% CO.sub.2.

(35) 2.2 Virus Culture

(36) RD cells were evenly plated on 10 cm cell culture plates, and the culture conditions included MEM medium containing 10% fetal bovine serum and glutamine, penicillin and streptomycin, 37° C., 5% CO.sub.2, and saturated humidity. When the cell confluence reached 90% or more, the cell culture medium was replaced with serum-free MEM medium, and each plate was inoculated with 10.sup.7 TCID50 of EV-D68-WT, EV-D68-HRV2, EV-D68-miR133&206T, EV-D68-GM-CSF or EV-D68-Anti-PD-1, the culture environment was changed to 33° C., 5% CO.sub.2, saturated humidity. After 24 hours, the EV-D68 or its modified form proliferated in RD cells and caused CPE in cells. When more than 90% of the cells turned contracted and rounded, showed increased graininess, and became detached and lysed, the cells and culture supernatants thereof were harvested. After freeze-thawing for three cycles, the culture supernatant was collected and centrifuged to remove cell debris, wherein the centrifuge conditions were 4000 rpm, 10 min, 4° C. Finally, the supernatant was filtered with a 0.22 μm disposable filter (Millipore Company) to remove impurities such as cell debris.

(37) 2.3 Determination of Virus Titer

(38) The RD cells were plated in a 96-well plate with a cell density of 10.sup.4 cells/well. After the cells adhered, the virus solution obtained in Example 2.2 was diluted 10-fold with serum-free MEM medium from the first 10-fold dilution. 50 μl of the dilution of virus was added to the wells with cells. After 7 days, the wells where CPE appeared were monitored and recorded, followed by calculation using Karber method, in which the calculation formula was lg.sup.TCID50=L−D (S−0.5), L: logarithm of the highest dilution, D: difference between the logarithms of dilutions, S: sum of proportions of positive wells. The unit of TCID50 thus calculated was TCID50/50 μl, which should be converted to TCID50/ml.

(39) 2.4 In Vitro Antitumor Experiment of Viruses

(40) Human tumor cells and normal cells were inoculated into 96-well plates at 10.sup.4 cells/well. After the cells adhered, the medium in each well was replaced with the corresponding cell culture medium without serum, and viruses were inoculated at an MOI of 0.1, 1, 10 or 100. Subsequently, CPE of the cells were monitored daily by a microscope.

(41) FIG. 1 shows micrographs of the human umbilical vein endothelial cell line HUVEC, the human esophageal cancer cell line TE-1, and the human thyroid cancer cell line SW-579 and TT, which were not infected with viruses (negative control group, Mock) or which were treated with EV-D68-WT at MOI=10 for 72 hours. The results showed that after 72 hours of infection at a multiplicity of infection (MOT) of 10, a significant reduction in the number of the tumor cells, marked shrinking and lysis and the like, were detected in the virus-infected groups; while as compared to the non-tumor cells in the Mock group, the non-tumor cells infected with the viruses showed almost no change in cell morphology. The above results demonstrated that EV-D68 had significant oncolytic effects on human tumor cell lines TE-1, SW-579 and TT, but did not have any effect on non-tumor cells HUVEC.

(42) After 72 hours of virus infection and culture, the cell survival rate was detected using Cell Counting Kit-8 (CCK-8 kit; Shanghai Biyuntian Biotechnology Co., Ltd.) and crystal violet staining method (only for adherent cells), and the specific method was as follows:

(43) (1) Cell survival rate detected by CCK8 method

(44) For adherent cells, the original medium in a 96-well cell culture plate was directly discarded; for suspension cells, the original medium in a 96-well cell culture plate was carefully discarded after centrifugation; and then 100 μl of fresh serum-free medium was added per well. 10 μl of CCK-8 solution was added to each of the wells inoculated with cells, and an equal amount of CCK-8 solution was also added to the blank culture medium as a negative control, followed by incubation at 37° C. in a cell culture incubator for 0.5-3 hours. The absorbance was detected at 450 nm using a microplate reader at 0.5, 1, 2, 3 hours, respectively, and the time point where the absorbance was within a suitable range was selected as a reference for cell survival rate. The CCK-8 test results of EV-D68-WT for each kind of cells were shown in Table 2, where “−” indicated that the cell survival rate after virus treatment was not significantly different from that of the MOCK group; “+” indicated that after virus treatment, the cell number was reduced, the survival rate was still greater than 50% but was significantly different from that of the MOCK group; “++” indicated that the cell survival rate after virus treatment was less than 50%, and was significantly different from that of the MOCK group.

(45) The calculation of cell survival rate was:

(46) $\mathrm{Cell\_survival}\mathrm{\_rate}\left(\%\right)=\frac{\begin{array}{c}(\mathrm{reading\_of}\mathrm{\_test}\mathrm{\_group}-\\ \mathrm{reading\_of}\mathrm{\_negative}\mathrm{\_group})\end{array}}{\begin{array}{c}(\mathrm{reading\_of}\mathrm{\_positive}\mathrm{\_group}-\\ \mathrm{reading\_of}\mathrm{\_negaive}\mathrm{\_group})\end{array}}\times 100\%.$

(47) (2) Cell survival rate detected by crystal violet staining method (only for adherent cells)

(48) After the cells were infected with viruses for 3 days, the culture supernatant in the 96-well cell culture plate was discarded, 100 μl of methanol was added to each well, followed by fixation in the dark for 15 min. Crystal violet powder (Shanghai Biotech Biotechnology Co., Ltd.) was weighed, and formulated as 2% (w/v) crystal violet methanol solution, which was stored at 4° C. An appropriate amount of 2% crystal violet methanol solution was taken and formulated with PBS solution to prepare 0.2% crystal violet working solution. After fixation for 15 minutes, the methanol fixation solution in the 96-well cell culture plate was discarded, and 100 μl of the crystal violet working solution was added to the plate and staining was performed for 30 min. After the crystal violet staining solution was discarded, PBS solution was used for washing for 3 to 5 times, until the excess staining solution was washed off, and air-drying was performed. ImmunSpot @S5 UV Analyzer (Cellular Technology Limited, USA) was used for photographing. FIG. 2 showed the crystal violet staining results of the human liver cancer cell lines HepG2, SMMC7721, BEL7404, BEL7402 and Huh7, the human cervical cancer cell lines Hela and Caski, the human lung cancer cell lines NCI-H1299 and A549, the human foreskin fibroblast cell line HFF-1, the human embryonic kidney cell line HEK-293, and the differentiated human hepatic progenitor cell line HepaRG in the control group (MOCK) and in the experimental groups (infected for 72 hours with EV-D68-WT at MOIs of 0.1, 1, and 10, respectively). As shown in the results, after 72 hours of infection at MOIs of 10, 1, and 0.1, the tumor cells in the experimental groups were significantly reduced as compared to the control group (MOCK) without addition of virus; while the number of non-tumor cells showed no significant change. The above results indicated that the EV-D68-WT had significant oncolytic effects on human tumor cell lines HepG2, SMMC7721, BEL7404, BEL7402, Huh7, Hela, Caski, NCI-H1299 and A549, but had no significant effect on non-tumor cell lines HFF-1, HEK-293 and the differentiated HepaRG.

(49) TABLE-US-00002 TABLE 2 Results of in vitro antitumor experiment of wild-type enterovirus EV-D68 Multiplicity of infection MOI Cell Line 0.1 1 10 100 RD ++ ++ ++ ++ Hela ++ ++ ++ ++ SiHa − − ++ ++ Caski − + ++ ++ C-33A − ++ ++ ++ SKOV-3/TR − − − + SKOV-3 − − ++ ++ Caov3 + ++ ++ ++ Hec-1-A − − − ++ Hec-1-B − + ++ ++ Ishikawa − − ++ ++ SPC-A-1 − + ++ ++ NCI-H1299 − ++ ++ ++ NCI-H1417 − − − + NCI-H1703 − − − + NCI-H1975 − ++ ++ ++ A549 + ++ ++ ++ NCI-H661 − − + ++ EBC-1 − − + ++ DMS114 ++ ++ ++ ++ MHCC97H + ++ ++ ++ C3A ++ ++ ++ ++ Hep3B − + + ++ HepG2 − ++ ++ ++ SMMC7721 + ++ ++ ++ BEL7402 ++ ++ ++ ++ BEL7404 + ++ ++ ++ Huh7 ++ ++ ++ ++ PLC/PRF/5 − + ++ ++ SK-Hep-1 − − + ++ A-498 + ++ ++ ++ 786-O − − + ++ Caki-1 ++ ++ ++ ++ SH-SY5Y − + ++ ++ SK-N-BE(2) − − − + U87-MG + ++ ++ ++ U118-MG ++ ++ ++ ++ MCF-7 − − − + BcaP37 − ++ ++ ++ BT-474 − − − + MDA-MB-231 ++ ++ ++ ++ MDA-MB-453 − − + ++ A-375 − + ++ ++ SK-MEL-1 + ++ ++ ++ MeWo − + ++ ++ PC-3 ++ ++ ++ ++ LNCap − + ++ ++ DU145 ++ ++ ++ ++ J82 − + ++ ++ 5637 − − − + Capan-2 − − + ++ HPAF-2 − + + ++ PANC-1 − ++ ++ ++ AGS − − + ++ SGC7901 − − − + BGC823 − + + ++ NCI-N87 − + ++ ++ DLD-1 − − − + SW1116 + + ++ ++ SW480 − + ++ ++ HCT-116 − − + ++ HT-29 + ++ ++ ++ TE-1 − + ++ ++ SW-579 − − ++ ++ TT − − ++ ++ Hep-2 − + ++ ++ 143B − − − + U2OS + + ++ ++ K562 − + + ++ U937 − − + ++ THP-1 + ++ ++ ++ Raji ++ ++ ++ ++ Daudi ++ ++ ++ ++ Jurkat ++ ++ ++ ++ MT-4 ++ ++ ++ ++ MRC-5 − − + ++ HEK-293 − − − − HFF-1 − − + + HaCat − − − − WPMY-1 − − − − HUVEC − − − − HepaRG − − − + Note: “−” indicated that there was no significant difference in cell survival rate between virus treatment group and MOCK group; “+” indicated that after virus treatment, the number of cells was reduced, the survival rate was greater than 50% but was significantly different from that of MOCK group; “++” indicated that the cell survival rate after virus treatment was less than 50%, and was significantly different from that of the MOCK group.

(50) It could be known from Table 2 that the wild-type enterovirus EV-D68 had a killing effect on the tested tumor cells, and therefore had a broad-spectrum anti-tumor activity. In particular, the virus had significant killing effects on liver cancer cell lines, glioma cell lines, prostate cancer cell lines, leukemia and lymphoma cell lines. On the other hand, the virus had little or no toxicity to the non-tumor cell lines tested, except that it was significantly toxic to human embryonic lung fibroblast MRC-5 at higher MOIs.

(51) In addition, in vitro antitumor experiments of EV-D68-HRV2, EV-D68-miR133&206T, EV-D68-GM-CSF and EV-D68-Anti-PD-1 showed that the four modified EV-D68s retained the broad-spectrum killing effect of the wild-type enterovirus EV-D68 on the tested tumor cells, and substantially retained the significant killing effect on the tested tumor cells of human hepatocellular carcinoma cell line, prostate cancer cell line, leukemia and lymphoma cell lines, wherein the CCK-8 test results of oncolytic effect of the four modified EV-D68s on cervical cancer cell line Hela, glioma cell line U118-MG, liver cancer cell line Huh7, prostate cancer cell line PC-3, and lymphoma cell line Raji were shown in Table 3.

(52) TABLE-US-00003 TABLE 3 Results of in vitro antitumor experiment of EV-D68-HRV2, EV-D68-miR133&206T, EV-D68-GM-CSF and EV-D68-Anti-PD-1 Multiplicity of infection MOI Cell Lines 0.1 1 10 100 EV-D68-HRV2 Hela + + ++ ++ U118-MG + ++ ++ ++ Huh7 ++ ++ ++ ++ PC-3 ++ ++ ++ ++ Raji − + + ++ EV-D68-miR133&206T Hela ++ ++ ++ ++ U118-MG ++ ++ ++ ++ Huh7 ++ ++ ++ ++ PC-3 ++ ++ ++ ++ Raji ++ ++ ++ ++ EV-D68-GM-CSF Hela ++ ++ ++ ++ U118-MG ++ ++ ++ ++ Huh7 ++ ++ ++ ++ PC-3 ++ ++ ++ ++ Raji ++ ++ ++ ++ EV-D68-Anti-PD-1 Hela ++ ++ ++ ++ U118-MG ++ ++ ++ ++ Huh7 ++ ++ ++ ++ PC-3 ++ ++ ++ ++ Raji ++ ++ ++ ++ Note: “−” indicated that there was no significant difference in cell survival rate between virus treatment group and MOCK group; “+” indicated that after virus treatment, the number of cells was reduced, the survival rate was greater than 50% but was significantly different from that of MOCK group; “++” indicated that the cell survival rate after virus treatment was less than 50%, and was significantly different from that of the MOCK group.

(53) In addition, the inventors unexpectedly found that EV-D68-HRV2 exhibited significantly improved killing activity on some tumors compared to EV-D68-WT, wherein the CCK-8 test results of the oncolytic activity of the human gastric cancer cell line AGS, the human endometrial cancer cell lines HEC-1-A and Ishikawa, the human cervical cancer cell line C-33A, and the human thyroid cancer cell line SW579 were shown in Table 4.

(54) TABLE-US-00004 TABLE 4 Comparison of the results of in vitro oncolytic experiment of EV-D68-WT and EV-D68-HRV2 on some tumor cells MOI Cell Line 0.01 0.1 1 10 EV-D68-WT AGS − − − + HEC-1-A − − − − Ishikawa − − − ++ C-33A − − ++ ++ SW579 − − − ++ EV-D68-HRV2 AGS ++ ++ ++ ++ HEC-1-A − + ++ ++ Ishikawa ++ ++ ++ ++ C-33A ++ ++ ++ ++ SW579 ++ ++ ++ ++ Note: “−” indicated that there was no significant difference in cell survival rate between virus treatment group and MOCK group; “+” indicated that after virus treatment, the number of cells was reduced, the survival rate was greater than 50% but was significantly different from that of MOCK group; “++” indicated that the cell survival rate after virus treatment was less than 50%, and was significantly different from that of the MOCK group.
2.5 Serial Passaging of EV-D68 for Adaptation

(55) In this example, EV-D68 was serially passaged for adaptation in a certain type of tumor cell to obtain a virus strain with enhanced killing activity to the tumor cell.

(56) The wild-type enterovirus EV-D68 was serially passaged for adaptation in the human cervical cancer cell line SiHa, human ovarian cancer cell line SKOV-3, human liver cancer cell line SK-hep-1, human pancreatic cancer cell line Capan-2, human gastric cancer cell line AGS or human colorectal cancer cell line HCT-116 on which the oncolytic effect of EV-D68 was not very significant, and the specific method was as follows:

(57) One kind of the above tumor cells was evenly plated on a 10 cm cell culture plate, and the culture conditions included a corresponding cell culture media containing 10% fetal bovine serum and glutamine, penicillin and streptomycin, 37° C., 5% CO.sub.2, and saturated humidity. When the cell confluence reached 90% or more, the cell culture medium was replaced with serum-free cell culture medium, each plate was inoculated with 10.sup.7 TCID50 of EV-D68, the culture environment was changed to 33° C., 5% CO.sub.2, saturated humidity. When EV-D68 proliferated in tumor cells and caused CPE in the cells (after infection for up to 3 days), the cells and their culture supernatant were harvested. After freeze-thawing for three cycles, centrifugation was performed at 4° C., 4000 rpm for 10 min. The centrifugation supernatant was taken and added onto new tumor cells with a cell confluence of more than 90% to complete one round of virus passage. The passage was repeated for more than 10 times, and a part of the virus solution was taken for virus titer detection in RD cells in each round of passage, and the specific method referred to Example 2.3. Generally, the virus replication ability would increase with the generation, and when a relatively high infectious titer was reached and the virus replication was stable in the tumor cell, the adapted strain of EV-D68 for the tumor cell was obtained.

(58) Subsequently, by the in vitro antitumor experimental method described in Example 2.4, the human tumor cell SiHa, SKOV-3, SK-hep-1, Capan-2, AGS, or HCT-116 was inoculated to a 96-well plate at 10.sup.4 cells/well. After the cells adhered, the medium in each well was replaced with the corresponding culture medium free of serum, followed by incubation at 37° C. for 30 min, and then the serially passaged EV-D68 virus strains (viral titers of which were detected on RD cells) adapted for each of the above kinds of cells at MOIs of 0.1, 1, 10, and 100 were inoculated. Subsequently, CPE of the cells were monitored daily by a microscope, and the cell survival rate was detected using CCK-8 method 72 hours after the infection and culture of viruses.

(59) The results were shown in Table 5, in which after serial passaging of the wild-type enterovirus EV-D68 in a certain kind of tumor cells on which EV-D68 had poor oncolytic effect, the killing activity thereof on the tumor cells was significantly enhanced, indicating that the serial passaging method could be used to obtain an EV-D68 adapted strain with enhanced oncolytic effect on the tumor cells.

(60) TABLE-US-00005 TABLE 5 Results of in vitro killing experiment of EV-D68 on a tumor cell after serial passaging for adaptation in the tumor cell Cell Line 0.1 1 10 100 SiHa + ++ ++ ++ SKOV-3 − ++ ++ ++ SK-hep-1 − ++ ++ ++ Capan-2 − + ++ ++ AGS + + ++ ++ HCT-116 − + ++ ++ Note: “−” indicated that there was no significant difference in cell survival rate between virus treatment group and MOCK group; “+” indicated that after virus treatment, the number of cells was reduced, the survival rate was greater than 50% but was significantly different from that of MOCK group; “++” indicated that the cell survival rate after virus treatment was less than 50%, and was significantly different from that of the MOCK group.
2.6 Evaluation of Oncolytic Effect of Genomic RNA of EV-D68

(61) In this example, a large amount of infectious live viruses of EV-D68 could be produced by transfecting the purified genomic RNA of EV-D68 into a certain kind of tumor cells, and thus kill the tumor cells.

(62) The viral genomic RNA was first obtained by in vitro transcription, and this method could be found in, for example, Hadac E M, Kelly E J and Russell S J. Mol Ther, 2011, 19(6): 1041-1047. Specifically, the infectious cloning plasmid of wild-type EV-D68 obtained in Example 1 was linearized, and the linearized plasmid was used as a template for in vitro transcription using MEGAscript™ T7 Transcription Kit (Thermo Fisher Scientific, AM1333) so as to produce a large amount of viral RNA. And the obtained viral RNA was purified using MEGAclear™ Transcription Clean-Up Kit (Thermo Fisher Scientific, AM1908) for next use. The RNA electropherograms of 4 parallel samples were shown in FIG. 3.

(63) Subsequently, according to the method of the in vitro antitumor experiment described in Example 2.4, the human cervical cancer tumor cell line Hela was inoculated to a 24-well plate at 10.sup.5 cells/well. After the cells adhered, the medium in each well was replaced with a corresponding cell culture medium free of serum, followed by incubation at 37° C. for 30 min. Then Hela cells were transfected with purified virus RNA at 1 μg per well using transfection reagent Lipofectamine® 2000 (Thermo Fisher Scientific, 11668019), and the negative control group was transfected with irrelevant RNA nucleic acid molecules. Subsequently, CPE of the cells were monitored daily by a microscope.

(64) The results showed that CPE began to appear in the Hela cells transfected with genomic RNA of EV-D68 about 8 hours after transfection, and then the cytopathy gradually increased. After 48 hours, the survival rate was measured using the CCK8 method, the Hela cells had almost all died and lysed. And the micrographs of Hela cells at 0, 24 and 48 hours after infection were shown in FIG. 4. The culture supernatant was inoculated into new Hela cells and CPE was quickly produced. The results indicated that the direct administration with the nucleic acid of EV-D68 also had good killing activity and could be used to treat tumors.

Example 3: In Vivo Antitumor Experiments of Enterovirus EV-D68 and its Modified Forms

(65) 3.1 Viruses, Cell Lines and Experimental Animals

(66) (1) Viruses: the EV-D68-WT (SEQ ID NO: 12), EV-D68-HRV2 (SEQ ID NO: 13), EV-D68-miR133&206T (SEQ ID NO: 14), EV-D68-GM-CSF (SEQ ID NO: 15) and EV-D68-Anti-PD-1 (SEQ ID NO: 16) provided in Example 1 were used in this example. The methods of virus culture and virus titer measurement could be seen in Examples 2.2 and 2.3, respectively.

(67) (2) Cell lines: human cervical cancer cell line Hela (ATCC® Number: CCL-2™), glioma cell line U118-MG (ATCC® Number: HTB-15™), and lymphoma cell line Raji (ATCC® Number: CCL-86™). Except that Raji was cultured with RPMI-1640 medium, the other Hela and U118-MG were all cultured with DMEM medium. These mediums were all supplemented with 10% fetal bovine serum, glutamine and penicillin-streptomycin. All the above cells were cultured under the standard conditions of 37° C. and 5% CO.sub.2.

(68) (3) Experimental animals: female C.B17 SCID mice aged 6-8 weeks were from Shanghai Slark Experimental Animal Co., Ltd.; the mice were raised under SPF conditions, according to the protocol approved by the Experimental Animal Center and Ethics Committee of Xiamen University.

(69) 3.2 In Vivo Antitumor Experiments of the Virus

(70) The tumor cells used for subcutaneous tumor formation in SCID mice were digested with 0.01% trypsin, and then resuspended into a single-cell suspension using a cell culture medium containing 10% fetal bovine serum. The cell density of the suspension was counted. The cells were precipitated by centrifugation under 1000 g for 3 min, and then the cells were resuspended with an appropriate volume of PBS to reach a concentration of about 10.sup.6-10.sup.7 cells/100 μl PBS. The tumor cells were subcutaneously inoculated in the back of SCID mice at 10.sup.6-10.sup.7 cells/100 μl PBS/site with a syringe. When the tumor cells grew into a tumor mass of about 100 mm.sup.3 under the skin of SCID mice after about 14-21 days, the tumor-bearing SCID mice were randomly divided into experimental groups (administrated with EV-D68-WT, EV-D68-HRV2, EV-D68-miR133&206T, EV-D68-GM-CSF or EV-D68-Anti-PD-1) and negative control group, with 4 mice (n=4) in each group. Oncolytic virus (EV-D68-WT, EV-D68-HRV2, EV-D68-miR133&2061, EV-D68-GM-CSF or EV-D68-Anti-PD-1) at 10.sup.6 TCID50/100 μl serum-free medium/tumor mass or equivalent amount of serum-free medium were intratumorally injected every two days, for a total of 5 treatments. The tumor size was measured with a vernier caliper and recorded every two days, and the method for calculating the tumor size was:
Tumor size (mm.sup.3)=tumor length value×(tumor width value).sup.2/2.

(71) The treatment results of EV-D68-WT for the above three tumors were shown in FIGS. 5A-5C. The results showed that after the challenge of EV-D68-WT, the growth of the three tested tumors of Hela (A), U118-MG (B) and Raji (C) gradually slowed down and arrested, and the tumors were even lysed and disappeared; by contrast, the tumors of the negative group (CTRL) maintained the normal growth, and their tumor sizes were significantly larger than those of the experimental groups.

(72) Table 6 showed the results obtained after a treatment of the Raji tumor model with EV-D68-WT, EV-D68-HRV2, EV-D68-miR133&206T, EV-D68-GM-CSF or EV-D68-Anti-PD-1 for 10 days. The results showed that the tumor volumes were significantly reduced after treatment with EV-D68-WT, EV-D68-HRV2, EV-D68-miR133&206T, EV-D68-GM-CSF, and EV-D68-Anti-PD as compared with the negative control group that was not treated with oncolytic virus, wherein similar reductions in tumor volume were detected after treatment with 4 oncolytic viruses EV-D68-WT, EV-D68-miR133&206T, EV-D68-GM-CSF and EV-D68-Anti-PD-1. The above results indicated that all of EV-D68-WT, EV-D68-HRV2, EV-D68-miR133&206T, EV-D68-GM-CSF and EV-D68-Anti-PD-1 showed remarkable and favorable antitumor activity in vivo.

(73) TABLE-US-00006 TABLE 6 Results of in vivo anti-tumor experiments of EV-D68- WT, EV-D68-HRV2, EV-D68-miR133&206T, EV-D68-GM-CSF and EV-D68-Anti-PD-1 on human lymphoma cell line Raji In vivo oncolytic effect Oncolytic virus on Raji after 10 days of treatment EV-D68-WT ++ EV-D68-HRV2 + EV-D68-miR133&206T ++ EV-D68-GM-CSF ++ EV-D68-Anti-PD-1 ++ Note: “+” indicated that after treatment, the tumor volume reducedand was greater than 50% of the negative control group, but was significantly different from that of the negative control group; “++” indicated that the tumor volume reduced to less than 50% of the negative control group after treatment, and was significantly different from that of the negative control group.

Example 4: Safety Evaluation of Oncolytic Virus

(74) 4.1 Viruses and Laboratory Animals Used

(75) (1) Virus: the EV-D68-WT (SEQ ID NO: 12) provided in Example 1 was used in this example. The methods for virus culture and virus titer measurement could refer to Examples 2.2 and 2.3, respectively.

(76) (2) Experimental animals: BALB/c pregnant mice were from Shanghai Slark Experimental Animal Co., Ltd.; according to the protocol approved by the Experimental Animal Center and Ethics Committee of Xiamen University, the mice were raised under clean conditions, and then the 1-day-old, 2-day-old, 3-day-old, 7-day-old and 14-day-old mice produced by the BALB/c pregnant mice were used for in vivo virulence evaluation of EV-D68.

(77) 4.2 Evaluation of the Safety of the Virus in Mice

(78) (1) BALB/c suckling mice aged 1 day were selected for challenge with EV-D68-WT by intraperitoneal injection, and the titer doses for challenge were 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, or 10.sup.7 TCID50/mouse. Then the survival rates and health scores for the BALB/c mice challenged with different doses were recorded daily, wherein the evaluation criteria of the health score were: score of 5, represents dying or died; score of 4 represents severe limb paralysis; score of 3 represents weakness or mild deformity of limb; score of 2 represents wasting; score of 1 represents lethargy, piloerection, and trembling; and score of 0 represents healthy.

(79) The results were shown in FIG. 6A. Within 20 days after challenge, all mice in the group with extremely high-dose of 10.sup.7 TCID50 became ill and died within 1 week; 80% of the mice in the group with high-dose of 10.sup.6 TCID50 eventually survived and only few mice became ill and died; in addition, no morbidity and death occurred in the mice of the challenge groups with other doses.

(80) (2) The 1-day-old, 2-day-old, 3-day-old, 7-day-old and 14-day-old BALB/c mice were injected with EV-D68-WT at an extremely high dose of 10.sup.7 TCID50/mouse, and then the survival rates and health scores for the BALB/c with different ages in days were recorded daily, wherein the evaluation criteria of the health score were the same as above.

(81) The results were shown in FIG. 6B. Within 20 days after challenge, the 1-day-old mice all died within 1 week; the 2-day-old mice resisted in a certain extent to EV-D68 toxicity, and eventually had a survival rate of 70%, but with a relatively high incidence of disease and relatively severe symptoms; the 3-day-old mice were already not vulnerable to EV-D68, and eventually had a survival rate of 90%, and with a low incidence of disease and mild symptoms; the 4- or more-day-old mice were fully tolerant to the high doses of EV-D68, and no morbidity and death occurred.

(82) The above results showed that the EV-D68-WT was less toxic to mice, and was only lethal to the 1- to 3-day-old BALB/c mice at an extremely high dose of 10.sup.7 TCID50/mouse, and had no effect on the 4- or more-day-old mice, thereby indicating good safety in vivo.

(83) Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that according to all the teachings that have been published, various modifications and changes can be made to the detail, and these changes are all within the protection scope of the present invention. The protection scope of the present invention is given by the appended claims and any equivalents thereof.