Patent ID: 12195517

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention is described in further detail below with reference to the specific embodiments. The examples are given for the purpose of illustration of the present invention only, and are not intended to limit the scope of the present invention.

Example 1 Construction of DNA Vaccine pVKD1.0-hLMN

The amino acid sequences of LAGE-1, MAGE-A3 and NY-ESO-1 are shown in SEQ ID NOs: 24-26, respectively. By means of an online codon optimization software, the nucleotide sequences for mammalian codon usage preference as shown in SEQ ID NOs: 27-29 respectively were obtained by optimization based on the above antigen amino acid sequences. The nucleotide sequences were synthesized by Shanghai Generay Biotech Co., Ltd., and then cloned between the multiple cloning sites Sal I and BamH I on the DNA vaccine vector pVKD1.0 (provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park) by a method well known in the art to construct the DNA vaccine vector pVKD1.0-hLMN capable of expressing the fusion protein as an antigen (the plasmid map is shown inFIG.1), which was stored after being sequenced for identification. The vector pVKD1.0-hLMN was identified by the restriction endonucleases Sal I and BamH I (the enzyme digestion system is shown in Table 1), and its enzyme digestion map for verification is shown inFIG.2.

TABLE 1Enzyme digestion system for identification of the plasmidpVKD1.0-hLMN (enzyme digestion at 37° C., 2 h)Enzyme digestion systemVolumePlasmid pVKD1.0-hLMN3 μL, about 1 μgSal I (Takara, Cat. No. 1080A)1 μLBamH I (Takara, Cat. No. 1010A)1 μLEnzyme digestion buffer1 μLddH2Oq.s. to 10 μL

Example 2 Construction of DNA Vaccine pVKD1.0-hLMN-CTB

The mammalian codon optimized sequence (SEQ ID NO: 31) of the amino acid sequence (SEQ ID NO: 30) of cholera toxin subunit B (CTB) and its eukaryotic expression vector pVKD1.0-CTB were provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park. The primers were designed by using pVKD1.0-CTB as a template (Table 2). The CTB gene fragment was amplified by PCR, and the corresponding fragment was then recovered from the gel. The CTB fragment was inserted into a corresponding position on the linearized vector pVKD1.0-hLMN by a homologous recombination method, to construct the DNA vaccine vector pVKD1.0-hLMN-CTB (the plasmid map is shown inFIG.3), which was stored after being sequenced for identification. The vector pVKD1.0-hLMN-CTB was identified by the restriction endonucleases Sal I and BamH I (the enzyme digestion system is shown in Table 3), and its enzyme digestion map for verification is shown inFIG.4.

TABLE 2Primers in Example 2PrimerSequence1F (SEQ IDTCCCTCAGGGCAGAGGCGCATCAAGCTGAAGTTCGGNO: 32)CGTG1R (SEQ IDGAAGGCACAGCAGATCTGGATCCTCAGTTGGCCATGNO: 33)CTGATGGC

TABLE 3Enzyme digestion system for identification of plasmidpVKD1.0-hLMN-CTB (enzyme digestion at 37° C., 2 h)Enzyme digestion systemVolumePlasmid pVKD1.0-hLMN-CTB3 μL, about 1 μgSal I (Takara, Cat. No. 1080A)1 μLBamH I (Takara, Cat. No. 1010A)1 μLEnzyme digestion buffer1 μLddH2Oq.s. to 10 μL

Example 3 Construction of DNA Vaccine pVKD1.0-CI-LMNB

The strong Th epitopes derived from cytomegalovirus (CMV) and influenza (Flu) virus (see Table 4) were obtained from an immune epitope database (IEDB), wherein the strong Th epitopes of CMV include pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, pp65-57, pp65-62, pp65-30, pp65-112 and pp65-104, and the strong Th epitopes of Flu virus include HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, NP221, HA434, HA440, NP324, M1-127 and M1-210. The selected epitopes in Table 4 cover most subtypes of MHC class II molecules in both human and mouse. The selected epitopes pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, and NP221 were then linked together in tandem to form an fusion peptide of CMV virus epitopes and Flu virus epitopes having the amino acid sequence shown in SEQ ID NO: 34. The epitope fusion peptide was subjected to mammal codon optimization to give the nucleic acid sequence shown in SEQ ID NO: 35, which was sent to Suzhou Synbio Technologies Co., Ltd for synthesis, and then inserted into the DNA vaccine vector pVKD1.0 (Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park) by a molecular biology method well known in the art to form the vector pVKD1.0-CI (the plasmid map is shown inFIG.5), and stored after being sequenced for identification. The vector pVKD1.0-CI was identified by the restriction endonucleases Pst I and Bgl II (the enzyme digestion system is shown in Table 5) and its enzyme digestion map for verification is shown inFIG.6.

TABLE 4Th epitopes in Example 3EpitopeNameSourceAmino acid sequencepp65-11CMVLLQTGIHVRVSQPSL (SEQ ID NO: 1)pp65-71CMVIIKPGKISHIMLDVA (SEQ ID NO: 2)pp65-92CMVEHPTFTSQYRIQGKL (SEQ ID NO: 3)pp65-123CMVAGILARNLVPMVATV (SEQ ID NO: 4)pp65-128CMVKYQEFFWDANDIYRI (SEQ ID NO: 5)pp65-57CMVKVYLESFCEDVPSGK (SEQ ID NO: 6)pp65-62CMVTLGSDVEEDLTMTRN (SEQ ID NO: 7)pp65-30CMVPLKMLNIPSINVHHY (SEQ ID NO: 8)pp65-112CMVACTSGVMTRGRLKAE (SEQ ID NO: 9)pp65-104CMVTERKTPRVTGGGAMA (SEQ ID NO: 10)HA203InfluNQRALYHTENAYVSVVS (SEQ ID NO: 11)NP438InfluSDMRAEIIKMMESARPE (SEQ ID NO: 12)NS1-84InfluALASRYLTDMTIEEMSR (SEQ ID NO: 13)M1-181InfluLASTTAKAMEQMAGSSE (SEQ ID NO: 14)HA375InfluSGYAADQKSTQNAINGITNKVN (SEQ IDNO: 15)NP24InfluEIRASVGKMIDGIGRFYI (SEQ ID NO: 16)NP95InfluPIYRRVDGKWMRELVLY (SEQ ID NO: 17)NP221InfluRMCNILKGKFQTAAQRAM (SEQ ID NO: 18)HA434InfluIWTYNAELLVLLENERT (SEQ ID NO: 19)HA440InfluELLVLLENERTLDFHDS (SEQ ID NO: 20)NP324InfluHKSQLVWMACNSAAFED (SEQ ID NO: 21)M1-127InfluCMGLIYNRMGAVTTESA (SEQ ID NO: 22)M1-210InfluRQMVQAMRAIGTHPSSSTGLKND (SEQ IDNO: 23)

TABLE 5Enzyme digestion system for identification of plasmidpVKD1.0-CI (enzyme digestion at 37° C., 2 h)Enzyme digestion systemVolumePlasmid pVKD1.0-CI3 μL, about 1 μgPst I (Takara, Cat. No. 1073A)1 μLBgl II (Takara, Cat. No. 1021A)1 μLEnzyme digestion buffer1 μLddH2Oq.s. to 10 μL

Finally, the primers were designed by using the vector pVKD1.0-hLMN-CTB in Example 2 as a template (Table 6). The target gene fragment hLMN-CTB was amplified by PCR, and then inserted between the restriction sites Not I and Bam HI on the pVKD1.0-CI vector by a molecular biology method well known in the art to construct the DNA vaccine vector pVKD1.0-CI-LMNB (the plasmid map is shown inFIG.7), which was stored after being sequenced for identification. The vector pVKD1.0-CI-LMNB was identified by the restriction endonucleases Bam HI and EcoR V (the enzyme digestion system is shown in Table 7), and its enzyme digestion map for verification is shown inFIG.8.

TABLE 6Primers in Example 3PrimerSequence3F (SEQ IDGCGCGGCCGCTGTCACCGTCGTCGACATGCAGGCCGNO: 36)AA3R (SEQ IDGCGATCCTCAGTTGGCCATGCTGATGGCGGCGATGNO: 37)

TABLE 7Enzyme digestion system for identification of plasmidpVKD1.0-CI-LMNB (enzyme digestion at 37° C., 2 h)Enzyme digestion systemVolumePlasmid pVKD1.0-CI-LMNB3 μL, about 1 μgBam HI (Takara, Cat. No. 1010A)1 μLEcoR V (Takara, Cat. No. 1042A)1 μLEnzyme digestion buffer1 μLddH2Oq.s. to 10 μL

Example 4 Construction of LMN Prokaryotic Expression Vector

The amino acid sequences of LAGE-1, MAGE-A3 and NY-ESO-1 are shown in SEQ ID NOs: 24-26, respectively. By means of an online codon optimization software, the nucleotide sequences forE. colicodon usage preference shown in SEQ ID NOs: 38-40 respectively were obtained through optimization based on the antigen amino acid sequences. The nucleotide sequences were synthesized by Suzhou Synbio Technologies Co., Ltd., and then inserted between the multiple cloning sites Nco I and Xho I on the prokaryotic expression vector pET-30a (+) (Novagen, Cat. No. 69909) by a molecular biology method well known in the art to construct the prokaryotic expression construct pET-30a (+)-LMN (the plasmid map is shown inFIG.9), which was stored after being sequenced for identification. The vector pET-30a (+)-LMN was identified by the restriction endonucleases Nco I and Xho I (the enzyme digestion system is shown in Table 8), and its enzyme digestion map for verification is shown inFIG.10.

TABLE 8Enzyme digestion system for identification of plasmid pET-30a(+)-LMN(enzyme digestion at 37° C., overnight)Enzyme digestion systemVolumePlasmid pET-30a(+)-LMN3 μL, about 1 μgNco I (Takara, Cat. No. 1160A)1 μLXho I (Takara, Cat. No. 1094A)1 μLEnzyme digestion buffer1 μLddH2Oq.s. to 10 μL

Example 5 Construction of LMN-CTB Prokaryotic Expression Vector

The amino acid sequence of cholera toxin subunit B (CTB) (SEQ ID NO: 30) and its prokaryotic codon optimized nucleic acid sequence (SEQ ID NO: 41) were provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park. The primers were designed (Table 9), and a nucleic acid fragment containing the CTB encoding sequence was amplified by a PCR method using the pET-30a(+)-CTB (Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park) as a template, and the instructions of Ex Taq Enzyme Reagent (Takara, Cat. No. RR001B) were referred to for the specific method. The nucleic acid fragment was then inserted into the pET-30a(+)-LMN vector by means of homologous recombination to construct the pET-30a(+)-LMN-CTB vector (the plasmid map is shown inFIG.11), which was stored after being sequenced for identification. The vector pET-30a(+)-LMN-CTB was identified by the restriction endonucleases Nco I and Xho I (the enzyme digestion system is shown in Table 10), and its enzyme digestion map for verification is shown inFIG.12.

TABLE 9Primers in Example 5PrimerSequence5F (SEQ IDGGTGGTGGTGGTGCTCGAGTTAGTTAGCCATAGAGANO: 42)5R (SEQ IDTCTGCGTGAAGGTGAAGAAGCTCAGGCTGAAGGTCGTGGNO: 43)

TABLE 10Enzyme digestion system for identification in Example5 (enzyme digestion at 37° C., overnight)Enzyme digestion systemVolumePlasmid pET-30a(+)-LMN-CTB3 μL, about 1 μgNco I (Takara, Cat. No. 1160A)1 μLXho I (Takara, Cat. No. 1094A)1 μLEnzyme digestion buffer1 μLddH2Oq.s. to 10 μL

Example 6 Construction of Prokaryotic Expression Vector Containing Fusion Protein of LMN-CTB and CMV Th Epitopes

Ten (10) CMV-derived Th epitopes pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, pp65-57, pp65-62, pp65-30, pp65-112 and pp65-104 were selected from Table 4, and linked together in tandem to form the amino acid sequence shown in SEQ ID NO: 44, wherein the sequence segment “EFELRRQ” in SEQ ID NO: 44 is formed due to the introduction of enzyme restriction site, which belongs to a common technique for fusion and construction. By means of an online codon optimization software, the nucleotide sequence forE. colicodon usage preference (SEQ ID NO: 45) was obtained through optimization based on the amino acid sequence of Th epitopes. The nucleotide sequence was synthesized by Shanghai Generay Biotech Co., Ltd., and then inserted between the multiple cloning sites Nco I and Xho I on the prokaryotic expression vector pET-30a (+) (Novagen, Cat. No. 69909) by a molecular biology method well known in the art to construct the prokaryotic expression construct pET-30a (+)-CMV Th (the plasmid map is shown inFIG.13) capable of expressing the fusion protein as an antigen, which was stored after being sequenced for identification. The vector pET-30a (+)-CMV Th was identified by the restriction endonucleases Mlu I and Xho I (the enzyme digestion system is shown in Table 11), and its enzyme digestion map for verification is shown inFIG.14.

As shown inFIG.13, CMV Th1 contains 5 CMV Th epitopes consisting of pp65-11, pp65-71, pp65-92, pp65-123 and pp65-128 in tandem, and CMV Th2 contains 5 CMV Th epitopes consisting of pp65-57, pp65-62, pp65-30, pp65-112 and pp65-104. Three restriction enzyme sites such as EcoR I, Sac I and Sal I were introduced between CMV Th1 and CMV Th2.

TABLE 11Enzyme digestion system for identification of plasmid pET-30a(+)-CMVTh (enzyme digestion at 37° C., overnight)Enzyme digestion systemVolumePlasmid pET-30a(+)-CMV Th3 μL, about 1 μgMlu I (Takara, Cat. No. 1071A)1 μLXho I (Takara, Cat. No. 1094A)1 μLEnzyme digestion buffer1 μLddH2Oq.s. to 10 μL

The primers were designed (Table 12), and a nucleic acid fragment containing the LMN-CTB encoding sequence was amplified by a PCR method using pET-30a(+)-LMN-CTB in Example 5 as a template, and the instructions of Ex Taq Enzyme Reagent (Takara, Cat. No. RR001B) were referred to for the specific method. The nucleic acid fragment was then inserted between Not I and Xho I on the pET-30a(+)-CMV Th vector in Example 6 by a molecular biology method well known in the art to construct the pET-30a(+)-CMV10-LMNB vector (the plasmid map is shown inFIG.15), which was stored after being sequenced for identification. The vector pET-30a(+)-CMV10-LMNB was identified by the restriction endonucleases BamH I and Xho I (the enzyme digestion system is shown in Table 13) and its enzyme digestion map for verification is shown inFIG.16. As shown inFIG.15, pET-30a(+)-CMV10-LMNB contains CMV Th1 and CMV Th2 fragments, i.e. all 10 CMV Th epitopes in Table 4. These epitopes are pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, pp65-57, pp65-62, pp65-30, pp65-112, and pp65-104.

TABLE 12Primer design in Example 6PrimerSequence6F (SEQ ID NO: 46)GCGCGGCCGCGACGACAAGGCCATGGCT6R (SEQ ID NO: 47)GCCTCGAGGTTAGCCATAGAGATAGC

TABLE 13Enzyme digestion system for identification of pET-30a(+)-CMV10-LMNB (enzyme digestion at 37° C., overnight)Enzyme digestion systemVolumePlasmid pET-30a(+)-CMV10-LMNB3 μL, about 1 μgBamH I (Takara, Cat. No. 1010A)1 μLXho I (Takara, Cat. No. 1094A)1 μLEnzyme digestion buffer1 μLddH2Oq.s. to 10 μL

Example 7 Construction of Prokaryotic Expression Vector Containing Fusion Protein of LMN-CTB and Influ Th Epitopes

Thirteen (13) Th epitopes derived from Flu virus, HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, NP221, HA434, HA440, NP324, M1-127 and M1-210 were selected from Table 4, and linked together in tandem to form the amino acid sequence shown in SEQ ID NO: 48.

By means of an online codon optimization software, the nucleotide sequence forE. colicodon usage preference (SEQ ID NO: 49) was obtained through optimization based on the amino acid sequence containing Flu virus Th epitopes. The nucleotide sequence was synthesized by Shanghai Generay Biotech Co., Ltd., and then inserted between the multiple cloning sites Nco I and Xho I on the prokaryotic expression vector pET-30a (+) (Novagen, Cat. No. 69909) by a molecular biology method well known in the art to construct the prokaryotic expression construct pET-30a (+)-Influ Th (the plasmid map is shown inFIG.17) capable of expressing the fusion protein as an antigen, which was stored after being sequenced for identification. The vector pET-30a (+)-Influ Th was identified by the restriction endonucleases Nco I and Xho I (the enzyme digestion system is shown in Table 14), and its enzyme digestion map for verification is shown inFIG.18.

As shown inFIG.17, Influ Th1 contains 8 Flu virus Th epitopes consisting of HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95 and NP221 in tandem, and Influ Th2 contains 5 Flu virus Th epitopes consisting of HA434, HA440, NP324, M1-127 and M1-210. Three restriction sites such as EcoR I, Sac I and Sal I were introduced between Influ Th1 and Influ Th2.

TABLE 14Enzyme digestion system for identification in Example7 (enzyme digestion at 37° C., overnight)Enzyme digestion systemVolumePlasmid pET-30a(+)-Influ Th3 μL, about 1 μgNco I (Takara, Cat. No. 1160A)1 μLXho I (Takara, Cat. No. 1094A)1 μLEnzyme digestion buffer1 μLddH2Oq.s. to 10 μL

The primers were designed (Table 15), and a nucleic acid fragment containing the LMN-CTB encoding sequence was amplified by a PCR method using pET-30a(+)-LMN-CTB in Example 5 as a template, and the instructions of Ex Taq Enzyme Reagent (Takara, Cat. No. RR001B) were referred to for the specific method. The nucleic acid fragment was then inserted between Not I and Sal I on the pET-30a(+)-Influ Th vector in Example 7 by a molecular biology method well known in the art to construct the pET-30a(+)-Influ8-LMNB vector (containing 8 Flu virus Th epitopes; the plasmid map is shown inFIG.19), which was stored after being sequenced for identification. The vector pET-30a(+)-Influ8-LMNB was identified by the restriction endonucleases BamH I and Xho I (the enzyme digestion system is shown in Table 16) and its enzyme digestion map for verification is shown inFIG.20.

As shown inFIG.19, the pET-30a(+)-Influ8-LMNB vector contains the Influ Th1 segment, i.e., 8 Flu virus Th epitopes including HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, and NP221 in Table 4.

TABLE 15Primers in Example 7PrimerSequence7F1 (SEQ ID NO: 50)GCGCGGCCGCGTTAGCCATAGAGATAGC7R1 (SEQ ID NO: 51)GCGTCGACAAGACGACAAGGCCATGGCTATGC

TABLE 16Enzyme digestion system for identification of plasmid pET-30a(+)-Influ8-LMNB (enzyme digestion at 37° C., overnight)Enzyme digestion systemVolumePlasmid pET-30a(+)-Influ8-LMNB3 μL, about 1 μgBamH I (Takara, Cat. No. 1010A)1 μLXho I (Takara, Cat. No. 1094A)1 μLEnzyme digestion buffer1 μLddH2Oq.s. to 10 μL

The primers were designed (Table 17), and a nucleic acid fragment containing the LMN-CTB encoding sequence was amplified by a PCR method using pET-30a(+)-LMN-CTB in Example 5 as a template, and the instructions of Ex Taq Enzyme Reagent (Takara, Cat. No. RR001B) was referred to for the specific method. This nucleic acid fragment was then inserted between Not I and Xho I on the pET-30a(+)-Influ Th vector in Example 6 by a molecular biology method well known in the art to construct the pET-30a(+)-Influ13-LMNB vector (containing 13 Flu virus Th epitopes; the plasmid map is shown inFIG.21), which was stored after being sequenced for identification. The vector pET-30a(+)-CMV10-LMNB was identified by the restriction endonucleases BamH I and Xho I (the enzyme digestion system is shown in Table 18) and its enzyme digestion map for verification is shown inFIG.22.

As shown inFIG.21, the pET-30a(+)-Influ13-LMNB vector contains both the Influ Th1 and Influ Th2 segments, i.e., 8 Flu virus Th epitopes including HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, and NP221 in Table 4, and 5 Flu virus Th epitopes including HA434, HA440, NP324, M1-127 and M1-210 in Table 4. The vector includes all 13 Flu virus Th epitopes in total in Table 4.

TABLE 17Primer design in Example 7PrimerSequence7F2 (SEQ ID NO: 52)GCCTCGAGGTTAGCCATAGAGATAGCA7R2 (SEQ ID NO: 53)GCGCGGCCGCGACGACAAGGCCATGGCTATG

TABLE 18Enzyme digestion system for identification in Example7 (enzyme digestion at 37° C., overnight)Enzyme digestion systemVolumePlasmid pET-30a(+)-Influ13-LMNB3 μL, about 1 μgBamH I (Takara, Cat. No. 1010A)1 μLXho I (Takara, Cat. No. 1094A)1 μLEnzyme digestion buffer1 μLddH2Oq.s. to 10 μL

Example 8 Expression and Purification of Fusion Protein

The prokaryotic expression vector pET-30a(+)-LMN constructed in Example 4, the prokaryotic expression vector pET-30a(+)-LMN-CTB constructed in Example 5, the prokaryotic expression vectors pET-30a(+)-CMV5-LMNB and pET-30a(+)-CMV10-LMNB constructed in Example 6, the prokaryotic expression vectors pET-30a(+)-Influ8-LMNB and pET-30a(+)-Influ13-LMNB constructed in Example 7 were respectively transformed into BL21 (DE3) competent cells (Tiangen Biotech (Beijing) Co., Ltd., Cat. No. CB105; the instructions of competent cells were referred to for the transformation method) to prepare the recombinant proteins LMN (its amino acid sequence is shown in SEQ ID NO: 59), LMNB (its amino acid sequence is shown in SEQ ID NO: 54), LMNB-C10 (its amino acid sequence is shown in SEQ ID NO: 58), LMNB-18 (its amino acid sequence is shown in SEQ ID NO: 55), and LMNB-I13 (its amino acid sequence is shown in SEQ ID NO: 56) according to the pET System Manual (TB055 8th Edition February 2099, Novagen), respectively, which were stored at −80° C. after subpackage.

The concentrations of the recombinant proteins prepared are 1 mg/mL, as detected by a BCA method (Beyotime Institute of Biotechnology, Cat. No. P0009), and the instructions of detection kit were referred to for the detection method. The contents of endotoxin in the prepared recombinant proteins were less than 1 EU/mg, as measured by a gel method (Chinese Horseshoe Crab Reagent Manufactory Co., Ltd., Xiamen, Cat. No. G011000), which meet the requirements of an animal experiment, and the instructions of horseshoe crab agent were referred to for the detection method.

Example 9 Animal Immunization Experiment

The information of the vaccines prepared in Examples 2, 3 and 8 is shown in Table 19. The DNA vaccine vector pVKD1.0 was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park, and the Flu antigen NP (NCBI reference sequence: YP_009118476.1) of the DNA vaccine pVKD1.0-NP (the expression is derived from the virus strain A/Shanghai/02/2013 (H7N9)) was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park, and the protein vaccine VP1 (VP1 protein of enterovirus 71, see the Chinese Patent Application No. 201310088364.5) was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park.

Sixteen (16) 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. The experimental animal grouping and vaccination schemes are shown in Table 20. All DNA vaccines were injected into the tibialis anterior muscle of the calf at 100 jug/animal. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), and injected subcutaneously into the back at 10 μg/animal. Two weeks after the last immunization, the mice were sacrificed, and their serums and splenocytes were collected for an enzyme-linked immunospot (ELISPOT) assay and an enzyme-linked immunosorbent assay (ELISA), respectively.

The mouse IFN-γ ELISPOT kit was purchased from BD, USA (Cat. No. 551083), and the instructions of IFN-γ ELISPOT kit from BD were referred to for the method. The stimulating peptide was NY-ESO-1 41 #peptide (WITQCFLPVFLAQPP) synthesized by Shanghai Science Peptide Biological Technology Co., Ltd., with a final stimulating concentration of 10 μg/mL. The positive stimuli phorbol-12-myristate-13-acetate (PMA) and ionomycin were purchased from Sigma, USA.

An ELISA method is well known for a person skilled in the art, and briefly described below. The 96-well ELISA plates were purchased from Jianghai Glass Instrument General Factory. Both the recombinant LMN and NY-ESO-1 were provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park. The plates were coated with the proteins in NaHCO3buffer (pH 9.6) at 4° C. overnight at a coating concentration of 10 μg/mL, followed by blocking with 0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) at 37° C. for 30 minutes and then washing 5 times with 0.5% Tween 20 in phosphate buffered saline (PBST). An incubation with the mouse serum at room temperature was carried out for 1 hour at an initial dilution of 1:100 and washed 5 times with PBST, and another incubation with goat anti-mouse HRP secondary antibody (Santacruz, USA) was carried out at 1: 5000 at 37° C. for 30 min. After being washing 5 times with PBST, the substrate was developed with 3,3,5,5-tetramethylbenzidine (TMB) at 37° C. for 15 min, the development was stopped with 2M dilute sulfuric acid, and then the absorbance (A) values were read at 450 nm using a microplate reader (Thermo, USA). A value which is 2.1 times greater than the negative control A value was judged to be positive, and the reciprocal of the highest dilution with respect to the positive values was defined as the serum antibody titer. A titer was defined as 50 when it was less than the initial dilution of 1: 100.

TABLE 19Vaccine informationVaccineAttributeAntigenCD4 Th epitopepVKD1.0-LMN-CTBDNALAGE-1,Epitope-freeMAGE-A3,NY-ESO-1,pVKD1.0-CI-LMNBDNALAGE-1,5 CMV epitopes,MAGE-A3,8 influenza virusNY-ESO-1epitopespVKD1.0-CIDNANone5 CMV epitopes,8 influenzavirus epitopespVKD1.0-NPDNANP5 influenza virusepitopesLMNBRecombinantLACE-1,Epitope-freeproteinMAGE-A3,NY-ESO-1LMNB-I8RecombinantLAGE-1,First 8 influenzaproteinMAGE-A3,virus epitopesNY-ESO-1LMNB-I13RecombinantLAGE-1,13 influenza virusproteinMAGE-A3,epitopesNY-ESO-1LMNB-C5RecombinantLAGE-1,First 5 CMVproteinMAGE-A3,epitopesNY-ESO-1LMNB-C10RecombinantLAGE-1,10 CMV epitopesproteinMAGE-A3,NY-ESO-1VP1RecombinantVP1Epitope-freeprotein

TABLE 20Grouping and immunization schemesWeek 0, 4, 8Week 12, 16, 20Week 24Week 28GroupingVaccineDoseVaccineDoseVaccineDoseVaccineDoseA(n = 4)pVKD1.0-NP100 μgpVKD1.0100 μgVP1/CFA100 μgVPI/IFA100 μgB(n = 4)pVKD1.0-NP100 μgpVKD1.0-LMNB100 μgLMNB/CFA100 μgLMNB/IFA100 μgC(n = 4)pVKD1.0-NP100 μgpVKD1.0-CI-LMNB100 μgLMNB-I8/CFA100 μgLMNB-I8/IFA100 μgD(n = 4)pVKD1.0-NP100 μgpVKD1.0-CI-LMNB100 μgLMNB-I13/CFA100 μgLMNB-I13/IFA100 μg

The results of cellular immune response assay are shown inFIG.23. Among them, the group primed with pVKD1.0-CI-LMNB DNA vaccine and boosted with LMNB-I13 protein (i.e., group D in Example 8) had the best immune effect, which was significantly higher than those of the parallel control group (group B) and the group boosted with LMNB-I8 (group C). Moreover, the level of cellular immune response in the group boosted with LMNB-I13 protein was nearly 3-fold higher than that in the parallel control group (group B). The results show that a load of 13 Flu virus Th epitopes (group D) can significantly increase the cellular immune response level of weak immunogens.

Example 10 Construction of Prokaryotic Expression Vector Containing Fusion Protein of LMN-CTB and CMV Th Epitope

The primers were designed (Table 21), and a nucleic acid fragment containing the LMN-CTB encoding sequence was amplified by a PCR method using pET-30a(+)-LMN-CTB in Example 5 as a template, and the instructions of Ex Taq Enzyme Reagent (Takara, Cat. No. RR001B) were referred to for the specific method. The nucleic acid fragment was then inserted between Not I and Sal I on the pET-30a(+)-CMV Th vector in Example 6 by a molecular biology method well known in the art to construct the pET-30a(+)-CMV5-LMNB vector (the plasmid map is shown inFIG.24), which was stored after being sequenced for identification. The vector pET-30a(+)-CMV5-LMNB was identified by the restriction endonucleases BamH I and Xho I (the enzyme digestion system is shown in Table 22) and its enzyme digestion map for verification is shown in FIG.25. As shown inFIG.24, pET-30a(+)-CMV5-LMNB contains a CMV Th1 fragment, i.e. the first 5 CMV Th epitopes in Table 4. These epitopes are pp65-11, pp65-71, pp65-92, pp65-123 and pp65-128.

TABLE 21Primer design in Example 10PrimerSequence7F1 (SEQ ID NO: 50)GCGCGGCCGCGTTAGCCATAGAGATAGC7R1 (SEQ ID NO: 51)GCGTCGACAAGACGACAAGGCCATGGCTATGC

TABLE 22Enzyme digestion system for identification of pET-30a(+)-CMV10-LMNB (enzyme digestion at 37° C., overnight)Enzyme digestion systemVolumePlasmid pET-30a(+)-CMV10-LMNB3 μL, about 1 μgBamH I (Takara, Cat. No. 1010A)1 μLXho I (Takara, Cat. No. 1094A)1 μLEnzyme digestion buffer1 μLddH2Oq.s. to 10 μL

Example 11 Expression and Purification of Fusion Protein

As described in Example 8, the prokaryotic expression vector pET-30a(+)-CMV5-LMNB constructed in Example 10 was transformed into BL21 (DE3) competent cells (Tiangen Biotech (Beijing) Co., Ltd., Cat. No. CB105; the instructions of competent cells were referred to for the transformation method) to prepare the recombinant protein LMNB-05 (its amino acid sequence is shown in SEQ ID NO: 57) according to the pET System Manual (TB055 8th Edition February 2099, Novagen), which was stored at −80° C. after subpackage.

The concentration of the recombinant protein prepared was 1 mg/mL, as detected by a BCA method (Beyotime Institute of Biotechnology, Cat. No. P0009), and the instructions of detection kit were referred to for the detection method. The content of endotoxin in the prepared recombinant protein was less than 1 EU/mg, as measured by a gel method (Chinese Horseshoe Crab Reagent Manufactory Co., Ltd., Xiamen, Cat. No. G011000), which met the requirements of an animal experiment, and the instructions of Horseshoe Crab agent were referred to for the detection method.

Example 12 Animal Immunization Experiment

The vaccine information is shown in Table 19. The DNA vaccine pVKD1.0-CI (Example 3) was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park.

Twenty (20) 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. The experimental animal grouping and vaccination schemes are shown in Table 23. All DNA vaccines were injected into the tibial anterior muscle of the calf at 100 μg/animal. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), and injected subcutaneously into the back at 10 μg/animal. Two weeks after the last immunization, the mice were sacrificed, and their serums and splenocytes were collected for an enzyme-linked immunospot (ELISPOT) assay and an enzyme-linked immunosorbent assay (ELISA), respectively.

The mouse IFN-γ ELISPOT kit was purchased from BD, USA (Cat. No. 551083), and the instructions of IFN-γ ELISPOT kit from BD were referred to for the method. The stimulating peptide was NY-ESO-1 41 #peptide (WITQCFLPVFLAQPP) synthesized by Shanghai Science Peptide Biological Technology Co., Ltd., with a final stimulating concentration of 10 μg/mL. The positive stimuli phorbol-12-myristate-13-acetate (PMA) and ionomycin were purchased from Sigma, USA.

An ELISA method is well known for a person skilled in the art and briefly described below. 96-well ELISA plates were purchased from Jianghai Glass Instrument General Factory. Both recombinant LMN and NY-ESO-1 were provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park. The plates were coated with the proteins in NaHCO3buffer (pH 9.6) at 4° C. overnight at a coating concentration of 10 μg/mL, followed by blocking with 0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) at 37° C. for 30 minutes and then washing 5 times with 0.5% Tween 20 in phosphate buffered saline (PBST). An incubation with mouse serum was carried out at room temperature for 1 hour at an initial dilution of 1: 100 and washed 5 times with PBST. Another incubation with goat anti-mouse HRP secondary antibody (Santacruz, USA) was carried out at 1: 5000 at 37° C. for 30 min, and washed 5 times with PBST. The substrate was then developed with 3,3,5,5-tetramethylbenzidine (TMB) at 37° C. for 15 min and the development was stopped with 2M dilute sulfuric acid, and the absorbance (A) values were read at 450 nm using a microplate reader (Thermo, USA). A value which is 2.1 times greater than the negative control A value was judged to be positive, and the reciprocal of the highest dilution with respect to the positive values was defined as the serum antibody titer. A titer was defined as 50 when it was less than the initial dilution of 1: 100.

TABLE 23Grouping and immunization schemesWeek 0, 4, 8Week 12, 16, 20Week 24Week 28GroupingVaccineDoseVaccineDoseVaccineDoseVaccineDoseA(n = 5)pVKD1.0-CI100 μgpVKD1.0100 μgVP1/CFA10 μgVP1/IFA10 μgB(n = 5)pVKD1.0-CI100 μgpVKD1.0-LMNB100 μgLMNB/CFA10 μgLMNB/IFA10 μgC(n = 5)pVKD1.0-CI100 μgpVKD1.0-CI-LMNB100 μgLMNB-C5/CFA10 μgLMNB-C5/CFA10 μgD(n = 5)PVKD1.0-CI100 μgpVKD1.0-CI-LMNB100 μgLMNB-C10/CFA10 μgLMNB-C10/CFA10 μg

The results of cellular immune response assay are shown inFIG.26. Among them, the group primed with the pVKD1.0-CI-LMNB DNA vaccine, boosted with the LMNB-05 protein (i.e. group C in Example 11) and boosted with the LMNB-C10 protein (i.e. group D in Example 11) had the best immune effect, which was significantly higher than that in the parallel control (group B). The results show that a load of 5 CMV virus Th epitopes (group C) and 10 CMV virus Th epitopes (group D) can significantly improve the cellular immune response of weak immunogens.

Example 13 Animal Experiment for Tumor Prevention

The information of vaccines prepared in Examples 2, 3 and 8 is shown in Table 19. The DNA vaccine vector pVKD1.0 was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park, and the Flu antigen NP (NCBI reference sequence: YP_009118476.1) of the DNA vaccine pVKD1.0-NP (the expression is derived from the virus strain A/Shanghai/02/2013 (H7N9)) was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park, and the protein vaccine VP1 (VP1 protein of enterovirus 71, see the Chinese Patent Application No. 201310088364.5) was provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park.

Sixty (60) 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. The experimental animal grouping and vaccination schemes are shown in Table 24. All DNA vaccines were injected into the tibials anterior muscle of the calf at 100 μg/animal. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), and injected subcutaneously into the back at 10 μg/animal. Two weeks after the last immunization, the mice were inoculated subcutaneously with the cell line transfected stably by 4T1-hNY-ESO-1 (provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park), at an inoculation dose of 1×105cells/mouse, and the tumor growth was continuously observed and measured after the inoculation. The tumor volume was calculated according to the following equation: tumor volume (mm3)=length×width/2. The mice were sacrificed when the tumor volume exceeded 2000 mm3.

TABLE 24Grouping and immunization schemesWeek 0, 4, 8Week 12, 16, 20Week 24Week 28GroupingVaccineDoseVaccineDoseVaccineDoseVaccineDoseA(n = 10)pVKD1.0-NP50 μgpVKD1.0100 μgVP1/CFA10 μgVP1/IFA10 μgpVKD1.0-CI50 μgB(n = 10)pVKD1.0-NP50 μgpVKD1.0-LMNB100 μgLMNB/CFA10 μgLMNB/IFA10 μgpVKD1.0-CI50 μgC(n = 10)pVKD1.0-NP50 μgpVKD1.0-CI-LMNB100 μgLMNB-I8/CFA10 μgLMNB-I8/IFA10 μgpVKD1.0-CI50 μgD(n = 10)pVKD1.0-NP50 μgpVKD1.0-CI-LMNB100 μgLMNB-I13/CFA10 μgLMNB-I13/IFA10 μgpVKD1.0-CI50 μgE(n = 10)pVKD1.0-NP50 μgpVKD1.0-CI-LMNB100 μgLMNB-I13/CFA10 μgLMNB-C10/IFA10 μgpVKD1.0-CI50 μgF(n = 10)pVKD1.0-NP50 μgpVKD1.0-CI-LMNB100 μgLMNB-C10/CFA5 μgLMNB-C10/IFA5 μgpVKD1.0-CI50 μgLMNB-I13/CFA5 μgLMNB-I13/IFA5 μg

The tumor growth of immunized mice in each group is shown inFIG.27. Among them, all the mice in the control group (group A) developed tumors on the 14th day after the tumor challenge (i.e. after the tumor inoculation), and the tumors grew rapidly. The tumor growth of mice in each immunization group lagged behind that in the control group, wherein the mice in the group boosted with LMNB-I13 (group D) and the mice in the group boosted with a mixture of LMNB-I13 and LMNB-C10 (group E) had the slowest tumor growth, so these two groups of vaccines had the best effects.

In addition, the analysis results of tumor-free survival for the mice are shown inFIG.28. The median tumor-free survival (TFS) of mice in the control group A was 14 days. The tumor-free survival of mice in each vaccine immunized group was significantly higher than that in the control group, indicating that all vaccines could increase the tumor-free survival of immunized mice. Among them, group D with the 113 epitope fusion peptide, and group E and F with the 113 and C10 epitope fusion peptide had the best effects, and the tumor-free survival of mice was doubled at the most. In the vaccine group with the 113 epitope fusion peptide (group D), the tumor-free survival was significantly increased by about 40% compared to the conventional vaccine group (group B), showing that a load of 13 Th epitopes of Flu virus or 10 Th epitopes of CMV could significantly improve the protection effect of tumor vaccine against tumor.

Finally, the analysis results of mouse overall survival are shown inFIG.29. Among them, the median overall survival (OS) of mice in control group A was 35 days. The overall survival of mice in each vaccine immunized group was significantly higher than that in the control group, indicating that all vaccines could increase the survival of mice after immunization. Among them, group D with 113 epitope fusion peptide and groups E and F with 113 and C10 epitope fusion peptide had the best effects, and the overall survival rate was increased by 83%. Compared with the conventional vaccine group (group B), the vaccine group with the 113 epitope fusion peptide (groups D and group F) significantly increased the tumor-free survival by 28% at the most, indicating that a load of thirteen (13) Flu virus Th epitopes or ten (10) CMV Th epitopes could greatly improve the tumor protection effect of tumor vaccine.

Example 14 Tumor Treatment Experiment

The vaccines involved are shown in Example 9. Thirty (30) 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. The experimental animal grouping and vaccination schemes are shown in Table 25. All DNA vaccines were injected into the tibialis anterior muscle of the calf at 100 μg/animal. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), and injected subcutaneously into the back at 10 μg/animal. Two weeks after the last immunization, the mice were inoculated subcutaneously with the cell line transfected stably by the tumor cells 4T1-hNY-ESO-1 (provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park), at a dose of 1×105cells/mouse, and the corresponding mice were vaccinated subcutaneously with the protein vaccines on day 1, 8 and 15 after the tumor cell inoculation, respectively. The tumor growth was continuously observed and measured after the vaccination. The tumor volume was calculated according to the following equation: tumor volume (mm3)=length×width/2. The mice were sacrificed when the tumor volume exceeded 2000 mm3.

TABLE 25Grouping and immunization schemesWeek 0, 4, 8Week 10Week 11, 12GroupingVaccineDoseVaccineDoseVaccineDoseA(n = 10)pVKD1.0-NP100 μgpVKD1.0100 μgVP1/CFA10 μgB(n = 10)pVKD1.0-NP100 μgLMNB/CFA10 μgLMNB/IFA10 μgC(n = 10)pVKD1.0-NP100 μgLMNB-I13/CFA10 μgLMNB-I13/IFA10 μg

The tumor growth of immunized mice in each group is shown inFIG.30. Among them, all the mice in the control group (group A) developed tumors on the 14th day after the tumor challenge (i.e. after the tumor inoculation), and the tumors grew rapidly. The mice in the group treated by the LMNB-I13 protein vaccine (group C) had the slowest tumor growth compared to the untreated control group (group A). Furthermore, the tumor size of mice in the group treated by the LMNB-I13 protein vaccine was significantly smaller than that in the control group (group A) on day 22 after the tumor challenge, and there was still a significant difference in tumor size between the two groups until day 30. By day 35, the tumor growth of mice began to accelerate in group C, which is possibly associated with the cease of vaccination with the LMNB-I13 protein vaccine. The results indicated that the LMNB-I13 protein vaccine could inhibit tumor growth in mice.

Example 15 Tumor Treatment Experiment

The vaccines involved are shown in Example 9. Thirty (30) 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. The experimental animal grouping and vaccination schemes are shown in Table 26. All DNA vaccines were injected into the tibialis anterior muscle of the calf at 100 μg/animal. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), and injected subcutaneously into the back at 10 μg/animal. Two weeks after the last immunization, the mice were inoculated subcutaneously with the cell line transfected stably by the tumor cells CT26-hLAGE-1 (provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park), at an inoculation dose of 1×105cells/mouse, and the corresponding mice were vaccinated subcutaneously with the protein vaccine on day 1, 8 and 15 after the tumor cell inoculation, respectively. The tumor growth was continuously observed and measured after the inoculation. The tumor volume was calculated according to the following equation: tumor volume (mm3)=length×width/2. The mice were sacrificed when the tumor volume exceeded 2000 mm3.

TABLE 26Grouping and immunization schemesWeek 0, 4, 8Week 10Week 11, 12GroupingVaccineDoseVaccineDoseVaccineDoseA(n = 10)pVKD1.0-NP100 μgpVKD1.0100 μgVP1/CFA10 μgB(n = 10)pVKD1.0-NP100 μgLMNB/CFA10 μgLMNB/IFA10 μgC(n = 10)pVKD1.0-NP100 μgLMNB-I13/CFA10 μgLMNB-I13/IFA10 μg

The tumor growth of immunized mice in each group is shown inFIG.31. Due to the failure of tumor inoculation of some mice in the untreated control group (group A) after the tumor challenge (i.e. after the tumor inoculation), such mice were not included for the analysis, and the parallel control vaccine group (group B) and the group treated by LMNB-I13 (group C) were compared. Compared with group B, the tumor growth of mice in group C was slower, and the tumor size of mice in the group treated by the LMNB-I13 protein vaccine was significantly smaller than that in parallel control vaccine group (group B) on day 22 after the tumor challenge. There was still a significant difference in tumor size between the two groups until day 30. Similarly, the increased tumor growth of mice in group C was also observed at a later stage in the CT26 mouse model, which is possibly associated with the cease of vaccination with the LMNB-I13 protein vaccine. These results indicated that the LMNB-I13 protein vaccine could inhibit tumor growth in mice.

Example 16 Construction of Shuttle Vector pSC65-LMNB-I13

The amino acid sequence of 113 (SEQ ID NO: 60) comprises 8 influenza virus Th epitopes including HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95 and NP221 in Table 4, and 5 influenza virus Th epitopes including HA434, HA440, NP324, M1-127 and M1-210 in Table 4. The eukaryotic codon optimized sequence of the amino acid sequence encoding 113 (SEQ ID NO: 61) was synthesized by Suzhou Genewiz Biotechnology Co., Ltd., and then inserted into the shuttle vector pSC65 (addgene, Cat. No. 30327) by a molecular biology method well known in the art to construct the shuttle vector pSC65-I13 comprising 13 influenza epitopes.

The vector pVKD1.0-hLMN-CTB expressing a fused triad of tumor antigens LAGE-1, MAGE-A3, NY-ESO-1 and cholera toxin subunit B was found in Example 2. Then the LMN-CTB fragment in the vector was digested by the enzymes Sal I and Kpn I for recovery and inserted into the aforesaid shuttle vector pSC65-I13 to construct the shuttle vector pSC65-LMNB-I13 (the plasmid map is shown inFIG.32) expressing a triad of tumor antigens, which was stored after being sequenced for identification, wherein the amino acid sequence of LMNB-I13 is shown in SEQ ID NO: 62, and the eukaryotic codon optimized sequence thereof is shown in SEQ ID NO: 63. The vector pSC65-LMNB-I13 was identified by the restriction endonucleases Sal I and Kpn I (the enzyme digestion system is shown in Table 27), and its enzyme digestion map for verification is shown inFIG.33.

TABLE 27Enzyme digestion system for identification of plasmidpSC65V3 (enzyme digestion at 37° C., 2 h)Enzyme digestion systemVolumePlasmid pSC65-LMNB-I133 μL, about 1 μgSal I (TaKaRa, Cat. No. 1080A)1 μLNot I (TaKaRa, Cat. No. 1166A)1 μLEnzyme digestion buffer1 μLddH2Oq.s. to 10 μL

Example 17 Construction of Recombinant Vaccinia Viral Vector rvv-LMNB-I13

The recombinant vaccinia viral vector was obtained from 143B cells according the following specific method. On the first day, 143B cells (provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park) were plated into a 6-well cell culture plate (JET, TCP-010-006), at 1×106cells/well, and incubated overnight in a carbon dioxide cell incubator at 37° C. On the second day, wild vaccinia virus strains (provided by Beijing Institute of Biological Products Co., Ltd.) were added with 0.05 MOI (i.e., 5×104PFU (plaque forming unit)/well), and then an incubation was conducted in the carbon dioxide cell incubator at 37° C. for 2 h, during which a shuttle vector/transfection reagent complex was prepared. The shuttle vector was pSC65-LMNB-I13 obtained in Example 16, the transfection reagent was Turbofect (Thermo Fisher Scientific, R0531), and the instructions of transfection reagent can be referred to for transfection dosage and complex method. After completion of the complex system, the supernatant of 143B cells 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. After 48 h of transfection, the supernatant was removed, and the cells were collected, re-suspended in 0.5 mL the maintenance medium and frozen-thawed repeatedly for three times. Then the recombinant cell lysates were introduced into new 143B cells (with 50 μg/mL BrdU), followed by an incubation at 37° C. for 1 to 2 days. During this period, the cytopathic effects were observed, and when an appropriate number of virus plaques appeared (less than 20 plaques/well), purification of single plaque was carried out.

Purification of Single Plaque

2% low melting agarose was heated in a microwave oven (medium to high heat for about 2 min) to boiling, then transferred to a water bath at 45° C. and allowed to cool and prevented from solidification.

An appropriate amount of 2×maintenance medium (1 mL/well) was pipetted, an X-gal stock was added at a volume ratio of 1:50, followed by preheating in a water bath at 45° C.

Low melting point agarose and 2×maintenance medium containing X-gal were mixed in a ratio of equal volume to prepare a solid medium for plaque spread, the cell supernatant was removed, and the solid medium for plaque spread was carefully added. Then, the 6-well plate was transferred into a refrigerator for solidification at 4° C. for 10 min, without being moved to prevent from a non-uniform solidification during this period.

After complete solidification, the 6-well plate was transferred to a cell incubator and incubated at 37° C. for 2 to 4 h (sometimes overnight) until blue plaques appeared.

After the blue plaques appeared, the blue plaques which were well-dispersed and deeper in color were preferentially picked by using a 1 mL pipette tip (the pipette tip was trimmed by scissors in advance). The cell layer below the solid culture medium must be picked during picking, and a plurality of blue plaques were picked per well and transferred into Ep tubes containing 0.5 mL the maintenance medium, respectively.

The Ep tubes containing the virus were mixed uniformly by shaking, frozen and thawed three times repeatedly (in a refrigerator at −80° C. for about 5 min, and at room temperature for about 2 min), and finally mixed uniformly by shaking, frozen and stored at −80° C.

Six rounds of purification of single plaque were repeated until the purity reached 100%.

Example 18 Amplification Preparation and Titration of Recombinant Vaccinia Viral Vector Rvv-LMNB-I13

The recombinant vaccinia viral vector rvv-LMNB-I13 constructed in Example 17, and wild strain of vaccinia virus were amplified in Vero cells (provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park), respectively. The amplification method is as follows:

The day before, Vero monolayer cells with 100% confluence were prepared for a total of 10 dishes (1×107cells/dish).

The supernatant was removed and replaced with the 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. A significant cytopathic effect was observed.

The cells were scraped off and collected, centrifuged at 1800 g for 5 min, and the supernatant was removed.

5 mL the maintenance medium was used for re-suspension, and an ultrasonic cell crusher was used to for ultrasonication on the ice. The ultrasonication conditions were as follows: 50 watts, 5 s ultrasonication/5 s interval, a total of 15 min.

Freezing and thawing were repeated two times (in a refrigerator at −80° C. for about 5 min, and at room temperature for about 2 min), followed by a final uniform mixing by shaking;

Aliquoting into 1.5 mL centrifuge tubes was carried out in a Class II biosafety cabinet, at 1 mL/tube, followed by freezing at −80° C.

The vaccinia virus prepared by amplification was subjected to a titration for infection titer on Vero cells, and the specific method was as follows.

The day before, Vero cells with 100% confluence were prepared in a 24-well plate, at 3×105cells/well.

The supernatant was removed, and 200 μL the maintenance medium was added into each well to prevent the cells from cell dryout.

100 μL of vaccinia virus to be tested was added into 900 μL the maintenance medium, and subjected to 10 fold dilutions to be serially diluted 101, 102, 103, up to 109folds. Note: with respect to the dilution, since it was conducted from a high concentration to a low concentration, the pipette tip should be replaced before each dilution to a lower concentration.

The virus was added into a 24-well plate at 400 μL dilution per well in duplicate with the concentration from low to high (109, 108, . . . , 104), and the measurements were carried out serially for 6 dilution folds. After addition, the 24-well plate was placed and incubated in a cell incubator at 37° C. for 2 days.

The numbers of viral plaques were counted under a microscope, and the number of more than 20 was recorded as 20+. A recombinant virus titer (PFU/mL) was calculated as: an average of the numbers of plaques that can be counted as less than 20 (inclusive) in two duplicate wells×2.5 (1000 μL/400 μL)×dilution fold of the corresponding wells.

Titration results of vaccinia viral vector titer are shown in Table 28.

TABLE 28Titrations of vaccinia virus vector titerVaccinia virusTiter (PFU/mL)Wild-type vaccinia virus rvv-wt1.5 × 108Recombinant vaccinia virus rvv-LMNB-I131.0 × 108

Example 19 Tumor Treatment Experiment 1

Twenty 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. On day 0, all the mice were inoculated subcutaneously with a cell line stably transfected by the tumor cells CT26-MAGE-A3 expressing the MAGE-A3 tumor antigen (provided Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park), at an inoculation dose of 2×105cells/mouse, and then randomly divided into two groups. The vaccinia viral vector prepared in Example 18 was vaccinated into the tibialis anterior muscle of calf of the corresponding mice on the 1st, 14th and 28th day after the tumor cell inoculation (the specific vaccination scheme is shown in Table 29). The tumor growth was continuously observed and measured after the vaccination. The tumor volume was calculated according to the following formula: tumor volume (mm3)=length×width/2. The mice were sacrificed when the tumor volume exceeded 10000 mm3.

TABLE 29Experimental animal grouping and vaccination schemesDay 1Day 14Day 28GroupingVaccineDoseVaccineDoseVaccineDoseControl Grouprvv-wt2 × 106rvv-wt2 × 106rvv-wt2 × 106(n = 10)PFUPFUPFUTreatmentrvv-LMNB-I132 × 106rvv-LMNB-I132 × 106rvv-LMNB-I132 × 106group (n = 10)PFUPFUPFU

The tumor growth of immunized mice in each group is shown inFIG.34. Among them, all the mice in the control group developed the tumor on the 15th day after the tumor inoculation, which grew rapidly. On day 30 after the tumor challenge, the average tumor size of mice in the treatment group was significantly smaller than that in the control group. The results show that the vaccinia viral vector vaccine rvv-LMNB-I13 can inhibit the growth of tumors with MAGE-A3 expression.

The results of survival status of mice are shown by the survival curves inFIG.35, and the overall survival of mice (the OS median: 36 days) in the group treated by the vaccinia viral vector vaccine rvv-LMNB-I13 was superior to that of mice in the control group (the OS median: 33 days, p<0.05). The results show that the vaccinia viral vector vaccine rvv-LMNB-I13 can improve the survival of mice having the tumors expressing MAGE-A3.

Example 20 Tumor Treatment Experiment 2

Twenty 6-8 weeks old female BAL B/c mice were purchased from the Laboratory Animal Center of Suzhou University and raised in the SPF animal house of the Laboratory Animal Center of Suzhou University. On day 0, all mice were inoculated subcutaneously with a cell line stably transfected by the tumor cells CT26-LAGE-1 expressing the LAGE-1 tumor antigen (provided by Vacdiagn Biotechnology Co., Ltd., Suzhou Industrial Park), at an inoculation dose of 1×105cells/mouse, and then randomly divided into two groups. The vaccinia viral vector prepared in Example 18 was vaccinated into the tibialis anterior muscle of calf of the corresponding mice on the 1st, 14th and 28th day after the tumor cell inoculation (the specific vaccination scheme is shown in Table 30). The tumor growth was continuously observed and measured after the vaccination. The tumor volume was calculated according to the following formula: tumor volume (mm3)=length×width/2. The mice were sacrificed when the tumor volume exceeded 10000 mm3.

TABLE 30Experimental animal grouping and vaccination schemesDay 1Day 14Day 28GroupingVaccineDoseVaccineDoseVaccineDoseControl Grouprvv-wt2 × 106rvv-wt2 × 106rvv-wt2 × 106(n = 10)PFUPFUPFUTreatmentrvv-LMNB-I132 × 106rvv-LMNB-I132 × 106rvv-LMNB-I132 × 106group (n = 10)PFUPFUPFU

The tumor growth of immunized mice in each group is shown inFIG.36. On day 26 after the tumor challenge, the tumor of mice in the control group grew rapidly and was significantly larger than that of mice in the treatment group until day 50. The results show that the vaccinia viral vector vaccine rvv-LMNB-I13 can inhibit the growth of tumors with LAGE-1 expression.

In addition, the results of survival status of mice are shown by the survival curves inFIG.37, and the overall survival of mice (the OS median: 75 days) in the group treated by the vaccinia viral vector vaccine rvv-LMNB-I13 was superior to that of mice in the control group (the OS median: 51 days, p<0.05). The results show that the vaccinia viral vector vaccine rvv-LMNB-I13 can improve the survival of mice having the tumors expressing LAGE-1.

A comparison between the immune effects of protein vaccines in Examples 13-15 and the treatment effects of recombinant vaccinia viral vectors in Examples 19-20 shows that the protein vaccines had a significant effect in preventing tumors (seeFIGS.27,28and29). Although the protein vaccines maintained a certain therapeutic effect in tumor treatment, the therapeutic effect was not particularly significant and could not be sustained (seeFIG.30andFIG.31). The recombinant vaccinia viral vector had achieved a particularly significant effect in tumor treatment, not only a sustained inhibition of tumor growth, but also a significant improvement in overall survival, which were prominent effects (seeFIG.34-37).