PARAMYXOVIRUS VACCINE STRAIN FOR NOVEL CORONAVIRUS PNEUMONIA AND CONSTRUCTION METHOD THEREOF

Abstract

A paramyxovirus vaccine strain for novel coronavirus pneumonia and a construction method thereof are provided. The method includes performing a recombination of N and F genes of Newcastle disease virus type VII of Paramyxoviridae with P, M, H, L genes of Canine distemper virus of Paramyxoviridae to obtain a recombinant virus, inserting S1 gene of the novel coronavirus between the P and M genes of the recombinant virus to obtain a recombinant vector. The vaccine strain constructed can stably and efficiently express the novel coronavirus S1 protein, and induce the body to produce antibodies; and the recombined virus vaccine strain can stimulate the human body to produce mucosal immunity, and the prepared vaccine can be vaccinated through a nasal spray. Moreover, the vaccine strain can be tested in poultry and dogs, saving time, reducing costs, and being more conducive to actual production due to large output.

Claims

1. A paramyxovirus vaccine strain for novel coronavirus pneumonia, wherein the paramyxovirus vaccine strain for novel coronavirus pneumonia is a recombinant paramyxovirus strain that expresses the novel coronavirus S1 protein (rCNDV-S), with an access number CCTCC NO: V202119, deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China.

2. A vaccine composition, comprising a pharmaceutically effective amount of the paramyxovirus vaccine strain for novel coronavirus pneumonia according to claim 1 and a pharmaceutically acceptable adjuvant.

3. The vaccine composition according to claim 2, wherein the pharmaceutically acceptable adjuvant is one or more selected from the group consisting of propolis, liposomes, lentinan, ganoderma polysaccharide, astragalus polysaccharide, gastrodia extract, cordyceps extract, sophora flavescens extract, and levamisole.

4-20. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0025] FIG. 1 shows a map of pBR322-CDV-NPF-S1 vector plasmid.

[0026] FIG. 2 shows a mass spectrometry analysis of S1 protein expressed by the paramyxovirus vaccine strain for novel coronavirus pneumonia.

[0027] FIG. 3 shows Western Blot (WB) identification of S1 protein expressed by the paramyxovirus vaccine strain for novel coronavirus pneumonia.

[0028] FIG. 4 shows a level analysis of anti-SARS-CoV-2 neutralizing antibody in the serum of Beagle dogs.

[0029] FIG. 5 shows a level analysis of anti-SARS-CoV-2 neutralizing antibody in the serum of rhesus monkey.

DESCRIPTION OF EMBODIMENTS

[0030] Unless otherwise specified, the terms used in the present disclosure generally have the meanings commonly understood by those of ordinary skill in the art.

[0031] The present disclosure will be described in further detail below in conjunction with specific examples and with reference to data. The following examples are only to illustrate the present disclosure, but not to limit the scope of the present disclosure in any way.

EXAMPLE 1

Construction Method of Paramyxovirus Vaccine Strain for Novel Coronavirus Pneumonia

[0032] (1) Establishment of Cell Line Expressing T7 RNA Polymerase (BHK21-T7)

[0033] BL21 strain was taken and shaken to prepare DNA, from which the T7 RNA polymerase gene fragment is amplified and obtained by PCR. Primers (the underlined indicated enzyme cutting site) are designed as follows:

TABLE-US-00001 T7-F: (SEQ ID NO: 1) 5′-CTGCTCGAGCCACCATGAACACGATTAACATCGCTAAGAACGAC-3′; and T7-R: (SEQ ID NO: 2) 5′-CTGTCTAGATTACGCGAACGCGAAGTCCGACTCTAAGATGT-3′;

[0034] The enzyme cutting sites were: upstream Xho I, and downstream Xba I (marked by the underline). The extracted BL21 DNA was used as a template for amplification and a target fragment was recovered, about 2.6 kb. The recovered target fragment and pCI-neo vector plasmid were subjected to double enzyme digestion with Xho I/Xba I respectively, and the target fragment was recovered by agarose gel electrophoresis, ligated with T4 ligase and transformed into DH5α competent cells. Plasmids were extracted in a small amount and identified by PCR and restriction enzyme digestion. Positive plasmids were selected for sequencing. If the sequence of the positive plasmids was proved to be correct a large number of recombinant plasmids containing the T7 RNA polymerase gene were prepared.

[0035] BHK-21 cells were cultured with DMEM containing 6% fetal bovine serum (Gibco) and inoculated with a 6-well plate. When the BHK-21 cells grew to about 70%, the cells were transfected with the prepared recombinant plasmid carrying the T7 RAN polymerase gene according to instructions of a liposome transfection kit (Invitrogen: 11668027). After 24 hours of transfection, the culture medium was replaced with DMEM containing G418 (700 ng/mL) for pressure screening. Cell RNA was extracted when the screened cells were passed continuously to the 25th generation, and then RT-PCR identification was performed after reverse transcription. Finally, the BHK-21 cell line (BHK21-T7) that can stably and continuously express the T7 RNA polymerase gene was obtained.

[0036] (2) Construction of Recombinant Expression Vector pBR322-CDV-NPF

[0037] The RNAs of CDV and NDV type VII were extracted, primers were designed for RT-PCR, and a whole genome sequence of a recombinant virus was constructed by enzyme digestion and homologous recombination. The whole genome sequence of the recombinant virus was divided into 8 segments for cloning, and the primers were as follows (the underline indicated the enzyme cutting site):

TABLE-US-00002 1-F: (SEQ ID NO: 3) 5′-GGAATTCTAATACGACTCACTATAGACCAAACAGAGAATCTGTGAG G-3′; 1-R: (SEQ ID NO: 4) 5′-GTAACACTGGTCTTGAATATTCAGTACCCCCAATCAGTGTCG-3′; 2-F: (SEQ ID NO: 5) 5′-CGACACTGATTGGGGGTACTGAATATTCAAGACCAGTGTTAC-3′; 2-R: (SEQ ID NO: 6) 5′-CGACGCGTCCGCGGGATGATGCGGGTTGCTTG-3′; 3-F: (SEQ ID NO: 7) 5′-CATCCCGCGGTAATCGGAAGG-3′; 3-R: (SEQ ID NO: 8) 5′-CGACGCGTGCCCATGGGGCTTGTCTGTTCG-3′; 4-F: (SEQ ID NO: 9) 5′-CCCCATGGGCTCCAAACCTTC-3′; 4-R: (SEQ ID NO: 10) 5′-GACGTGTGACCAGATTGCTTCATGCTCTCGTGGTGGCTCTC-3′; 5-F: (SEQ ID NO: 11) 5′-GAGAGCCACCACGAGAGCATGAAGCAATCTGGTCACACGTC-3′; 5-R: (SEQ ID NO: 12) 5′-CGACGCGTGATAAGCTTGTCCAGTGCATCTC-3′; 6-F: (SEQ ID NO: 13) 5′-GACAAGCTTATCGGGGCCAAAATTG-3′; 6-R: (SEQ ID NO: 14) 5′-CTAAAGCTTCATCCTCGTCCTG-3′; 7-F: (SEQ ID NO: 15) 5′-GAAGCTTTAGCCGCTTTCTTGATG-3′; 7-R: (SEQ ID NO: 16) 5′-CGACGCGTCTGCTAGCAGATATCTGGCTTAG-3′; 8-F: (SEQ ID NO: 17) 5′-CTGCTAGCAGTCTTGGATTGATTC-3′; 8-R: (SEQ ID NO: 18) 5′-CGACGCGTACGCGTCAAAAAACCCCTCAAGACCCGTTTAGAGGCCC CAAGGGGTTATGCTAGACCAGACAAAGCTGGGTATGATAAC-3′.

[0038] Among them, the first segment (1-F, 1-R) and the fourth segment (4-F, 4-R) primers were amplified using NDV type VII as a template; the rest segments were amplified using CDV as a template. After the amplification was completed, each fragment was recovered by agarose gel electrophoresis, and the whole genome sequence of the recombinant virus was constructed from sequences of fragments by enzyme digestion and homologous recombination. Furthermore, a promotor sequence of T7 was added upstream of the whole genome sequence and a terminator sequence of T7 was added downstream thereof, and a pBR322 vector was introduced to construct a recombinant plasmid pBR322-CDV-NPF.

[0039] (3) Construction of Auxiliary Plasmid

[0040] The pBR322-CDV-NPF plasmid constructed above was used as a template to clone the NP, P, and L genes, respectively. PCR primers for each gene are as follows (the underline indicated the enzyme cutting site):

TABLE-US-00003 NP-F: (SEQ ID NO: 19) 5′-GCTCTAGAATGTCGTCTGTTTTCGACGAATAC-3′; NP-R: (SEQ ID NO: 20) 5′-TCCCCCGGGTCAGTACCCCCAATCAGTGTC-3′; P-F: (SEQ ID NO: 21) 5′-GCTCTAGAATGGCAGAGGAGCAGGCCTATC-3′; P-R: (SEQ ID NO: 22) 5′-TCCCCCGGGTCAGTACCCCCAATCAGTGTCG-3′; L-F: (SEQ ID NO: 23) 5′-GCTCTAGAATGGACGCCGTCTCAGTGAAC-3′; L-R: (SEQ ID NO: 24) 5′-TCCCCCGGGTCAGTGGTTTCTAATCAGTGC-3′;

[0041] PCR products recovered by agarose gel electrophoresis and pCI-neo plasmid were subjected to double enzyme digestion with Xba I/Sma I respectively, and the digested products were recovered. The digested products were ligated by T4 DNA ligase at 16° C. for 4 hours. The ligated products were transformed into XL-10 chemocompetent cells, incubating overnight at 37° C. A single clone was picked up and inoculated in a liquid medium containing ampicillin, incubating overnight at 37° C. and 220 rpm. A single colony was identified by PCR, and the positive clones were sequenced and analyzed by the Illumina NextSeq 500 system to obtain target plasmids, which were named as pCI-neo-NP, pCI-neo-P, and pCI-neo-L respectively.

[0042] (4) Construction of the Whole Genome Recombinant Vector of the Novel Coronavirus Pneumonia Paramyxovirus

[0043] According to the SARS-CoV-2-S1 gene sequence MN908947.3 published in Genbank, a pUC57 plasmid containing the SARS-CoV-2 S1 protein encoding gene was synthesized by Shanghai Shenggong Biotechnology Co., Ltd., where the nucleotide sequence of SARS-CoV-2 S1 gene is SEQ ID NO: 25.

TABLE-US-00004 SEQ ID NO: 25: atgttcgtgt tcctggtgct gctgcctctg gtgagctccc agtgcgtgaa cctgaccacc   60 agaacccagc tgccacctgc ctacaccaac tcctttacca ggggcgtgta ttaccctgac  120 aaggtgttca gaagctccgt gctgcactcc acacaggacc tgttcctgcc attcttttcc  180 aacgtgactt ggttccacgc catccatgtg tctggcacca atggcacaaa gaggtttgac  240 aatccagtgc tgcctttcaa tgacggcgtg tattttgcct ccacagagaa gtctaatatc  300 atcagaggct ggatcttcgg cacaaccctg gattctaaga cccagtccct gctgatcgtg  360 aataatgcca ccaacgtcgt gatcaaggtg tgtgagtttc agttctgcaa cgaccctttc  420 ctgggcgtgt actaccacaa gaataacaag tcttggatgg agtctgagtt ccgggtgtac  480 tctagcgcta ataactgtac cttcgagtac gtgagccagc cctttctgat ggacctggag  540 ggcaagcagg ggaactttaa gaacctgagg gagttcgtgt tcaagaacat tgacggctac  600 tttaagatct attctaagca cacaccaatc aacctggtga gggatctgcc tcagggcttc  660 tccgccctgg agcccctggt ggacctgcct atcggcatca acatcaccag attccagacc  720 ctgctcgctc tgcatagaag ctacctgacc cccggcgaca gctcttccgg atggaccgct  780 ggcgctgccg cctactacgt gggctacctg cagccccgga ccttcctgct gaagtacaat  840 gagaacggaa ccatcaccga cgccgtggac tgtgctctgg accccctgag cgagaccaag  900 tgcactctga agtccttcac cgtggagaag ggcatctacc agacttctaa ctttcgggtg  960 cagcctaccg agtccattgt gcggttccct aacatcacta atctgtgccc attcggcgag 1020 gtgtttaacg ccacccggtt cgctagcgtg tacgcctgga acaggaagcg catctccaac 1080 tgtgtggccg actactctgt gctgtacaat tctgccagct tttctacctt caagtgttac 1140 ggcgtgagcc caacaaaact gaacgacctg tgctttacca acgtgtacgc cgacagcttc 1200 gtgatcaggg gcgatgaggt gagacagatc gcccctggcc agaccggcaa gatcgccgac 1260 tataactaca agctgcctga tgatttcacc ggatgcgtga tcgcctggaa ctctaacaat 1320 ctggatagca aggtgggcgg gaattacaac tacctgtaca gactgttcag aaagtccaac 1380 ctgaagcctt tcgagcggga catctccacc gagatctacc aggctggatc aaccccctgc 1440 aacggcgtgg agggctttaa ttgttacttt cccctgcagt cttacggctt ccagcccacc 1500 aacggcgtgg gctaccagcc ttatagagtg gtggtgctga gctttgagct gctgcacgcc 1560 cctgccaccg tgtgcggccc taagaagtct acaaacctgg tgaagaataa gtgcgtgaac 1620 tttaatttca acggcctgac cggcaccggc gtgctgaccg agagcaacaa gaagttcctg 1680 cccttccagc agttcggaag ggacatcgcc gatacaaccg acgccgtgag agacccacag 1740 accctggaga tcctggacat caccccttgt agctttggcg gcgtgagcgt gatcacccct 1800 ggcaccaaca cctccaacca ggtggccgtg ctgtaccagg acgtgaattg cactgaggtg 1860 cctgtggcta tccacgctga ccagctgacc cctacctgga gagtgtatag caccggatcc 1920 aacgtgttcc agaccagagc tggctgcctg atcggcgctg agcacgtgaa taacagctat 1980 gagtgtgaca tccccatcgg cgccggcatc tgcgctagct accagaccca gaccaactcc 2040 tga  2043

[0044] A specific procedure included the following steps:

[0045] according to the SARS-CoV-2 S1 protein coding sequence of SEQ ID NO: 25, designing the amplification primers using a pUC57 cloning vector containing the sequence as a template, where the primer sequences were as follows:

TABLE-US-00005 nCoV2019-S1-F: (SEQ ID NO: 26) 5′-GTTTAAACTTAAGAAAAAACACGGGTAGAAATGTTCGTGTTCCT GGTGC-3′; nCoV2019-S1-R: (SEQ ID NO: 27) 5′-GTTTAAACTCAGGAGTTGGTCTGGGTC-3′; and

[0046] performing PCR amplification of SARS-CoV-2 S1 gene according to the following procedure: pre-denaturing at 98° C. for 2min; 30 cycles (denaturing at 98° C. for 10 s; annealing at 60° C. for 30 s; extending at 72° C. for 2 min); and extending at 72° C. for 10 min. The gel containing positive fragments identified by agarose (1%) gel electrophoresis were recovered and purified to obtain the SARS-CoV-2 S1 gene fragment. The pBR322-CDV-NPF vector was subjected to single enzyme digestion with Pme I and was ligated with the SARS-CoV-2 S1 gene fragment at 16° C. overnight. The ligated product was transformed into XL-10 chemocompetent cells and incubated at 37° C. overnight. A single clone was picked up and inoculated in a liquid medium containing ampicillin at 37° C. and at 220 rpm overnight. A single colony was identified by PCR, and the positive clones were sequenced and analyzed by the Illumina NextSeq 500 system to obtain an expression vector plasmid correctly ligated with S1 gene, where the expression vector plasmid is named as pBR322-CDV-NPF-S1, as shown in FIG. 1.

[0047] (5) Cell Transfection and Virus Rescue

[0048] The plasmid pBR322-CDV-NPF-S1 constructed in step (4) and the auxiliary plasmids pCI-neo-NP, pCI-neo-P, and pCI-neo-L constructed in step (3) were transferred into the cultured BHK21-T7 cells by liposome transfection. After culturing for 48-72 hours, the cells and cell supernatant were collected, and were frozen and thawed once at −20° C. The supernatant was collected aseptically after centrifugation and filtered at 0.22 μm to obtain a filtrate. The filtrate was inoculated aseptically into 9-11-day-old SPF chicken embryos, and continued to incubate for 2-4 days. An allantoic fluid was collected aseptically and tested for its hemagglutination (HA) titer. Through RT-PCR identification and sequence analysis, an allantoic fluid containing SARS-CoV-2-S1 protein and having a hemagglutination titer 7-8 log2, RT-PCR positive and correct sequence, was obtained, indicating that the virus rescue was successful.

[0049] The above-mentioned successfully rescued virus was deposited in the China Type Culture Collection, Wuhan University, Wuhan, China, on Feb. 11, 2021, with a deposit number of CCTCC NO: V202119 and a deposit name of a recombinant paramyxovirus strain expressing novel coronavirus S1 Protein (rCNDV-S).

EXAMPLE 2

Expression and Biological Activity Identification of S1 Protein of Paramyxovirus Vaccine Strain for Novel Coronavirus Pneumonia

[0050] Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis

[0051] The LC-MS/MS analysis was performed by the following steps: after enzymatic hydrolysis of each sample, an appropriate amount of enzymatically hydrolyzed peptide segments were taken and subjected to chromatographic separation through a nanoliter flow rate Easy nLC 1200 chromatography system (Thermo Scientific), wherein a buffer solution includes solution A: 0.1% of formic acid in water, and solution B: 0.1% of formic acid and 80% of acetonitrile in water. The column was balanced with 100% of the solution A. The sample was injected into the Trap Column (100 μm×20 mm, 5 μm, C18, Dr. Maisch GmbH) and then passed through a chromatographic analysis column (75 μm*150 mm, 3 μm, C18, Dr. Maisch GmbH) for gradient separation at a flow rate of 300 nl/min. A liquid phase separation gradient was used as follows: 0-2 min, solution B with a linear gradient from 2% to 5%; 2-44 min, solution B with a linear gradient from 5% to 28%; 44-51 min, solution B with a linear gradient from 28% to 40%; 51-53 min, solution B with a linear gradient from 40%-100%; and 53-60 min, solution B of 100%. After separation of the peptide segments, the Q-Exactive HF-X mass spectrometer (Thermo Scientific) was used for DDA mass spectrometry analysis, where analysis time: 60 min; detection mode: positive ion; parent ion scan range: 350-1800m/z; resolution of primary mass spectrometer: 60,000@m/z 200; AGC target: 3e6; primary maximum IT: 50 ms. Secondary mass spectrometry analysis of peptide segments was performed according to the following method: after each full scan, a secondary mass spectrum of 20 highest-intensity parent ions was triggered and collected (MS2 scan), where resolution of the secondary mass spectrum: 15,000@m/z 200; AGC target: 1e5; Level 2 Maximum IT: 50 ms; MS2 Activation Type: HCD; Isolation window: 1.6 m/z; Normalized collision energy: 28. After searching and analyzing via MaxQuant 1.6.1.0, the results showed that: LC-MS/MS mass spectrometry analysis showed specific peptide segments, and the mass spectrometry results were as shown in FIG. 2. It showed that the SARS-CoV-2 S1 gene was expressed in recombinant cells of BHK21-T7/pBR322-CDV-NPF-S1.

[0052] Western Blot (WB) identification:

[0053] The culture supernatant of the constructed recombinant cells, containing the SARS-CoV-2 S1 protein gene, of BHK21-T7/pBR322-CDV-NPF-S1 was subjected to Western Blot analysis and LC-MS/MS analysis. The Western Blot analysis step includes: SDS-PAGE gel electrophoresis was performed on a sample group, a control group and a protein marker, the electrophoresis was stopped when the bromophenol blue dye moved to the end of the gel, and the protein was transferred from the gel to a nitrocellulose membrane. After transferring to the membrane, it is blocked with a skimmed milk powder for 1 hour. An appropriate amount of mouse anti-SARS-CoV-2 S1 protein monoclonal antibody was added as a primary antibody for reaction, followed by incubating at 37° C. for 1 hour, and washing with TBST for 4 times. A FITC-labeled goat anti-mouse IgG secondary antibody was then added for reaction, followed by incubating at 37° C. for 1 hour, washing with TBST for 4 times, then adding a luminescent substrate solution for developing. WB results showed that the supernatant control group of empty vector cells showed no band, while the sample group had obvious bands, as shown in FIG. 3.

EXAMPLE 3

Animal Immunogenicity of Paramyxovirus Vaccine Strain for Novel Coronavirus Pneumonia

[0054] (1) 12 1-month-old beagle dogs were taken and randomly divided into a control groups and a test group, with 6 dogs in each group. The control group was immunized by nasal spray with sterile normal saline, and the test group was immunized by nasal spray with a sample vaccine of 10.sup.8.3 EID.sub.50/0.1 ml prepared from rCNDV-S virus cultured in SPF chicken embryo, once a day, 200 μl every time, continuous immunization for 3 days. On the 14th day (D14) after immunization, blood was collected from the forelimb veins of dogs in the control and test groups, for separating serum. The level of the neutralizing antibody was determined by blocking ELISA. As shown in FIG. 4, only dogs in the test group produced specific antibodies, and the antibody level was significantly higher than that in the control group (P<0.05). It showed that rCNDV-S virus cultured from the allantoic fluid of chicken embryo can be used as an antigen to prepare a novel coronavirus vaccine, and the beagle dogs can be immunostimulated by nasal spray to produce specific antibodies.

[0055] (2) 12 experimental rhesus monkeys of 3-4 kg were taken and divided into a control group and a test group randomly, with 6 animals in each group. The control group was immunized by nasal spray with sterile normal saline, and the test group was immunized by nasal spray with a sample vaccine of 10.sup.8.3 EID.sub.50/0.1 ml prepared from rCNDV-S virus cultured in SPF chicken embryo, once a day, 200 μl every time, continuous immunization for 3 days. On the 14th day (D14) and 28th day (D28), blood was collected from the forelimb veins of all experimental rhesus monkeys for separating serum. The level of the neutralizing antibody was determined by blocking ELISA. As shown in FIG. 5, only rhesus monkeys in the test group produced specific antibodies, and the level of the antibody was significantly higher than that in the control group (P<0.05). It showed that rCNDV-S virus cultured from the SPF chicken embryo can be used as an antigen to prepare a novel coronavirus vaccine, and the rhesus monkeys can be immunestimulated by nasal spray to produce specific antibodies.

EXAMPLE 4

Stability of Paramyxovirus Vaccine Strain for Novel Coronavirus Pneumonia

[0056] The lung tissues of the New Zealand white rabbits immunized with the method in Example 3 were ground and diluted with normal saline to obtain diluted tissue solution. Another five healthy New Zealand white rabbits were randomly selected to inoculate with the diluted tissue solution of 100 μL for each rabbit, numbered as Nos. 1-5. After 7 days, the lung tissues of rabbits Nos. 1-5 were ground and diluted with normal saline, and underwent aseptic treatment, to randomly inoculate another 5 healthy rabbits, numbered as Nos. 6-10. In such way, the lung tissues of rabbits Nos. 25-30 were ground and diluted with normal saline, to inoculate the last 5 healthy rabbits, numbered as Nos. 31-35. All the 7 groups of rabbits were tested for recombinant virus. Meanwhile, RNA was extracted and reverse transcribed into cDNA, and S1 gene was amplified by RT-PCR. If there was a positive gene, it would be sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing.

[0057] Stability test of the vaccine strain using Beagle dogs as an animal model was the same as above.

[0058] Experimental results: recombinant virus can be detected in 35 rabbits and 35 beagle dogs in animal stability test. By analyzing and comparing of the sequence, it was found that the sequencing results of the S1 gene was the same as the S1 gene sequence which was inserted into the vector, indicating that the recombinant virus has not undergone mutations, the passage is stable, and there was no the virulence enhancement of virus.

[0059] The above are only preferred examples of the present disclosure, and are not intended to limit the present disclosure in other forms. Any person familiar with the profession may use the technical content disclosed above to make a change or modification to obtain the equivalent examples. However, without departing from the content of the technical solution of the present disclosure, any simple modifications, and equivalent changes and modifications made to the above examples based on the technical essence of the present disclosure still belong to the protection scope of the technical solution of the present disclosure.