Patent Application
20250066740
The contents of the electronic sequence listing (Substitute Sequence Listing.txt; Size: 174,474 bytes; and Date of Creation: Jul. 11, 2023) is herein incorporated by reference in its entirety.
The invention relates to the technical field of genetic engineering and the field of immunology, in particular to a novel chimpanzee adenovirus vector and a construction method and application thereof.
Adenovirus is a common virus causing upper respiratory tract and lung diseases, and the human immune system will have a strong immune response to adenovirus. Therefore, it is necessary to transform adenovirus into harmless vaccine vector. After decades of research and development, human adenovirus type 5 vaccine vector is widely used. Adenovirus is very popular as vaccine virus, usually, the adenovirus vector used in gene therapy is mainly human adenovirus type 5 (AdHu5), but neutralizing antibodies against AdHu5 are common in the population, and about one third of the population has become immune to it in adulthood. The immune system of these populations will destroy vaccines instead of producing antibodies against viruses carrying inserted genes. Moreover, the Hexon protein of AdHu5 binds to coagulation factor X (FX) in blood, which leads to the accumulation of adenovirus in liver and affects the tumor targeting of AdHu5. Therefore, the efficacy and targeting of adenovirus type 5 need to be further improved and perfected.
Chimpanzee adenovirus provides us with another feasible method. Because chimpanzee adenoviruses such as AdC7 and AdC23 are rarely prevalent in people, there are generally no corresponding neutralizing antibodies in people, and they will not be neutralized by antibodies against human adenovirus type 5 (AdHu5). At the same time, Hexon protein of chimpanzee adenovirus does not bind to FX. Therefore, chimpanzee adenovirus vector can be developed into an ideal adenovirus vector platform, which can be used to prepare higher quality vaccine reagents, such as Novel Coronavirus vaccine, and maintain inherent safety.
COVID-19, which began to break out in large numbers all over the world in 2019, caused serious diseases and caused huge losses. Coronavirus is a large virus family, which is known to cause colds and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). Novel Coronavirus is a new strain of coronavirus that has never been found in humans before.
Novel Coronavirus expressed 29 proteins, of which 16 were related to its replication, and 4 were structural proteins, Spike protein (S protein), Nucleocapsid protein (N protein) and Envelope protein (E protein). S protein is the most important surface protein of coronavirus. S protein can bind to human ACE2 (host cell receptor angiotensin converting enzyme 2), and its affinity is much higher than that of severe acute respiratory syndrome coronavirus (SARS-COV). S protein is the key to enter cells and the target of antibody drugs. N protein is abundant in coronavirus, which is a highly immunogenic protein and participates in genome replication and cell signaling pathway regulation.
The antibodies in novel coronavirus pneumonia patients will disappear within a few months, and so may the antibodies provided by vaccines, which means that people can't get vaccinated once and for all, and the antibodies will fade in a short time. The sooner or later of fading depends on the antibody level at the highest point, which determines how long the antibodies can stay in the body. Therefore, how to improve the antibody level of novel coronavirus pneumonia virus in patients is the primary problem to be considered in preparing Novel Coronavirus vaccine.
After testing, the serum of the population does not contain the pre-stored antibody of chimpanzee adenovirus, so the immune system will not destroy the vaccine, which is conducive to the effective expression of the target antigen and improve the level of antibody. Therefore, chimpanzee adenovirus is a good platform as a vaccine vector. The titer of chimpanzee adenovirus is generally low, and the process of constructing vaccine vector is complex and difficult to be industrialized. Therefore, it is urgent to find a chimpanzee adenovirus vector with higher titer and form a complete process platform, which can be used for the preparation of various vaccines such as Novel Coronavirus vaccine.
To solve traditional problems, the invention provides a brand-new chimpanzee adenovirus with higher virus titer. The defective chimpanzee adenovirus vector prepared by the invention can be used for preparing various vaccines such as Novel Coronavirus vaccine. The chimpanzee adenovirus vaccine vector provided by the invention has no preexisting antibody in the population, and the E1 region of the novel chimpanzee adenovirus vector is very different with the E1 region of human adenovirus type 5 in 293 cells, which can greatly avoid recovery mutation; at the same time, the novel chimpanzee adenovirus vector has the characteristics of higher stability, and can induce strong humoral immunity and cellular response in mouse model.
One of the first aspects, the present invention provides a new chimpanzee adenovirus comprising a hyper variable region HVR, and the hyper variable region HVR comprises a nucleotide sequence represented by any one or more of sequence represent by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14 or a nucleotide sequence having more than 50% homology with the above sequences including SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14.
Human adenoviruses belong to adenoviridae and mammalian adenoviruses. Up to now, 52 serotypes of human adenoviruses have been isolated, belonging to a ˜G7 subgenera. At present, virus isolation technology, antibody detection technology and molecular biology technology (such as polymerase chain reaction, restriction fragment length polymorphism analysis technology, in situ hybridization technology, real-time fluorescence quantitative PCR technology) are mainly used in the diagnosis and typing of adenovirus infection.
Chimpanzee adenovirus also has different serotypes; it's gene sequence includes the sequence of hypervariable region HVR1-9 and relatively conservative sequence. The relatively conservative sequence can be used to distinguish different adenoviruses (such as chimpanzee adenovirus or human adenovirus), while the hypervariable region sequence can be used to distinguish different serotypes of adenoviruses, that is to say, the sequence of hypervariable region determines the specific serotype of adenovirus.
The sequence diagram of HVR1-9 is shown in
The gene sequence of the novel chimpanzee adenovirus provided by the invention is mainly based on the sequence of the hexon gene hypervariable region HVR1-6 of the new chimpanzee adenovirus. The sequence of each region in the sequence of the hexon gene hypervariable region HVR1-6 of the chimpanzee adenovirus provided by the present invention is completely or significant different from the sequence of the serotype HVR1-6 of the known chimpanzee adenovirus in the database (genebank). The HVR1-6 sequence of the hexon gene hypervariable region of the new chimpanzee adenovirus comprises six mutation sequences respectively located in each sequence of the HVR1-6 sequence: HVR1 is the sequence indicated by the sequence number SEQ ID NO. 9, HVR2 is the sequence indicated by the sequence number SEQ ID NO. 10, HVR3 is the sequence indicated by the sequence number SEQ ID NO. 11, HVR4 is the sequence indicated by the sequence number SEQ ID NO. 12, HVR5 is the sequence indicated by the sequence number SEQ ID NO. 13, and HVR6 is the nucleotide sequence indicated by the sequence number SEQ ID NO. 14. The sequence of the adenovirus of the present invention at the position of HVR7-9 in the hypervariable region is not mutated, as shown in the sequence SEQ ID NO. 15.
The person with ordinary skill in the present invention will appreciate that the sequences of these hypervariable regions, once published, may be modified, artificially synthesized, or other unrelated sequences added to these sequences. These sequences, which have been modified, artificially synthesized, or added to 3 or 5 segments of the sequences consolidated by the invention, Thus forming a new sequence, Although these new sequences are not 100% the same as those published in the present invention, However, the scope of the present invention can still cover sequences having homology with the sequences of the above hypervariable regions 1-6, These new sequences are also within the scope of protection of the present invention, where the new sequence homology may be a sequence homology of a region in a single hypervariable region, for example, a sequence having more than 50% homology with the sequence shown in sequence number Seq ID NO. 9, for example, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 95%, or 99%. In addition, sequences such as those having 50% homology with the sequences one of the hypervariable regions 1-6 of the present invention, such as 55%, 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 95%, or 99%, are also possible, and these sequences are also one of the scope of the present invention. In addition, if the sequence of the present invention or the protein sequence encoded by the sequence having more than 50% homology with the sequence of the present invention can be encoded, it is also within the scope of protection of the present invention, that is, the protein or amino acid sequence encoded by these sequences is also within the scope of protection of the present invention.
Of course, these new sequences may include one or more of the nucleic acid sequences in the hypervariable regions of the invention, for example it includes one or more of the nucleic acid sequences shown in hypervariable regions 1-6, the latter comprising sequences having more than 50% homology with each of the nucleic acid sequences in the hypervariable regions 1-6 of the invention, for example 55%, 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 95%, or 99% homology.
Further, in some embodiments, the adenovirus comprises a region of the hypervariable region HVR7-9 having a nucleotide sequence as shown in the sequence as presented by SEQ ID NO. 15.
Further, the hypervariable region HVR has a nucleotide sequence as shown in the sequence SEQ ID NO. 7, or a nucleotide sequence having more than 50% homology with this sequence.
Further, the hypervariable region HVR has a nucleotide sequence as shown in the sequence SEQ ID NO. 7, or a nucleic acid sequence having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 95%, or 99% homology therewith.
Further, the chimpanzee adenovirus also includes a conserved region.
On the second aspect, the invention provides a chimpanzee adenovirus, which has a preservation number of CCTCC NO: V202120 and a strain of sAd-AY01.
In some ways, the present invention provides an adenovirus comprising a nucleotide sequence as shown in the sequence table Seq ID NO. 1, or a sequence having more than 50% homology with the sequence.
The invention provides a brand-new chimpanzee adenovirus. The whole genome of the chimpanzee adenovirus genome DNA is sequenced to determine the final virus genome sequence. The chimpanzee adenovirus was deposited in China Typical Culture Collection Center on Apr. 25, 2021. It was classified as orangutan adenovirus name as sAd-AY01, the collection number was CCTCC NO: V202120, and the collection address was Wuhan University, China. The genome of this new chimpanzee adenovirus sAd-AY01 is 36687 bp.
The brand-new chimpanzee adenovirus provided by the invention, It has a hypervariable region HVR sequence completely different from the existing inhibitory serotype. Compared with the known chimpanzee adenovirus partial serotype HVR1-6 sequence, monkey adenovirus partial serotype HVR1-6 sequence and human adenovirus partial serotype HVR1-6 sequence in gene bank, the homology is between 49% and 91%.
Based on the sequence of HVR1-6 in the hypervariable region of its hexon gene, it is compared with the known chimpanzee adenovirus partial serotype HVR1-6 sequence, monkey adenovirus partial serotype HVR1-6 sequence and human adenovirus partial serotype HVR1-6 sequence in genebank, the homology is between 49% and 91%. The phylogenetic tree was constructed by DNAstar software (
Since the discovery and successful isolation of adenoviruses in 1950s, more than 100 serotypes have been found one after another, among which there are at least 52 human adenoviruses, which are divided into seven subgroups: A, B, C, D, E, F and G. Human adenoviruses type 2 and type 5 commonly used in gene therapy belong to subgroup C in serological classification, and have 95% homology in DNA sequence. According to blood cell agglutination characteristics for chimpanzee adenovirus, twenty-two virus strain have been divided into three categories, each virus strain has an adenovirus complement immobilized antigen (Hillis W D, Goodman R (1969) Serological classification of chimpanzee adenoviruses by hemagglutination and hemagglutination inhibition.), the type-specific epitopes of adenovirus that can be recognized by neutralizing antibodies are mainly located on the hexon surface. The epitopes of hexon protein are plotted into two loops, L1 and L2, and are divided into seven hypervariable regions, HVR1-6 (L1) and HVR7 (L2). The hypervariable regions can lead to abnormal recombination. In most cases, HVR1-6 sequence sequencing can obviously distinguish serotypes. Molecular typing based on the hexon gene HVR1-7 and the discovery of new adenovirus variants have been widely used. (Casas A, Avellon M, Mosquera O, et al. Molecular typing of human adenoviruses by PCR and sequencing of a partial region of the hexon gene. Lu X, Erdman D D. Molecular typing of human adenoviruses by PCR and sequencing of a partial region of the hexon gene. Arch Viro 1, 2006, 151:1587-1602.).
According to the reference of Complete nucleotide sequences and genome organization of four chimpanzee adenoviruses, chimpanzee adenovirus serotypes SAdV-22, SAdV-23, SAdV-24 (simian adenovirus 22, simian adenovirus 23, simian adenovirus 24) were classified into subtype E (as shown in
The highest homology with the orangutan adenovirus sAd-AY01 provided by the invention is Pan troglodytes adenovirus clone 17.2 major capsid protein gene and Pan troglodytes adenovirus clone 17.2 major capsid protein gene. The two known chimpanzee adenoviruses are not preserved by any preservation institution at present, and only 2000 bp sequences are disclosed.
The inventor has proved through a large number of studies that the novel chimpanzee adenovirus sAd-AY01 provided by the invention has very high virus titer, and the virus titer can reach 5×108 FFU/mL, which is far higher than the virus titer of the existing chimpanzee adenovirus and human adenovirus.
On the third aspect, the invention provides a new chimpanzee adenovirus vector psAd. The chimpanzee adenovirus vector psAd containing a HVR sequence in a hypervariable region is prepared by constructing a shuttle plasmid psAd-shuttle and carrying out homologous recombination with the chimpanzee adenovirus gene as described above after linearization.
In some embodiments, the plasmid contains a fluorescent labeled nucleic acid or a nucleic acid containing a fluorescent protein; the shuttle plasmid psAd-shuttle has a nucleotide sequence as shown in the sequence SEQ ID NO. 3, including replicon (pACYC), resistance gene (kanamycin), green fluorescent expression frame (RSV promoter, EGFP, BGHpolyA) nucleic acid.
In some embodiments, the cyclic plasmid psAd has a nucleotide sequence as shown in the sequence SEQ ID NO. 2, including a replicon (pACYC), a resistance gene (kanamycin), and a green fluorescent expression box (RSV promoter, EGFP, BGHpolyA) nucleic acid.
The chimpanzee adenovirus (sAd) genome is a linear DNA larger than 36 kb in length, which is difficult to edit effectively. Therefore, it is necessary to connect the replication-related elements in bacteria, such as replicon (pACYC) and resistance gene (kanamycin), to the chimpanzee genome to form a circular plasmid.
The existing method of constructing cyclic plasmid is usually cosmid (phage), which needs to buy corresponding kits, and phage may also pollute the laboratory.
Shuttle plasmids are usually used as expression vectors to carry genes to be expressed into target plasmids. The invention creatively constructs a chimpanzee adenovirus vector ring plasmid by constructing a shuttle plasmid psAd-shuttle comprising a replicon (pACYC), a resistance gene (kanamycin), a green fluorescent expression frame (RSV promoter, EGFP, BGHpolyA), and then carries out homologous recombination with the chimpanzee adenovirus genome after linearization to form a chimpanzee ring plasmid, which can effectively replicate in bacteria; at the same time, in order to monitor the transfection efficiency of plasmid in cells, green fluorescent expression frames (RSV promoter, EGFP, BGHpolyA) were added, and the transfection efficiency of vector could be judged by the amount of fluorescence in TP0 generation after transfection into cells. It can be understood that these are not necessary for shuttle plasmids, such as resistance gene (kanamycin), green fluorescent expression box (RSV promoter, EGFP, BGHpolyA), and of course, green fluorescent expression box (RSV promoter, EGFP, BGHpolyA) is the least necessary.
The method for constructing the circular plasmid of the invention has the advantages that: 1, no need to purchase a reagent kit and avoid the possible contamination of the laboratory by using bacteriophage; 2. The transfection efficiency of TP0 can be tested by adding EGFP fluorescence sequence; 3. It is more flexible and can build the components it needs according to its own needs.
In yet another aspect, the present invention provides a replication-defective chimpanzee adenovirus vector based on the chimpanzee adenovirus described above, conserved region deletion gene.
It can be understood that on the basis of the circular plasmid prepared by the chimpanzee adenovirus sAd-AY01 provided by the invention, the replication-defective chimpanzee adenovirus vector can be prepared by deleting any gene.
In some ways, the deletion gene is associated with chimpanzee adenovirus replication, and the vector after deletion of the gene is a replication-deficient chimpanzee adenovirus vector.
In some ways, the deletion gene includes any one or more of E1, E2A, E2B, E3, E4. For example, deletion of one of E1, E2A, E2B, E3 and E4 regions, or deletion of any combination of multiple regions, such as E1 and E3; E1, E3 and E4; E1, E2A, E3 and E4; E1, E2A, E2B, E3, E4 and so on.
As shown in
In some embodiments, replication-defective chimpanzee adenovirus vectors are missing genes in the E1 and E3 regions.
In some embodiments, replication-deficient chimpanzee adenovirus vectors can be constructed by deleting E2A, E2B and E4 regions in addition to E1 and E3 regions.
In some embodiments, the E1 and E3 genes are deleted and have a nucleotide sequence as shown in the sequence SEQ ID NO. 4.
Further, the invention provides a replication-defective chimpanzee adenovirus vector psAd ΔE1 ΔE3, which is based on the chimpanzee adenovirus described above, knocks out its E1 and E3 genes to obtain a chimpanzee adenovirus vector psAd ΔE1ΔE3 deletion of E1 and E3 genes.
The replication-defective chimpanzee adenovirus vector comprises the sequence of the hypervariable region, the sequence of one region of the latter's six adjacent hypervariable regions or the region represented by a plurality of sequences.
The replication-deficient chimpanzee adenovirus vector psAdΔE1ΔE3 has a nucleotide sequence as shown in the sequence SEQ ID NO. 4.
Wild-type chimpanzee adenovirus vector can replicate and amplify in ordinary cells. In application, this vector needs to lack its replication-related elements, and construct replication-deficient adenovirus vector, so that it can only replicate and package in specific complementary cell lines, but cannot replicate and package in ordinary cells. Therefore, the invention specifically knocks out the E1 and E3 genes of the chimpanzee adenovirus vector psAd, so that it can only be packaged in a specific cell line (such as the 293 cell line) and named as psAdΔELΔE3. It will be understood that the adenovirus of the present invention also knocks out other sites, or adenoviruses with deletions of other sites, such as deletions of E2A, E2B, E4 sites, to obtain replication-defective adenovirus vectors.
In another aspect, the invention provides a method for constructing a replication-defective chimpanzee adenovirus vector, comprising the following steps:
Further, the shuttle plasmid psAd-shuttle in step 1) has a nucleotide sequence as shown in the sequence SEQ ID NO. 3, and the cyclic plasmid psAd has a nucleotide sequence as shown in the sequence SEQ ID NO. 2.
Further, in step 2), the E1 gene of psAd is digested by CRISPR/Cas9 enzyme, and an HRPT sequence containing a PsiI enzyme digestion site is inserted, and then purified and linked to obtain psAdΔE1; the HRPT sequence has a nucleotide sequence as shown in the sequence SEQ ID NO. 5.
Further, step 3) through CRISPR/Cas9 enzyme digesting psAdΔE1, the obtained product and the fusion fragment pVIII-U-exon are seamlessly cloned to obtain psAdΔE1ΔE3; the fusion fragment pVIII-U-exon has a nucleotide sequence as shown in the sequence SEQ ID NO. 6.
Shuttle plasmids are widely used in the construction of adenovirus vectors, and it is necessary to find a single enzyme cleavage site. The invention creatively adopts CRISPR/cas9 to construct recombinant adenovirus vector, selects appropriate E1, E3, E4 and E2A knockout sites through comparison, selects CRISPR sites according to the positions of E1, E3, E4 and E2A sequences and the number of knockout gene bases, and designs the best gRNA, thereby completing the construction of recombinant adenovirus vector.
In yet another aspect, the present invention provides a vaccine comprising a target gene in a gene deletion region of the replication-defective chimpanzee adenovirus vector as described above.
It can be understood that the nucleic acid sequence inserted into the deletion region can be any nucleic acid sequence, such as infectious disease, tumor and the like, which can be obtained or expressed by the vector to prepare vaccine reagents.
In some embodiments, the gene deletion region includes any one or more of E1, E2A, E2B, E3, E4.
In some embodiments, the target gene is a virus, a bacterium, a tumor gene or a gene fragment.
In some embodiments, the target gene is the Novel Coronavirus S gene.
In some embodiments, the gene of interest is located in the E1 region of a defective chimpanzee adenovirus vector, which is shown to have a nucleotide sequence as shown in sequence SEQ ID NO. 4.
In some embodiments, the invention provides a novel coronavirus vaccine, which is obtained by constructing a recombinant chimpanzee adenovirus vector expressing novel Coronavirus S gene and packaging it with 293 cells; The S gene is expressed in the E1 region of the defective chimpanzee adenovirus vector as described above to form a recombinant adenovirus vector psAd-S.
In another aspect, the invention provides a construction method of a recombinant chimpanzee adenovirus vector expressing novel Coronavirus S gene, comprising the following steps:
Further, the WPRE element is shown to have a nucleotide sequence as shown in the sequence SEQ ID NO. 17.
In another aspect, the present invention provides a titer determination method of recombinant chimpanzee adenovirus, which detects the titer of chimpanzee adenovirus by detecting the change of HVR antigen epitope of hypermutant region by specific antibody prepared from hexon hypermutant region HVR gene fragment, said HVR gene having nucleotide sequence as shown in sequence SEQ ID NO. 7.
Further, the chimpanzee adenovirus is the chimpanzee adenovirus as described above, or the replication defective chimpanzee adenovirus vector, or the vaccine as described above, or any recombinant chimpanzee adenovirus vector, etc.
The novel chimpanzee adenovirus of the invention is quite different from the traditional human type 5 adenovirus and the traditional chimpanzee adenovirus. After detection, the titer detection of the chimpanzee adenovirus of the invention needs to prepare its unique antibody. The main coat proteins of the new chimpanzee adenovirus are hexon, penton and fiber, and the hypervariable region HVR of hexon is the key region to identify the serotype of adenovirus. Therefore, expressing HVR protein and preparing mouse serum can detect the titer of chimpanzee adenovirus.
The detection method of chimpanzee adenovirus titer provided by the invention can be used for detecting the titer of recombinant chimpanzee adenovirus psAd-S vaccine provided by the invention.
In another aspect, the invention provides a packaging method of a recombinant chimpanzee adenovirus vector, wherein the vector of psAd-S was digested with I-sceI enzyme, 293 cells were transfected with the linearized plasmid, and cell suspension was collected.
The beneficial effects of the invention are mainly embodied in:
Preferred embodiments of the invention are described in further detail below in conjunction with the accompanying drawings and it should be noted that the embodiments described below are intended to facilitate understanding of the invention without any limiting effect thereon.
Anal swabs from chimpanzees were collected, and chimpanzee adenovirus was cultured by HEK293 cells (purchased from ATCC, batch number 70009859).
First, the collected anal swab is melted on ice. Until room temperature, Put it in a 5 ml cryopreservation tube, Add 3 ml DMEM, Vortex 30 s, 4° C., 2000 rpm, Centrifuge for 5 min, The supernatant is filtered through a 0.2 μm pinhole filter membrane for about 200 μm into a sterile 48-well plate for later use, Then inoculate HEK293 cells with 48 wells prepared in advance, and observe their pathological changes every day. If the cells are full in 48 hours, collect the cell suspension, freeze and thaw twice, and then freeze at −20° C. If it is not full, collect the cell suspension on the 5th day, and continue to inoculate HEK293 cells until the pathological changes, and extract the virus genome for PCR amplification and identification. The Hirt Virial DNA Extract method was used to further expand the identified virus strains (the main steps were collecting 3.5 ml infected cells, freezing and thawing for 3 times, adding 20% SDS 87.5 ul, 20 mg/ml Proteinase K 87.5 ul, incubating at 55° C. for 4 h; Add 1 ml of 5 mol/L NaCl solution into the centrifuge tube dropwise, mix well, incubate at 55° C. for 4 h; Chimpanzee adenovirus genome was extracted with phenol: chloroform: isoamyl alcohol (25:24:1) and chloroform: isoamyl alcohol (24:1). The upper water phase was extracted by adding 1/10 volume of 3 mol/L NaAc and 2 times volume of absolute ethanol at −20° C. for 30 min, centrifuging at 4° C. at 12,400 r/min for 5 min, slowly discarding the supernatant, naturally drying the residual alcohol, and then adding 50 μL Elution Buffer to dissolve DNA. The genomic DNA sequence of chimpanzee adenovirus was confirmed by agarose gel electrophoresis, and the results are shown in
The chimpanzee adenovirus genome DNA obtained in Example 1 was sequenced in the whole genome, and Shenggong Bioengineering (Shanghai) Co., Ltd. was responsible for determining the final virus genome sequence. The chimpanzee adenovirus was deposited in China Typical Culture Collection Center, classified and named as orangutan adenovirus sAd-AY01, and the preservation number was CCTCC NO: V202120. The genome of orangutan adenovirus sAd-AY01 is 36687 bp.
Human adenoviruses belong to adenoviridae and mammalian adenoviruses. Up to now, 52 serotypes of human adenoviruses have been isolated, belonging to a ˜G7 subgenera. At present, virus isolation technology, antibody detection technology and molecular biology technology (such as polymerase chain reaction, restriction fragment length polymorphism analysis technology, in situ hybridization technology, real-time fluorescence quantitative PCR technology) are mainly used in the diagnosis and typing of adenovirus infection.
The invention is based on the comparison of the sequence of HVR1-6 of the hypervariable region of the hexon gene with the sequence of HVR1-6 of the known serotype in the database (reference: Rika M O, Yasushi S, Tsunetada K, et a 1. Quantitative Detection and Rapid Identification of Human Adenoviruses. J Clin Microbiol. 2007. 4 5:9 5 8. 967.). The comparison results are shown in
The chimpanzee adenovirus (sAd) genome is a linear DNA larger than 36 kb in length, which is difficult to edit effectively. Therefore, it is necessary to connect the replication-related elements in bacteria, such as replicon (pACYC) and resistance gene (kanamycin), to the chimpanzee genome to form a circular plasmid.
Therefore, it is necessary to construct a circular plasmid with a size of about 6000 bp from replicon (pACYC), resistance gene (kanamycin), left arm (˜ 1400 bp) and right arm (˜ 1400 bp) of chimpanzee genome, and then transform it into BJ5183 competence together with chimpanzee adenovirus genome after linearization, and carry out homologous recombination to form a circular plasmid of chimpanzee, which can make it replicate effectively in bacteria. At the same time, in order to monitor the transfection efficiency of plasmids in cells, green fluorescent expression frames (RSV promoter, EGFP, BGHpolyA) were added. After transfection into cells, the transfection efficiency of large vectors was judged by the amount of fluorescence in TP0 generation.
3.1 Construction of Adenovirus Genomic DNA Cyclic Shuttle Plasmid psAd-Shuttle
Primer Design of Chimpanzee Adenovirus sAd-AY01 Left Arm, Right Arm and Bacterial Element
The colonies were cultured in 5 mL LB liquid medium containing Kan for 12˜16 h at 37° C., and the plasmid was extracted for enzyme digestion verification. The results are shown in
4.1 the E1 Region of Chimpanzee Adenovirus Vector psAd was Excised by CRISPR/Cas9 Method to Obtain the Vector Plasmid psAd E1 with E1 Knockout, and the Digestion Site PsiI was Introduced
The target sequence of E1 gene knockout was determined by using Thermofisher.com/crisprdesign software.
The vector plasmid psAd was obtained by digesting example 3 with sAd-E1A-gRNA, sAd-E1B-gRNA and cas9. The reaction system was Cas9 protein 3 μg, sAd-E1A-gRNA 3 ug, sAd-E1B-gRNA 3 ug, psAd vector plasmid 3 ug, NEB buffer 3.5 uL and supplemented water to 50 uL.
The reaction was incubated overnight at 37° C. and detected by agarose gel electrophoresis. The results are shown in
The amplified sequence is:
PCR amplified fragment, as shown in
The target sequence of E1 gene knockout was identified using the Thermofisher.com/crisprdesign software GeneArt™ CRISPR Search and Design tool (thermofisher.com/crisprdesign)
Designing Upstream and Downstream Primers of DNA Template for Amplification of sAd-E3A-gRNA and sAd-E3B-gRNA
The psAdΔE1 plasmid was digested with sAd-E3A-gRNA, sAd-E3B-gRNA and cas9. The reaction system was Cas9 protein 3 ug, sAd-E3A-gRNA 3 ug, sAd-E3B-gRNA 3 ug, psAdΔE1 vector plasmid 3 ug, NEB buffer 3.15 μL, and water supplementation to 50 μL.
Enzyme digestion reaction was incubated at 37° C. overnight. The carrier was purified using Axygen Gel Recovery Kit. The results of enzyme digestion are shown in
The excised pVIII and U-exon regions were amplified by PCR, and the glue was recovered;
The amplification primers are:
The amplified fragments are:
E3A-F and E3A-R were used as a pair of primers to amplify partial fragments of pVIII; E3B-F and E3B-R were used as a pair of primers to amplify partial fragments of U-exon. As shown in
The pVIII-U-exon fragment was amplified by E3A-F and E3B-R fusion PCR, and the glue was recovered; As shown in
The new chimpanzee adenovirus vector is 35 kb long and has many restriction sites, so it is difficult to operate directly on the vector. It is necessary to construct its special shuttle plasmid and integrate the foreign genes to be expressed into the adenovirus vector by homologous recombination.
S protein is the main antigen protein of Novel Coronavirus, which will cause the main immune response in the process of infection. Therefore, COVID-19 vaccine, a new chimpanzee vector, chose S gene of COVID-19 as the expressed foreign gene. Therefore, it is designed to use CMV promoter to drive expression in E1 region, which can produce a large number of antigens and cause strong immune response.
5.1 Construction of a New Shuttle Plasmid pSsAdE1 for Chimpanzee Adenovirus Vector E1 Region
To construct the shuttle plasmid of chimpanzee adenovirus vector, plasmid-related elements (replicon puc origin, resistance gene, eukaryotic system exogenous gene expression frame CMV-MCS-SV40 polyA) and the left arm and right arm of E1 region were connected in a suitable sequence.
The primer design is as follows
PCR amplification system: left-arm-F and left-arm-R were used as a pair of primers to amplify the left arm of the shuttle plasmid, puc-F and Amp-R were used as a pair of primers to amplify the replicon and resistance genes needed by the plasmid, right-arm-F and right-arm-R were used as a pair of primers to amplify the right arm of the shuttle plasmid, and CMV-F and SV40-R were used as a pair of primers to amplify the foreign gene expression frame CMV-MCS-SV40 polyA (Seq ID NO. 16).
The amplification results are shown in
The above four fragments were purified by Axygen gel recovery kit;
After screening, the extracted plasmid was digested and identified; Results As shown in
5.2 Adding WPRE Element to Shuttle Plasmid to Obtain pSsAdE1-WPRE Element
The WPRE and the digested pSsAdE1 gum were recovered and purified;
6.1 Homologous Recombination of Shuttle Plasmid pSsAdE1-WPRE-S and Adenovirus Vector Plasmid psAd ΔE1ΔE3
The reaction condition is 37° C., 1 h; 65° C., 20 min inactivation.
The psAd-S vector plasmid was packaged using 293 cells (purchased from ATCC, lot number 70009859) as follows:
Transfection: The linearized 2 μg plasmid and PEI were diluted with 100 μL serum-free medium respectively; Plasmid diluent was added to PEI diluent, and then absorbed repeatedly for 5 times or swirled for 10 seconds, and then incubated at room temperature for 10 minutes to form transfection complex. During incubation, the cell culture medium was gently sucked from the culture plate, and 2 mL of fresh growth medium was added. After 10 minutes, the transfection complex was added to the cells changed from fresh medium.
Cell culture: 293 cells after transfection were cultured in 37° C., 5% CO2 incubator for 72-96 hours; After 72-96 hours of viral plasmid transfection, 6-well plate cell suspension was collected and placed in 15 ml centrifuge tube, i.e. TP0.
293 cells were prepared. The cells grew well in T75 culture flask, discarded the supernatant, washed with PBS, digested with 0.25% trypsin, then added 10 ml DMEM fresh medium containing 10% fetal bovine serum to stop digestion, then blew and mixed evenly, inoculated into 6-well plate (5×105/ml, 2 ml per well), and stood in a 5% CO2 and CO2 incubator at 37° C. After 24 hours, when the cells grew into monolayer cells, the culture medium was discarded, and the recombinant adenovirus was continuously diluted by 10-2˜10-6 times with serum-free DMEM maintenance solution. Each dilution was inoculated into 2 wells with 250 uL per well. After 1 hour of infection, the supernatant was discarded, and the complete culture medium was supplemented, and then the culture was static in a 5% CO2 and CO2 incubator at 37° C. After 24 hours, the supernatant was discarded and the cells were washed with PBS, 1 mL per well. After discarding PBS, the cells were fixed with 1 mL cold formaldehyde at room temperature for 10 min. After discarding formaldehyde, the cells were washed with PBS, 1 mL per well, and then incubated with chimpanzee adenovirus HVR antibody, 1 ml per well. After 1 h at room temperature, the cells were washed with PBS again, 1 mL per well, and the sheep anti-rabbit-FITC second antibody was incubated. After 1 h at room temperature, the supernatant was discarded and the cells were washed with PBS again, and counted under fluorescence microscope (200 times, 10 continuous visual fields). Calculation: Virus titer (FFU/mL)=average×1013×4×10(−n). The FFU of psAd-S virus is 5×108 FFU/ml.
The adenovirus stored by our company and HAd26, HAd5 and HAd28 produced in the market are used to detect the virus titer. The results are shown in Table 1. sAd-AY01 is a new chimpanzee adenovirus provided by the invention, and its titer is the highest, which can reach 5×108 FFU/ml, providing the most basic guarantee for the industrial production of chimpanzee adenovirus vaccine.
The 293 cells were prepared. The cells grew well in T75 culture flask, discarded the supernatant, washed with PBS, digested with 0.25% trypsin, then added 10 ml of DMEM fresh medium containing 10% fetal bovine serum to stop digestion. Then, the 293 cells were planted in a 6-well plate (5×105 cells/ml, 2 ml/well), incubated at room temperature for 1 hour to make them adhere to the wall. After incubation, the adherence degree was examined under microscope. The infection was carried out with psAd-S virus particles, and the titer of infection was 2 MOI/well. After the 293 cells became pathological changes 48 hours later, the cells were collected, frozen and thawed repeatedly for 3 times, then centrifuged at 2000 g, and the supernatant was collected. FFU was detected in the collected supernatant, and then the new 293 cells were re-infected until 30 generations. The virus fluids collected from the 5th, 10th, 15th, 20th, 25th and 30th generations were detected, and it was found that the virus genome was still intact, which indicated that replication-defective psAd-S virus could be stably packaged in 293 cells.
The psAd-S virus RCA detection method is as follows:
RCA was calculated using the equation as observed under a 10-fold fluorescence microscope
The judging standard was that the level of RCA was less than 1 RCA/3×1010 vp. After statistics, the RCA level of psAd-S is less than 1 RCA/3×1010 vp, which indicates that the replication-defective psAd-S virus prepared by the invention can be stably packaged in 293 cells and cannot be converted into wild type or has low probability of being converted into wild type.
The 293 cells were prepared one day in advance and placed in a 12-well cell culture plate. The 293 cells were infected with psAd-S virus and the cells became diseased 48 hours later. All 1 ml of cells were collected and washed with PBS for sample preparation for Western Blot test. Spike's RBD protein antibody was used as the secondary antibody to detect the target protein. The RBD protein antibody was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. The results of Western Blot test are shown in
Neutralizing antibody against AdHu5 is common in the population, and about one-third of the population has been immunized against AdHu5 in adulthood, while the population does not have the antibody against the novel chimpanzee adenovirus. Therefore, injection of the novel chimpanzee adenovirus vector vaccine into human body will not be neutralized by pre-existing immunity, and sufficient antibody can be produced. The result can be verified by neutralize antibody experiments. The experimental design was shown in Table 1, and the experimental steps were as follows:
The neutralizing antibody experiments were conducted with chimpanzee adenovirus sAd-AY01 and human adenovirus type 5, respectively. The results showed that none of the dilutions of the positive serum sickness venom was pathological changes, and all of the dilutions of the negative serum sickness venom were pathological changes. The experiments were established. The specific results are shown in Table 2 and Table 3,
The results showed that, as shown in Table 2 and
Twenty SPF-grade mice (6-8 weeks of age) were randomly divided into three groups, five for each group. Mice were immunized with psAd-S vaccine according to the groupings shown in Table 4. The injection method was as follows: intramuscular injection was performed on the medial aspect of the posterior thigh. Injection dose: 100 μl.
Blood was collected 14 days after immunization and serum was separated from SARS-COV-2 (2019-nCOV) Spike S1 antibody titer detection kit (mouse), Art. No. KIT007, for detecting anti-SARS-COV-2 (2019-nCOV) Spikes1 antibody level in mouse serum samples.
The detection principle was as follows: an indirect ELSA method was used to coat novel coronavirus S1 protein on a solid phase carrier, and a sample to be detected was added for incubation. The anti-novel coronavirus antibody in the sample was combined with the protein on the solid phase carrier, and after plate washing, the anti-mouse IgG antibody labeled with horseradish peroxidase (HRP) was added to form an antigen-antibody-enzyme labeled antibody complex. After coloration with the substrate TMB, TMB is converted to blue under the catalysis of peroxidase and finally yellow under the action of acid, and the absorbance (OD value) at 450 m is read, and the OD value at 450 m is positively correlated with the anti-novel coronavirus antibody content in the sample.
Calculate an enzyme label strip required for detect a sample, taking out that enzyme label strip from the aluminum foil bag, putting the rest of the enzyme label strip back into the aluminum foil bag and seal the bag mouth, preserving at low temperature, washing the plate with 1*washing buffer solution and soaking Imin at 300 mu/hole, patting the enzyme label plate for the next cleaning, adding the sample to be detected into the plate for five times of washing, diluting serum with 1*dilution buffer solution at 1:2000, incubating at 100/hole for 2 hours at room temperature, discarding the liquid in the hole, patting the enzyme label plate for drying, washing the plate with 1*washing buffer solution and soaking at 300 uL/hole for 1 min, and patting for drying The anti-mouse IgG antibody, −HRP1:100, was diluted with 1× dilution buffer, and 100 μl/well was added to the ELISA plate, mixed, and incubated for 1 hour at room temperature. The liquid in the wells was discarded, and the ELISA plate was pat dry. The plate was washed with 1× washing buffer solution, and Imin was immersed in 300 μl/well. The ELISA plate was pat dry for the next washing, and the plate was washed five times in total. The pre-prepared substrate solution was added into the ELISA plate at 200 μl/well, mixed evenly, and incubated for 20 minutes at room temperature in the dark. Add 50/well stop solution to the ELISA plate and gently shake the plate until uniform color development is achieved. Read the light absorption value for OD450 mm.
The test results are shown in
Ten SPF-grade mice (6-8 weeks of age) were randomly divided into two groups, five for each group. Mice were immunized with psAd-S vaccine according to the groupings shown in Table 5. The injection method was as follows: intramuscular injection was performed on the medial aspect of the posterior thigh. Injection dose: 100 μl.
The mice were sacrificed 14 days after immunization, and the splenic lymphocytes were isolated. BHK21 cells transfected with the shuttle plasmid pSsAdE1-WPRE-S were stimulated and cultured for 6 hours, and cytokine secretion was blocked by adding a protein secretion blocker. After 6 hours, Fc receptors were blocked, dead cells and cell surface molecular markers were stained, and intracellular cytokines were stained after the cells were fixed and perforated. Cell surface markers included CD4 and CD8, and intracellular cytokines included IFNγ, IL2 and TNF. The expression levels of IFNγ, IL2 and TNF in CD4+T cells and CD8+T cells stimulated by the target protein were analyzed by flow cytometry (CyExpert).
psAd-S-induced CD8+T-cell and CD4+T-cell immune responses are shown in
SARS-COV-2 pseudovirus neutralization test kit is purchased from Nanjing Kingsri Biotechnology Co., Ltd. on the principle that the envelope glycoprotein in the lentivirus vector is replaced by Covid-19 S protein to form a pseudovirus simulating Covid-19 infection. Pseudoviruses infect target cells through surface S proteins and express reporter luciferase genes. Neutralizing agents (such as antibodies) can block the binding of S protein and ACE2, thereby preventing pseudovirus infection of the host cells. By detecting the expression level of the reporter gene luciferase, the degree to which the virus is blocked can be deduced for screening or verification of the neutralizing agent.
The experimental steps were as follows: The mouse serum, positive control and negative control collected in the cell immune response test were diluted and mixed with the diluted HRP-RBD in a 1:1 manner and incubated at 37 C for 30 minutes. Incubate 100 U of the mixture in the corresponding well plate for 15 minutes at 37 C; Wash the well plates 4 times with 260 μl of 1× Wash buffer; Then 100 μl TMB buffer was added and incubated for 15 min at 20-25 C in the dark. Adding 50 μl of termination buffer; OD450 reads the absorbance. The result are shown in table 6:
Among them: quality control: positive control <0.3, negative control >1.0; Inhibition rate=(1−sample OD value/negative control OD value)*100%; Judgement: Positive inhibition rate ≥20% and negative inhibition rate <20%.
In summary, after mice were injected with the total dose of 10{circumflex over ( )}8 FFU vaccine (diluted with PBS buffer) and 10{circumflex over ( )}7 FFU (diluted with PBS buffer), they could induce strong humoral and cellular immune responses, which were significantly different from those in the control group. At the same time, the collected serum can play a blocking role in the simulated neutralization antibody experiment. The results showed that our psad-S vaccine could protect Covid-19, which had guiding significance for the development of vaccine in COVID-19.
Although the present invention has been disclosed as above, the present invention is not limited thereto. Various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present invention, and the scope of the present invention should be determined by the scope of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010642745.3 | Jul 2020 | CN | national |
| 202010945335.6 | Sep 2020 | CN | national |
The present application claims priority to a U.S. National Stage of International Application No. PCT/CN2021/104641 filed on Jul. 6, 2021, a Chinese prior application No. 2020106427453 and filed on Jul. 6, 2020, and a Chinese prior application No. 202010945335.6 and filed on Jul. 6, 2020; the entire contents of all of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/104641 | 7/6/2021 | WO |
