Vector system for packaging recombinant poxviruses and use thereof

Abstract

Disclosed in the present invention is a vector system for packaging recombinant poxviruses. The vector system comprises a first plasmid, a second plasmid, a third plasmid, a fourth plasmid, and a fifth plasmid which respectively carry five segments that are obtained by sequential segmentation on the basis of the full-length genome of a poxvirus, wherein the fifth plasmid carries an inverted terminal repeats RITR fragment and LITR fragment of the genome of the poxvirus, and each plasmid has, at two ends thereof, overlapping fragments which perform homologous recombination with adjacent plasmids, wherein the 5' end of the first plasmid is adjacent to the 3' end of the fifth plasmid, and the 3' end of the fourth plasmid is adjacent to the 5' end of the fifth plasmid. Also disclosed in the present invention are a method for using the recombinant poxvirus vector system to package recombinant poxviruses, and a use of the recombinant poxvirus vector system in preparation of a poxvirus vector vaccine. The recombinant poxviruses have the effects of large load capacity and good immunization effect stimulation.

Description

A vector system for packaging recombinant poxviruses and its application Technical Field

[0001] This invention relates to a vector system for packaging recombinant poxviruses, particularly viral vectors in which exogenous antigens are inserted into various backbone plasmids V1, V2, V3, and V4, and their applications. These systems can be used in research on human poxvirus vaccines, poxvirus epidemiology, and poxvirus vector vaccines. Background Technology

[0002] The vaccinia virus genome is 130-375 kb in length, with 10 kb inverted terminal repeats (ITRs) at both ends. The genome is huge and complex, encoding more than 200 proteins.

[0003] In 1982, vaccinia virus was first used to express exogenous genes, and it has since become a popular expression system, gradually expanding to attenuated variants of vaccinia virus and other poxvirus strains such as fowlpox virus. Modified Vaccinia virus Ankara (MVA, ATCC accession VR-1508) is an attenuated variant produced by passaged more than 570 times of the replicating vaccinia virus Ankara in chicken embryo fibroblasts. During continuous passage, approximately 12% of the genome is lost, thus preventing replication in most mammalian cells. Today, MVA has been developed as a third-generation smallpox vaccine and, like canarypox virus, is frequently used as a recombinant poxvirus vaccine vector in research. Compared to other viral vector systems, the MVA poxvirus vector has advantages such as a large capacity for inserting exogenous genes, no safety risks associated with intracytoplasmic replication, high exogenous gene expression levels, and the ability of the expression product to induce a prolonged humoral and cellular immune response. In 2020, Janssen Pharmaceuticals' Ebola vaccine Ad26.ZEBOV and MVA-BN-Filo two-dose immunization regimen were approved for marketing by the European Commission. In 2022, the MVA-BN-RSV vaccine, developed based on the MVA-BN platform and containing five different respiratory syncytial virus antigens, received Breakthrough Therapy Designation from the US FDA for active immunization in people aged 60 and older. This vaccine received Phase III clinical trial IND approval in China in November 2022.

[0004] Poxvirus vectors possess large genome sequences, capable of accommodating foreign gene insertions up to 25 kb. Most gene engineering strategies targeting poxviruses involve transfecting DNA fragments with homologous segments at both ends of the insertion site into poxvirus-infected cells to induce homologous recombination. This is a natural process during poxvirus replication, but homologous recombination requires a complex selection process. Therefore, a rapid poxvirus packaging system is needed that facilitates the insertion of foreign gene fragments and rapidly yields recombinant poxviruses. The purpose of this invention is to provide a plasmid based on synthetic biology research methods, consisting of five MVA poxvirus genome fragments assembled from chemically synthesized poxvirus DNA fragments. Transfecting cells with this plasmid can rapidly yield recombinant poxviruses and can be used for rapid antigen insertion and efficient gene editing in recombinant poxvirus or related vector vaccines. Technical solutions

[0005] To achieve the above objectives, this invention first provides a recombinant MVA vaccinia virus vector five-plasmid system (MVA-Fast). This recombinant vaccinia virus vector system includes plasmids 1, 2, 3, 4, and 5, each carrying a sequentially segmented vaccinia virus genome based on the full-length genome. Plasmid 5 carries an inverted terminal repeat (RITR) fragment and a LITR fragment from the vaccinia virus genome. Each plasmid has overlapping segments at both ends that allow for homologous recombination with adjacent plasmids. Specifically, the 5' end of plasmid 1 is adjacent to the 3' end of plasmid 5, and the 3' end of plasmid 4 is adjacent to the 5' end of plasmid 5. In this invention, plasmids 1, 2, 3, 4, and 5 are named V1, V2, V3, V4, and V5, respectively.

[0006] In a preferred embodiment, the poxvirus is the Modified Vaccinia virus Ankara (MVA) strain, and the ATCC accession number of the MVA strain is VR-1508.

[0007] In a more preferred embodiment, the first, second, third, and fourth plasmids each carry a segment of four sequentially cut segments based on the full-length vaccinia virus genome (Genbank: U94848.1), each with a length between 30 kb and 64 kb.

[0008] Preferably, the first, second, third, and fourth plasmids carry four segments sequentially segmented from the full-length vaccinia virus genome, with lengths of approximately 47kb, 64kb, 43kb, and 34kb, respectively. These segments are located between 9730 bp and 56760 bp, 52893 bp and 106124 bp, 102100 bp and 144689 bp, and 134789 bp and 168360 bp, respectively, within the vaccinia virus genome. The LITR and RITR fragments are approximately 9.8kb each, with the LITR located between 1 bp and 9794 bp, and the RITR located from 168295 bp to the end, being inversely complementary to the LITR. V1 and V2 share approximately 3.8kb of homologous region (52893~56760 bp), and V2 and V3 share 4kb of homologous region (102100~106124 bp). V3 and V4 share a 9.9kb homologous region (134789~144689 bp), while V5 shares a 65bp (9730~9794 bp) homologous region with V1 and a 66bp (168295~168360 bp) homologous region with V4.

[0009] More preferably, the length of the homologous region segment adjacent to the first plasmid and the fifth plasmid is 65 bp (sequence shown in SEQ ID NO.1), and the length of the homologous region segment adjacent to the fourth plasmid and the fifth plasmid is 66 bp (sequence shown in SEQ ID NO.2).

[0010] In another preferred embodiment, the first, second, third, and fourth plasmids each have either a built-in or introduced restriction enzyme sites for inserting foreign genes.

[0011] In a more preferred embodiment, the first, second, third, and fourth plasmids respectively have or have introduced restriction enzyme sites for inserting foreign genes: EagI, AsisI, PacI, and EagI.

[0012] In a preferred embodiment, plasmids 1, 2, 3, and 4 are all constructed using BACYAC plasmid as the starting plasmid, and plasmid 5 is constructed using PET plasmid as the starting plasmid. In this invention, plasmids 1, 2, 3, 4, and 5 are also named BACYAC-F01-F10 (V1), BACYAC-F10-F21 (V2), BACYAC-F21-F30 (V3), BACYAC-F29-F35 (V4), and PET-LITR-RITR (V5), respectively.

[0013] In one embodiment of the invention, each poxvirus genome fragment is linked to the BACYAC backbone plasmid via AsisI and AscI single-restriction sites, allowing for linearization via enzyme digestion. Each adjacent linearized fragment shares homologous regions. When the fragment is transfected into cells infected with helper poxviruses, the fragments can undergo sequential homologous recombination in the host cells, splicing together to form the complete MVA poxvirus genome sequence. In some embodiments, the helper poxvirus may be fowlpox virus, vaccinia virus, or rabbit fibroma virus.

[0014] Secondly, this invention provides a method for constructing the above-mentioned recombinant vaccinia virus vector system, the method comprising the following steps:

[0015] (1) The genome segments covered by plasmids V1 to V4 were divided into 7 to 12 5kb sub-segments, and the genome segment covered by V5 was divided into two sub-segments, LITR and RITR, and synthesized separately. Homologous recombination regions were introduced into the sub-segments adjacent to the plasmid (BACYAC plasmid or PET plasmid), and restriction enzyme sites (AscI and AsisI) for linearization were introduced. Homologous recombination was used, and the V1 to V4 plasmids and BACYAC plasmid were co-transfected into competent yeast cells. Circular closed plasmids were obtained by homologous recombination in the host cells. The V5 plasmid was obtained by in vitro ligation of the two fragments contained therein with the PET plasmid using Gibson and transformation into E. coli.

[0016] (2) Linearized plasmids V1~V5;

[0017] (3) Linearized plasmids V1~V5 were transfected into BHK-21 cells infected with fowlpox virus FPV;

[0018] (4) Maintain culture for 3-5 days, freeze and thaw the cell culture medium, and passage continuously until the cells become persistently diseased to obtain stable passaged recombinant MVA poxvirus.

[0019] Third, this invention provides the application of the above-mentioned recombinant vaccinia virus vector system in the preparation of vaccinia virus vaccines. In this application, an antigen gene expression cassette is inserted as a foreign gene into the restriction enzyme site of the above-mentioned foreign gene, for example, EagI of plasmid 1, AsisI of plasmid 2, PacI of plasmid 3, or EagI of plasmid 4. This allows for the packaging of a recombinant vaccinia virus carrying the antigen gene into host cells with the assistance of a helper virus. The vaccinia virus can then be prepared as a vaccine to induce a specific immune response against the antigen.

[0020] In one specific embodiment of the present invention, an expression cassette of the HA protein of influenza virus H1N1 was inserted into the PacI restriction enzyme site inherent in the third plasmid, and an MVA-HA recombinant vaccine was prepared by packaging recombinant vaccinia virus using a recombinant vaccinia virus vector system.

[0021] Finally, the present invention provides a method for packaging recombinant vaccinia virus using the above-described recombinant vaccinia virus vector system, the method comprising the following steps:

[0022] (1) Insert the foreign gene into the non-essential regions of viral replication in plasmids V1-V4 of the recombinant vaccinia virus vector system;

[0023] (2) Transfect the recombinant poxvirus vector system obtained in step (1) into the host cell, and at the same time assist the virus in infect the host cell;

[0024] (3) Culture the host cells obtained in step (2);

[0025] (4) Identify and obtain recombinant poxviruses packaged with exogenous genes.

[0026] In a preferred embodiment, the non-essential viral replication region in step (1) is located between F14L and F15L of the MVA gene (37266 bp in Genbank:U94848.1), between Thymidine nucleoside kinase TK gene region (75560 bp~76093 bp in Genbank:U94848.1), between A26L and A27L of the MVA gene (130950 bp in Genbank:U94848.1), and between hemagglutinin HA gene region (149416 bp~150363 bp in Genbank:U94848.1).

[0027] In one specific embodiment of the present invention, a gene fragment “Loxp-p7.5-eGFP-Loxp” (SEQ ID NO:3) is inserted into the TK gene region of F15, which can be used to express eGFP.

[0028] In a more preferred embodiment, the helper virus in step (2) is fowlpox virus (FPV). This invention provides five plasmids assembled from five artificially synthesized MVA genome fragments: BACYAC-F01-F10 (V1), BACYAC-F10-F21 (V2), BACYAC-F21-F30 (V3), BACYAC-F29-F35 (V4), and PET-LITR-RITR (V5), which contain the entire MVA genome. When packaging the recombinant MVA virus, the five linearized fragments are transfected into BHK-21 cells infected with fowlpox virus. The virus is harvested three days after transfection, and progeny viruses are obtained through continuous passage in BHK-21 cells. The recombinant MVA poxvirus obtained using this method exhibits characteristics consistent with the wild-type virus, including peak titer, growth curve, host range, and plaque size. Beneficial effects

[0029] The MVA-Fast poxvirus vector system constructed in this invention comprises five backbone plasmids. The non-essential regions for poxvirus replication in plasmids V1-V4 contain single restriction enzyme sites for the insertion of foreign genes. The foreign genes are directly embedded into the restriction enzyme sites of the backbone plasmids via a one-step in vitro recombination process, yielding full-length recombinant plasmids for packaging. This system greatly leverages the high capacity advantage of the MVA poxvirus vector, providing a rapid, efficient, multi-site gene editing, and multi-regional genome modification vector tool for viral genomes. It shortens the preparation cycle of recombinant poxviruses. During virus packaging, a highly safe and distantly related fowlpox virus is used as an auxiliary virus, avoiding non-specific recombination. It is fast, efficient, low-cost, and can be used for batch preparation, exhibiting significant application advantages. It is an ideal vector system for preparing recombinant poxviruses. Attached Figure Description

[0030] Figure 1 shows a schematic diagram of the MVA-Fast system recombinant plasmid. The wild-type MVA genome (U94848.1) was divided into 37 DNA segments and synthesized. These segments were then recombined using TAR to form BACYAC-F01-F10 (V1), BACYAC-F10-F21 (V2), BACYAC-F21-F30 (V3), and BACYAC-F29-F35 (V4). PET-LITR-RITR (V5) was then recombined using Gibson. The vector regions and fragments were linked by AsisI and AscI restriction sites. The V1 to V5 fragments shared homologous regions of 65 bp, 3.8 kb, 4.0 kb, 9.9 kb, and 66 bp, respectively.

[0031] Figure 2 shows a schematic diagram of the BACYAC-F01-F10 (V1) plasmid. V1 is formed by linking chemically synthesized F01~F10 with the BACYAC plasmid via AscI and AsisI. The EagI single enzyme restriction site of F06 is located between F14L and F15L of the MVA gene (37266 bp), which can be used for the insertion of foreign genes.

[0032] Figure 3 shows a schematic diagram of the splicing of the BACYAC-F10-F21 (V2) plasmid. V2 was obtained by splicing chemically synthesized F10~F21 with the BACYAC-Loxp-p7.5-eGFP-Loxp plasmid. BACYAC and Loxp-p7.5-eGFP-Loxp are linked by homologous primers with added AsisI, which can be used for the insertion of foreign genes. F10 and F21 are linked by AscI for plasmid linearization. The "Loxp-p7.5-eGFP-Loxp" gene fragment can be used to express eGFP.

[0033] Figure 4 shows a schematic diagram of the splicing of the BACYAC-F21-F30 (V3) plasmid. V3 is formed by linking the chemically synthesized F21~F30 plasmids with the BACYAC plasmid via AscI and AsisI. The PacI single enzyme restriction site of F28 is located between A26L and A27L of the MVA gene (130950 bp), which can be used for the insertion of foreign genes.

[0034] Figure 5 shows a schematic diagram of the splicing of the BACYAC-F29-F35 (V4) plasmid. V4 is formed by linking chemically synthesized F29~F35 plasmids with the BACYAC plasmid via AscI and AsisI. The EagI single enzyme restriction site of F32 is located in the hemagglutinin HA gene region (149416 bp~150363 bp), which can be used for the insertion of foreign genes.

[0035] Figure 6 is a schematic diagram of the PET-LITR-RITR (V5) plasmid assembly. V5 is composed of chemically synthesized LITR and RITR connected to a PET vector via AsisI and AscI.

[0036] Figure 7 shows the agarose gel electrophoresis images of the five plasmids. BACYAC-F01-F10 (V1), BACYAC-F21-F30 (V3), BACYAC-F29-F35 (V4), and PET-LITR-RITR (V5) were linearized using AsisI and AscI, and BACYAC-F10-F21 (V2) was linearized using AscI. The enzyme digestion results were detected by agarose gel electrophoresis.

[0037] Figure 8 illustrates the process of packaging MVA poxvirus into MVA using the MVA-Fast system. BHK-21 cells infected with fowlpox virus FPV were transfected with plasmids V1-V5 linearized with AsisI and AscI, and recombinant MVA poxvirus was packaged through homologous recombination of DNA fragments containing homologous regions.

[0038] Figure 9 shows microscopic images of eGFP fluorescent protein expression observed after continuous passage of recombinant MVA poxvirus packaged by the MVA-Fast system in BHK-21 cells, as well as electron microscopy results of MVA-syn isolated and purified from sucrose in BHK-21 cells.

[0039] Figure 10 shows the PCR identification results of recombinant MVA poxvirus.

[0040] Figure 11 shows the growth curves of MVA-WT and three MVA virus plaque screening strains MVA-syn1, MVA-syn4, and MVA-syn-5 obtained by packaging using the method described in this invention on BHK-21 and DF-1 cells. MVA-syn and MVA-WT showed similar growth performance in BHK-21 and DF-1 cells.

[0041] Figure 12 shows the plaque characteristics of MVA-WT and MVA-syn1, MVA-syn4, and MVA-syn5. The plaques produced by MVA-syn and MVA-WT after infecting BHK-21 cells were similar in size, indicating that MVA-syn and MVA-WT have similar infection characteristics in the same allowable cells.

[0042] Figure 13 is a schematic diagram of the MVA-HA recombinant vaccine packaged using the MVA-Fast system. Using poxvirus mH5 as the promoter and the HA protein of influenza virus H1N1 as the antigen, the constructed complete protein expression cassette was inserted into the PacI site of the BACYAC-F21-F30 (V3) plasmid to obtain the recombinant V3-HA plasmid. The recombinant V3-HA plasmid was linearized by AsisI and AscI and co-transfected with other linearized four plasmids into FPV-infected BHK-21 cells infected with fowlpox virus FPV. The recombinant MVA-HA vaccine was packaged by homologous recombination of DNA fragments containing homologous regions.

[0043] Figure 14 shows the results of MVA-HA genome PCR identification and protein expression identification. The target protein gene sequence was successfully inserted into the MVA poxvirus genome, and HA protein expression was detected in BHK-21 cells infected with MVA-HA at MOI=1 for both 12 h and 24 h.

[0044] Figure 15 shows the antibody detection results after immunization of BALB / c mice with the recombinant MVA-HA candidate vaccine. MVA-HA was administered twice, on day 0 and day 21, via intramuscular injection and subcutaneous injection in the neck and back of the neck, with 100 μL injected each time. A high-dose group (5 × 10⁶) was established based on the immunization dose. 7 IFU and low-dose group 5×10 6 Two groups of IFU were used. The serum-specific antibody levels induced by the candidate vaccine in mice were detected by ELISA at 21 days (before the second dose) and 28 days (7 days after the second dose). Embodiments of the present invention

[0045] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0046] Unless otherwise specified, the starting plasmids, enzymes, and related reagents used in the following examples are all available from commercially available companies, and the primers used are synthesized by synthetic companies.

[0047] Example 1. Construction of the recombinant MVA poxvirus vector five-plasmid system (MVA-Fast)

[0048] The poxvirus vector five-plasmid system (MVA-Fast) contains the entire MVA genome. The MVA genome U94848.1 was divided into 37 chemically synthesized DNA fragments. The location, length, and shared homologous regions of each DNA fragment are shown in Table 1. Plasmids V1 to V4 were linked with multiple adjacent fragments to the BACYAC plasmid (purchased from Invitrogen) via yeast transformation-associated recombination (TAR recombination). TAR recombination is currently the most efficient method for assembling large DNA fragments and has a significant advantage in synthesizing ultra-large genomes. By designing genomic DNA with homologous arms to form a circular plasmid with a yeast artificial chromosome, the recombinant plasmid can replicate autonomously in yeast and E. coli. During recombination, it does not need to integrate into the yeast chromosome. It can be separated from the yeast chromosome and transfected into E. coli for replication. The E. coli plasmid was extracted to obtain a large number of recombinant plasmids. LITR and RITR were spliced ​​using the Gibson method (kit purchased from NEB). The kit contains T5 exonuclease, DNA polymerase, and DNA ligase. T5 exonuclease digests the synthesized DNA fragment or fragments excised from the plasmid, producing 3'-single-stranded overhangs. The exposed homologous sequences are specifically annealed, and the exonuclease is gradually inactivated by heat. Then, DNA polymerase fills in the gaps and removes the single-stranded overhangs. Finally, DNA ligase ligates the grown DNA molecules (see Figure 1 for a schematic diagram). This method is convenient, rapid, and efficient, achieving seamless splicing.

[0049] Table 1. Information on fragment locations, lengths, and shared homologous region locations of MVA genome segments.

[0050]

[0051] 1. Construction of BACYAC-F01-F10 (V1)

[0052] BACYAC-F01-F10 (V1) is 56.393 kb in length and contains MVA genome (U94848.1) fragments from 9730 bp to 56760 bp. It was synthesized by homologous recombination of 10 chemically synthesized MVA genome fragments (F01~F10) with the BACYAC plasmid, co-transfected into VL6-48N yeast competent cells (purchased from Shanghai Zeye Biotechnology Co., Ltd.). The homologous regions of F01 and BACYAC, as well as the AsciI restriction site, were provided using the PYE-Link-F01 primer pair, namely PYE-Link-F01-F (SEQ ID NO. 4) and PYE-Link-F01-R (SEQ ID NO. 5). The homologous regions of F10 and BACYAC, as well as the AsciI restriction site, were provided using the F10-Link-PYE primer pair, namely F10-Link-PYE-F (SEQ ID NO. 5). NO.6) and F10-Link-PYE-R (SEQ ID NO.7).

[0053] The specific method for TAR recombination is as follows: 100 ng of purified linearized BACYAC vector, 200 ng of purified DNA fragments (F01~F10) after 10 PCR amplifications, 500 ng of 2 pairs of primers, and 20 μL of Yeastmaker Carrier DNA were added to 200 µL of VL6-48N yeast competent cells (purchased from Shanghai Zeye Biotechnology Co., Ltd.). The mixture was gently mixed, followed by 800 μL of PEG / LiAc. The cells were incubated at 30℃ for 45 min, with gentle shaking every 15 min. 160 µL of fresh DMSO was added, and the cells were incubated at 42℃ for 20 min, with gentle shaking every 10 min. The cells were centrifuged at 700 g for 5 min at room temperature. The cells were resuspended in 3 mL of YPD Plus medium (purchased from Beijing Cooler Master Technology Co., Ltd.), cultured on a shaker at 30℃ for 90 min, centrifuged at 700 g for 5 min at room temperature, and the supernatant was discarded. The cells were resuspended in 500 μL of 0.9% (w / v) NaCl solution. 100 μL of the resuspended cells were then collected. µL was plated on SD-trp agar plates (purchased from Takara) and incubated at 30°C for 3–5 days. Yeast DNA mixtures that were positive for PCR identification from yeast colonies were extracted and electrically stimulated to transform DH10B competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.). A large number of recombinant plasmids were obtained through plasmid extraction. AsisI and AscI restriction sites were introduced at the intersection of homologous arms. During virus packaging, the BACYAC plasmid was linearized with the single restriction sites AsisI and AscI connected to both ends of the genome (see Figure 2).

[0054] 2. Construction of BACYAC-F10-F21 (V2)

[0055] BACYAC-F10-F21 (V2) is 63.724 kb in length and contains MVA genome (U94848.1) fragments from 52893 bp to 106124 bp. It is formed by homologous recombination of 12 chemically synthesized MVA genome fragments (F10~F21) and BACYAC plasmids in VL6-48N yeast competent cells. The homologous regions of F21 and F10 and the AscI restriction site are provided by the primer pair F21-LINK-F10, namely F21-LINK-F10-F (SEQ ID NO.8) and F21-LINK-F10-R (SEQ ID NO.9).

[0056] The homologous regions of F14 and BACYAC are provided by primer pairs of F14-LINK-PYE, namely F14-LINK-PYE-F (SEQ ID NO.10) and F14-LINK-PYE-R (SEQ ID NO.11).

[0057] The homologous regions of F15 and BACYAC are provided by the F15-LINK-PYE primer pair, F15-LINK-PYE-F (SEQ ID NO.12) and F15-LINK-PYE-R (SEQ ID NO.13). In this pair, the TK gene region of F15 contains an insertion of the sequence "Loxp-p7.5-eGFP-Loxp" at 275 bp. The sequence is shown in SEQ ID NO. NO:3, the poxvirus early and late promoter P7.5 is added in front of eGFP. The two loxP sites are located at both ends of the eGFP expression cassette and are oriented in the same direction. The sequence between LoxP can be knocked out by Cre recombinase. One end of BACYAC is linked to F14, and the other end is linked to the "Loxp-p7.5-eGFP-Loxp" sequence through the single restriction site AsisI. The single restriction site AsisI introduced by the primer pair that provides the homologous region can be used for the insertion of foreign gene expression cassette. F10 and F21 are linked head to tail. When packaging the virus, the F10 and F21 linkage site is cut by AscI to linearize the plasmid restriction and expose the homologous fragment (see Figure 3).

[0058] 3. Construction of BACYAC-F21-F30 (V3)

[0059] BACYAC-F21-F30 (V3) is 51.952 kb in length and contains MVA genome (U94848.1) fragments from 102100 bp to 144689 bp. It is assembled from 10 chemically synthesized (F21~F30) MVA genome fragments and BACYAC plasmids via TAR recombination.

[0060] The homologous regions of F21 and BACYAC are provided by primer pairs of F21-Link-PYE, namely F21-Link-PYE-F (SEQ ID NO.14) and F21-Link-PYE-R (SEQ ID NO.15).

[0061] The homologous regions of F30 and BACYAC are provided by primer pairs F30-Link-PYE, namely F30-Link-PYE-F (SEQ ID NO.16) and F30-Link-PYE-R (SEQ ID NO.17). One end of BACYAC is linked to F21 via AsisI, and the other end is linked to F30 via AscI. During virus packaging, the BACYAC plasmid is linearized through the single restriction enzyme sites AsisI and AscI linked to both ends of the genome (see Figure 4).

[0062] 4. Construction of BACYAC-F29-F35 (V4)

[0063] BACYAC-F29-F35 (V4) is 42.934 kb in length and contains MVA genome (U94848.1) fragments from 102,100 bp to 144,689 bp. It is assembled by TAR recombination of seven chemically synthesized MVA genome fragments (F29~F35) with the BACYAC plasmid. The homologous regions of F29 and BACYAC are provided by the primer pairs F29-Link-PYE, namely F29-Link-PYE-F (SEQ ID NO.18) and F29-Link-PYE-R (SEQ ID NO.19).

[0064] The homologous regions of F35 and BACYAC are provided by the PYE-Link-F35 primer pair, F35-Link-PYE-F (SEQ ID NO.20) and F35-Link-PYE-R (SEQ ID NO.21). One end of BACYAC is linked to F29 via AsisI, and the other end is linked to F35 via AscI. During virus packaging, the BACYAC plasmid is linearized through the single restriction enzyme sites AsisI and AscI linked to both ends of the genome (see Figure 5).

[0065] 5. Construction of PET-LITR-RITR (V5)

[0066] PET-LITR-RITR (V5) was assembled using Gibson splicing. V5 was formed by ligating chemically synthesized F36 (LITR) and F37 (RITR) with a PET plasmid (preserved in our laboratory) via AsisI and AscI. A 21bp sequence was added to the 3' end of the chemically synthesized RITR sequence to the 5' end of the LITR sequence to provide the necessary homologous region for homologous recombination during Gibson splicing. Linearized fragments of LITR and RITR were prepared and linearized using reserved single-restriction enzyme sites XmaI and AsisI at the ends of the chemically synthesized LITR and RITR genes. The PET vector fragment was amplified. The primers for adding the 3' homologous arm of LITR to the PET vector fragment were PET-LITR-F (SEQ ID NO. 22), and the primers for adding the 5' homologous arm of RITR were PET-RITR-R (SEQ ID NO. 23). The amplified product length was 2540. After purification of the fragments obtained from the above enzyme digestion and PCR amplification, the Gibson recombination system (NEB) was used for splicing. The specific method was as follows: 50 ng of purified vector fragment, approximately 100 ng of multiple purified fragments for integration (molar ratio of 2:1 with the vector), 10 μL of Gibson Mix assembly solution, and 20 μL of sterile water were added. The mixture was incubated at 50°C for 15 min, followed by an ice bath for about 3 min. 3 μL of the recombination system was transformed into 50 μL of TOP10 chemocompetent cells, incubated for 40-60 min, plated, and cultured overnight. After positive colony identification, the recombinant plasmid was extracted. After splicing, AsisI and AscI restriction sites were introduced at the intersection of homologous arms for plasmid linearization (see Figure 6).

[0067] This invention provides five plasmids assembled from five artificially synthesized MVA genome fragments, including BACYAC-F01-F10 (V1), BACYAC-F10-F21 (V2), BACYAC-F21-F30 (V3), BACYAC-F29-F35 (V4), and PET-LITR-RITR (V5), which contain the entire MVA genome (see Figures 2-6).

[0068] Figure 7 shows the agarose gel electrophoresis images of the five plasmids. BACYAC-F01-F10 (V1), BACYAC-F21-F30 (V3), BACYAC-F29-F35 (V4), and PET-LITR-RITR (V5) were linearized using AsisI and AscI, and BACYAC-F10-F21 (V2) was linearized using AscI. The enzyme digestion status was detected using 1% agarose gel, and the lengths of the obtained plasmids were consistent with expectations.

[0069] Example 2. Packaging of recombinant MVA-synpoxvirus

[0070] Figure 8 shows a schematic diagram of the packaging process of this invention. Linearized plasmids V1-V5 were transfected into BHK-21 cells infected with fowlpox virus (FPV). Recombinant MVA poxvirus was packaged through homologous recombination of DNA fragments containing homologous regions. Specifically, BHK-21 cells (preserved in our laboratory) were selected as the packaging cell line. After infecting BHK-21 cells (preserved in our laboratory) with FPV at an MOI of 0.1-1 for 1 hour, the linearized fragments were transfected into the BHK-21 cells using Turbofect transfection reagent. The cells were maintained in culture for 3-5 days, and the cell culture medium was frozen and thawed. The cells were then continuously passaged until persistent cytopathic effects occurred, thus obtaining stably passaged recombinant MVA-syn virus. We completed the packaging of recombinant MVA-syn poxvirus using the MVA-Fast vector system. Infection of BHK-21 cells with this MVA-syn recombinant virus revealed sustained eGFP expression (Figure 9A). Electron microscopy was used to examine the structure of the recombinant virus particles. The diameter of the virus particles ranged from 220 to 450 nm, and they had a typical oval or brick-like structure, which was consistent with the expected results (Figure 9B). This indicates that the system can obtain recombinant MVA-synpoxvirus with normal structure and stable expression of exogenous genes.

[0071] Example 3. PCR identification results of recombinant MVA poxvirus

[0072] To identify the recombinant MVA-syn poxvirus genome, five recombinant MVA-syn poxvirus single clones (MVA-syn1~5) were isolated using plaque purification. The genomes of MVA-syn1~5, MVA-WT, and FPV fowlpox viruses were extracted using a viral genomic DNA / RNA rapid extraction kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.). Fragment-specific amplification primers were designed for BACYAC, F01, F06, F16, F26, F35, and FPV, respectively. Primers were used to detect the BACYAC plasmid (SEQ ID NO. 24-25), F01 (SEQ ID NO. 26-27), F16 (SEQ ID NO. 28-29), F26 (SEQ ID NO. 30-31), F35 (SEQ ID NO. 32-33), and MVA ITR (SEQ ID NO. 34-35). The PCR products were analyzed by agarose gel electrophoresis. The PCR results (see Figure 10) revealed that the recombinant MVA-syn poxvirus contained the BACYAC plasmid and a PCR fragment of the same length as MVA-WT, but did not contain the PCR fragment of FPV fowlpox virus. This indicates that the five plasmid fragments of the MVA-Fast system have been successfully recombined. The recombinant MVA-syn virus can be isolated from FPV helper virus through passage or plaque purification.

[0073] Example 4. Growth curve of recombinant MVA poxvirus

[0074] MVA poxviruses cannot replicate on most mammalian cells, but they can replicate on BHK-21 and DF-1 cells. Three PCR-positive recombinant MVA-syn poxviruses, MVA-syn1, MVA-syn4, and MVA-syn5, were randomly selected for comparison with MVA-WT. BHK-21 and DF-1 cells (purchased from Beina Biotechnology Co., Ltd.) were infected with MOI=0.01, and viral growth curves were plotted at 0, 12, 24, and 72 h to compare the growth characteristics of MVA-syn and MVA-WT. The growth curve results showed that MVA-syn and MVA-WT exhibited similar growth kinetics on BHK-21 and DF-1 cells (see Figure 11).

[0075] Example 5. Analysis of plaque characteristics of recombinant MVA poxvirus

[0076] MVA-WT and MVA-syn were used to infect monolayer BHK-21 cells in 6-well plates with an MOI of 0.001. After infection, the culture medium was discarded, the cells were washed three times with PBS, covered with low-temperature agar, and cultured at 37°C for 72 h. The culture medium was then discarded, and sufficient 4% PFA tissue cell fixation medium (purchased from Beijing Regen Biotechnology Co., Ltd.) was slowly added along the well wall. The cells were fixed at room temperature for 12 h, stained with crystal violet, and the well plates were scanned using a Celigo Nexcelom full-field cell analyzer. The plaque area was measured using the scanned digital images. The results showed that the plaques produced by MVA-syn and MVA-WT infection of BHK-21 cells were similar in size, and their ability to spread in the same allowable cells was similar (see Figure 12).

[0077] Example 6. Construction and characterization of recombinant MVA poxvirus vector MVA-HA vaccine

[0078] As shown in Figure 13, the HA protein expression cassette was inserted into the PacI site of the BACYAC-F21-F30 (V3) plasmid using the Gibson method. The specific method is as follows: the V3 plasmid was digested with PacI, and the digestion product was purified using ethanol precipitation (Takara). The HA protein expression cassette (sequence shown in SEQ ID NO:36) was amplified by PCR using primers HA-F (sequence shown in SEQ ID NO:37) and HA-R (sequence shown in SEQ ID NO:38). The two fragments were then assembled using the Gibson in vitro one-step recombination method (specific method shown in Example 1) to obtain the V3-HA recombinant plasmid with successfully inserted HA protein. The recombinant MVA-HA influenza vaccine was packaged using the MVA-Fast system (specific method shown in Example 2). The viral genomes of MVA-HA, MVA-WT, and MVA-syn4 were extracted (see Example 3 for specific methods). Primers HA-Part-F (sequence shown in SEQ ID NO:39) and HA-Part-R (sequence shown in SEQ ID NO:40) were designed to identify the HA protein gene. Agarose gel electrophoresis results of the PCR amplification products showed that MVA-HA successfully inserted the HA protein gene sequence (see Figure 14). BHK-21 cells were infected with MVA-HA at MOI=1, and cell samples were collected after 12 h and 24 h of culture. Western blot results showed that BHK-21 cells infected with MVA-HA could express the HA protein normally within 12 h and 24 h.

[0079] Example 7. Immunogenicity of MVA-HA vaccine

[0080] To determine the immunogenicity of the HA antigen vectored by MVA poxvirus, we used 5 × 10⁻⁶ cells / mL. 7 IFU / only 5×10 6Balb / c mice were immunized with IFU / mouse. The mice were immunized twice, once on day 0 and once on day 21, by intramuscular injection and once by subcutaneous injection in the neck and back, respectively, with a volume of 100 μL / mouse. Serum was collected on day 21 and day 28. The level of HA protein binding antibody was detected by ELISA. The specific method is as follows: HA protein (purchased from Sinocare Biotechnology Co., Ltd.) was diluted to 1 μg / mL and 100 μL / well was added to microplates. The plates were coated overnight at 4 ℃. After washing the plates three times with washing buffer, 100 μL / well of blocking buffer was added and the plates were blocked at 37 ℃ for 1 h. The liquid in the plates was discarded and the plates were washed three times with a plate washer. Serum samples were diluted with a specific initial dilution (1:100, 1.5 μL + 148.5 μL) and then serially diluted 4-fold. Seven dilutions were set for each sample. Twelve control wells without serum were set for each plate. After serum dilution, the plates were incubated at 37°C for 1 h. After washing, HRP-conjugated anti-mouse IgG secondary antibody (Santa Cruz, sc-2005) diluted 1:20000 was added and the plates were incubated for another 1 h. The plates were then developed using TMB single-component chromogenic buffer (solarbio, PR1200) for 6 minutes. 50 μL of 2M sulfuric acid was added to each well to stop the reaction. The absorbance at 450 nm was measured using a ELISA reader (MolecμLar Devices, Spectra Max). The antibody detection results (see Figure 15) showed that both intramuscular and subcutaneous immunizations after two doses stimulated high-titer antibody responses in mice, with a significant dose-dependent antibody response level, indicating that the antigen delivery using this vector can elicit excellent immunization effects.

Claims

1. A vector system for packaging recombinant poxvirus, characterized in that, The vector system for packaging recombinant vaccinia virus includes plasmids 1, 2, 3, 4, and 5, each carrying a segment of the full-length vaccinia virus genome sequentially divided into five regions. Plasmid 5 carries an inverted terminal repeat (RITR) fragment and a LITR fragment of the vaccinia virus genome. Each plasmid has homologous regions at both ends for homologous recombination with adjacent plasmids. The 5' end of plasmid 1 is adjacent to the 3' end of plasmid 5, and the 3' end of plasmid 4 is adjacent to the 5' end of plasmid 5.

2. The vector system for packaging recombinant poxvirus according to claim 1, characterized in that, The poxvirus in question is the Ankara strain of poxvirus, ATCC accession number VR-1508.

3. The vector system for packaging recombinant poxvirus according to claim 2, characterized in that, The first, second, third, and fourth plasmids each carry a segment of the vaccinia virus genome that is sequentially divided into four regions, each ranging in length from 30 kb to 64 kb.

4. The vector system for packaging recombinant poxvirus according to claim 3, characterized in that, The first, second, third, and fourth plasmids carry four segments, sequentially divided from the full-length vaccinia virus genome, with lengths of 47 kb, 64 kb, 43 kb, and 34 kb, respectively. These segments are located between 9730 bp and 56760 bp, 52893 bp and 106124 bp, 102100 bp and 144689 bp, and 134789 bp and 168360 bp, respectively. The LITR and RITR segments are both 9.8 kb, with the LITR segment located from 1 bp to 9794 bp and the RITR segment located from 168295 bp to the end, and are inversely complementary to the LITR segment.

5. The vector system for packaging recombinant poxvirus according to claim 4, characterized in that, The first plasmid shares a 3.8 kb homologous region with the second plasmid, the second plasmid shares a 4 kb homologous region with the third plasmid, and the third plasmid shares a 9.9 kb homologous region with the fourth plasmid. The sequences of the homologous regions of the first plasmid and the fifth plasmid are shown in SEQ ID NO.1, and the sequences of the homologous region fragment lengths of the fourth plasmid and the fifth plasmid are shown in SEQ ID NO.

2.

6. The vector system for packaging recombinant poxvirus according to claim 2, characterized in that, The first, second, third, and fourth plasmids each contain restriction enzyme sites for inserting foreign genes.

7. The vector system for packaging recombinant poxvirus according to claim 6, characterized in that, The first, second, third, and fourth plasmids contain restriction enzyme sites for inserting foreign genes: EagI, AsisI, PacI, and EagI, respectively.

8. A method for constructing a vector system for packaging recombinant poxvirus according to any one of claims 1-7, characterized in that, The method includes the following steps: (1) The genome segments covered by plasmids 1-4 were divided into 7-12 5 kb sub-segments, and the genome segment covered by plasmid 5 was divided into LITR and RITR sub-segments, which were synthesized separately. Homologous recombination regions were introduced into the sub-segments adjacent to the plasmids, and restriction enzyme sites for linearization were introduced. Using homologous recombination, plasmids 1-4 and BACYAC plasmid were co-transfected into competent yeast cells. Circular closed plasmids were obtained by homologous recombination in the host cells. Plasmid 5 was obtained by in vitro ligation of the two fragments contained therein with PET plasmid through Gibson and transformation into Escherichia coli. (2) Linearized plasmids 1-5 were digested with enzymes; (3) Linearized plasmids 1-5 were transfected into BHK-21 cells infected with fowlpox virus FPV. (4) Maintain culture for 3-5 days, freeze and thaw the cell culture medium, and passage continuously until the cells become persistently diseased to obtain stable passaged recombinant MVA poxvirus.

9. The use of the vector system for packaging recombinant poxvirus according to any one of claims 1-7 in the preparation of a poxvirus vector vaccine.

10. The application according to claim 9, characterized in that, The poxvirus vector vaccine is a poxvirus vector vaccine based on the HA protein of influenza virus H1N1.

11. A method for packaging recombinant vaccinia virus using the vector system for packaging recombinant vaccinia virus according to any one of claims 1-7, characterized in that, The method includes the following steps: (1) Inserting foreign genes into the non-essential regions of viral replication in a recombinant poxvirus vector system; (2) Transfect the recombinant poxvirus vector system obtained in step (1) into the host cell, and at the same time assist the virus in infect the host cell; (3) Culture the host cells obtained in step (2); (4) Identify and obtain recombinant poxviruses with inserted foreign genes.

12. The method according to claim 11, characterized in that, The non-essential region for viral replication described in step (1) is located between F14L and F15L of the MVA gene, the TK gene region of the thymidine nucleoside kinase, between A26L and A27L of the MVA gene, or the coding region of the hemagglutinin HA gene.

13. The method according to claim 11, characterized in that, The helper virus mentioned in step (2) is fowlpox virus.

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