Poxvirus-based vectors made from natural or synthetic DNA and their uses

Abstract

This invention provides a method for producing poxvirus-based vectors or recombinant poxvirus-based vectors. [Solution] A method is provided for producing a poxvirus-based vector or a recombinant poxvirus-based vector from naturally derived DNA fragments, chemically synthesized DNA fragments, or a combination thereof. One or more antigens, one or more DNA sequences encoding their subunits or fragments, or other heterogeneous gene sequences are inserted into one or more poxvirus insertion sites in one or more DNA fragments. The method includes transfecting one or more circular or linear DNA fragments into a host cell such that the poxvirus or recombinant poxvirus is reconstituted in the host cell and the reconstituted poxvirus or recombinant poxvirus contains the genome of a desired poxvirus.

Description

[Technical Field]

[0001] Priority Claim This application claims priority under U.S. Provisional Patent Application No. 62 / 969,628 filed 3 February 2020 and U.S. Provisional Patent Application No. 63 / 113,803 filed 13 November 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is incorporated in its entirety herein by reference. The ASCII copy, created on February 2, 2021, is filenamed 8200WO00_SequenceListing.txt and is 652 kilobytes in size. [Background technology]

[0003] background Poxviruses are large, double-stranded DNA viruses with an envelope that completely replicate in the cytoplasm of infected cells by encoding their own enzymes for DNA transcription and replication. 20、27 One of the greatest achievements in medical history was the eradication of the smallpox pathogen (varicella-zoster virus) through mass vaccination with replicable, attenuated vaccinia virus vaccine strains. Vaccinia viruses have been used as prototype members for studying poxvirus replication and have been further developed to produce recombinant vaccines and oncolytics.

[0004] Modified Vaccinia Ankara (MVA) is a highly attenuated orthopoxvirus obtained from the parent strain Chorioallantois Vaccinia Ankara (CVA) through 570 passages in chicken embryo fibroblasts (CEF). 1As a result of the attenuation process, MVA acquired six major genomic deletions (Del1-6) resulting in gene fragmentation, shortening, short internal deletions, and amino acid substitutions, as well as several shorter deletions, insertions, and point mutations. 2 While all of these mutations likely contribute to the highly attenuated phenotype of MVA, the exact genetic determinants associated with MVA assembly deficiency remain unclear. 2、3 MVA has a strictly limited host cell tropism, enabling productive assembly only in chicken cells, e.g., CEF cells and baby hamster kidney (BHK) cells; in human and most other mammalian cells, MVA assembly fails due to late-stage inhibition of viral assembly. 4、5 As demonstrated in various animal models and in humans, MVA is nonpathogenic and highly attenuated, yet retains excellent immunogenicity. 6、7 In the later stages of the smallpox eradication program, MVA was used as a priming vector for replicable vaccinia-based vaccines in over 120,000 individuals in Germany, with no adverse events reported. 8 For the past several decades, MVA has been developed as a standalone smallpox vaccine and is now being pursued by the U.S. government as a safer alternative to existing vaccinia-based vaccine stocks as a preventative measure in the event of a smallpox epidemic. 9~11 The FDA approved MVA under the brand name Jynneos (Bavarian Nordic) on September 24, 2019, to prevent both smallpox and monkeypox. Previously, the same MVA vaccine, under the brand name Imvamune, had been approved in Europe as a smallpox vaccine.

[0005] All MVA vectors and their derivatives currently in use are licensed or owned by academic, commercial, or governmental institutions, severely limiting their use in developing MVA-based vaccine vectors. Therefore, there is a need to develop alternative MVA vectors for various research, prophylactic, and therapeutic uses. Furthermore, novel technologies are urgently needed to accelerate the development of recombinant poxvirus vectors for pathogen preparedness and disease prevention. [Overview of the project]

[0006] overview In one aspect, the present disclosure relates to a method for producing a poxvirus vector or a recombinant poxvirus vector. The method comprises the step of transfecting one or more DNA fragments into a host cell, wherein the one or more DNA fragments include the entire genomic DNA sequence of the poxvirus so that a desired poxvirus is reconstituted in the host cell. In certain embodiments, two or more DNA fragments are co-transfected into a host cell, and each DNA fragment includes a partial sequence of the poxvirus genome so that the two or more DNA fragments are sequentially assembled by homologous recombination and, when reconstituted in the host cell, include the full-length sequence of the poxvirus genome. In certain embodiments, the method further comprises the step of infecting the host cell with a helper virus before, during, or after the transfection of one or more DNA fragments in order to initiate transcription of one or more DNA fragments. In certain embodiments, the helper virus is fowlpox virus (FPV), sheep fibroma virus, vaccinia virus, or cowpox virus. In certain embodiments, one or more DNA fragments are cyclized before transfection or transfected into host cells in a circular form. In certain embodiments, one or more DNA fragments are cloned in a plasmid vector or a bacterial artificial chromosome (BAC) vector. In certain embodiments, one or more DNA fragments are linearized before cotransfection or transfected into host cells in a linearized form. In certain embodiments, one or more DNA fragments are naturally occurring DNA fragments, chemosynthetic DNA fragments, or a combination of naturally occurring and chemosynthetic DNA fragments. In certain embodiments, the poxvirus genome sequence includes a sequence with modified vaccinia ankara (MVA) accession number #U94848 or #AY603355. In certain embodiments, the poxvirus genome sequence includes a sequence of vaccinia virus genome. In certain embodiments, two adjacent DNA fragments have a duplicate sequence to facilitate homologous recombination. In certain embodiments, the duplicate sequence is about 100 bp to about 5000 bp long.In certain embodiments, one or more DNA fragments further comprise an inverted terminal repeat (ITR) region. In certain embodiments, one or more DNA fragments further comprise a poxvirus hairpin loop (HL) sequence, a poxvirus genome resolution (CR) sequence, or both, wherein the HL sequence or the CR sequence is added to one or both ends of the DNA fragment as a single-stranded or double-stranded DNA sequence in the sense or antisense orientation. In certain embodiments, one or more DNA fragments further comprise one or more HL sequences and one or more CR sequences. In certain embodiments, each HL sequence is adjacent to two CR sequences at both ends of the HL sequence. In certain embodiments, only a subset of the one or more DNA fragments comprises an HL sequence or a CR sequence. In certain embodiments, one or more DNA fragments further comprise one or more antigens, one or more DNA sequences encoding subunits or fragments thereof, or other heterologous DNA sequences. In certain embodiments, two or more DNA fragments comprise DNA sequences of the same antigen, subunits or fragments thereof, or the same heterologous DNA sequence. In certain embodiments, two or more DNA fragments comprise DNA sequences of different antigens, subunits or fragments thereof, or other heterologous DNA sequences. In certain embodiments, the DNA sequences of the antigens, subunits or fragments thereof, or other heterologous DNA sequences are codon-optimized for expression in a host cell. In certain embodiments, one or more DNA fragments further comprise a viral promoter upstream of the DNA sequence of the antigen, subunit or fragment thereof, or other heterologous DNA sequence, or a transcription termination signal downstream of the DNA sequence of the antigen, subunit or fragment thereof, or other heterologous DNA sequence, or both. In certain embodiments, the DNA sequences encoding the antigens, subunits or fragments thereof, or other heterologous DNA sequences are inserted into one or more poxvirus insertion sites, such as intergenic regions, non-essential genes and regions, and deletion sites.

[0007] In another aspect, the following expression systems are disclosed herein: (i) a single DNA fragment containing the entire genome of a desired poxvirus, or two or more DNA fragments, each containing a partial sequence of the genome of a desired poxvirus, which, when transferred to a host cell by cotransfection, are sequentially assembled to contain the full sequence of the poxvirus genome and enable the reconstruction of the poxvirus; and (ii) one or more DNA sequences, or other heterologous DNA sequences, that are inserted into one or more insertion sites of the poxvirus and encode one or more antigens, subunits or fragments thereof, or other heterologous DNA sequences, which are expressed in the host cell by transfection of one or more poxvirus DNA fragments and reconstruction of the poxvirus. In certain embodiments, one or more DNA fragments are cyclized before transfection or transfected to the host cell in a circular form. In certain embodiments, one or more DNA fragments are cloned into a plasmid vector or a BAC vector. In certain embodiments, one or more DNA fragments are linearized before transfection or transfected into host cells in a linearized form. In certain embodiments, one or more DNA fragments are naturally occurring DNA fragments, chemosynthetic DNA fragments, or a combination of naturally occurring and chemosynthetic DNA fragments. In certain embodiments, the poxvirus genome sequence includes the sequence with MVA accession number #U94848 or #AY603355. In certain embodiments, two adjacent DNA fragments have a duplicate sequence to facilitate homologous recombination. In certain embodiments, the duplicate sequence is about 100 bp to about 5000 bp in length. In certain embodiments, one or more DNA fragments further include a terminal inversion (ITR) region.In certain embodiments, one or more DNA fragments further comprise a poxvirus terminal hairpin loop (HL) sequence, a poxvirus genome isolation (CR) sequence, or both, wherein the HL or CR sequence is appended to one or both ends of the DNA fragment as a sense-directed or antisense-directed single-stranded or double-stranded DNA sequence. In certain embodiments, one or more DNA fragments further comprise one or more HL sequences and one or more CR sequences. In certain embodiments, each HL sequence is flanked by two CR sequences at both ends of the HL sequence. In certain embodiments, only a subset of one or more DNA fragments comprises HL sequences or CR sequences. In certain embodiments, one or more DNA fragments further comprise a viral promoter upstream of the DNA sequence encoding the antigen, its subunit or fragment, or upstream of another heterologous DNA sequence, or further comprises a transcription termination signal downstream of the DNA sequence encoding the antigen, its subunit or fragment, or downstream of another heterologous DNA sequence, or both. In certain embodiments, one or more antigens, DNA sequences encoding their subunits or fragments, or other heterologous DNA sequences are inserted into one or more poxvirus insertion sites.

[0008] In another aspect, (i) a single DNA fragment containing the entire genome of a desired poxvirus, or two or more DNA fragments that, when cotransfected into a host cell, are sequentially assembled and contain the full-length sequence of the poxvirus genome and enable reconstitution of the poxvirus, wherein each of the two or more DNA fragments contains a partial sequence of the genome of the desired poxvirus, and (ii) one or more DNA sequences encoding one or more antigens, subunits or fragments thereof, or other heterologous DNA sequences inserted at one or more insertion sites of the poxvirus, wherein the antigen, subunit or fragment thereof, or other heterologous DNA sequence is expressed in the host cell by transfection of one or more DNA fragments of the poxvirus and reconstitution of the poxvirus, a vaccine composition for preventing or treating cancer or infectious diseases is disclosed herein. In certain embodiments, the antigen, subunit or fragment thereof, or other heterologous DNA sequence is inserted at one or more poxvirus insertion sites. In certain embodiments, the vaccine composition further comprises a pharmaceutically acceptable carrier, adjuvant, additive, or combination thereof.

[0009] In another aspect, a method for preventing or treating cancer or viral infection in a subject is disclosed herein, comprising the step of administering to the subject a prophylactic or therapeutically effective amount of a vaccine composition, wherein the vaccine comprises (i) a single DNA fragment containing the entire genome of a desired poxvirus, or two or more DNA fragments, each containing a partial sequence of the genome of a desired poxvirus, such that when transferred into a host cell by cotransfection, two or more DNA fragments are sequentially assembled to contain the full sequence of the poxvirus genome and enable the reconstruction of the poxvirus; and (ii) one or more DNA sequences or other heterologous DNA sequences encoding one or more antigens, subunits or fragments thereof, which are inserted into one or more insertion sites of the poxvirus, and which are expressed in a host cell by transfection of one or more DNA fragments of the poxvirus and reconstruction of the poxvirus. In certain embodiments, an antigen, its subunits or fragments, or other heterologous DNA sequences are inserted into one or more poxvirus insertion sites. [Brief explanation of the drawing]

[0010] This application includes at least one drawing made in color. A copy of this application, including the color drawing, will be provided by the authorities upon request and payment of the required fees. [Figure 1]Figures 1A-1C show the design of the sMVA construction. Figure 1A: Schematic diagram of the MVA genome. The MVA genome is approximately 178 kbp long and contains a unique region (UR) adjacent to a large terminal inversion sequence (ITR) of approximately 9.6 kbp. Figure 1B: Schematic diagram of sMVA fragments. Each of the three sMVA fragments is approximately 60 kbp long. sMVA fragment 1 (F1) contains the sequence of the left portion of the MVA genome, including the left ITR; sMVA fragment 2 (F2) contains the sequence of the central portion of the MVA genome; and sMVA fragment 3 (F3) contains the sequence of the right portion of the MVA genome, including the right ITR. sMVA F1 and F2, and sMVA F2 and F3 share approximately 3 kbp of overlapping sequences (marked with dotted "x"s) for homologous recombination. The approximate locations of commonly used MVA insertion sites, such as Del2, IGR44 / 45(44 / 45), and IGR64 / 65(69 / 70) in sMVA F1, IGR69 / 70(69 / 70) and TK insertion sites in sMVA F2, and Del3 in sMVA F3 are shown. Figure 1C: Schematic diagram of terminal HL and CR sequences. Each sMVA fragment contains a sequence composition at both ends that includes double-stranded copies of the MVA terminal HL and adjacent CR sequences to facilitate genome isolation and packaging. Each sMVA fragment contains SfiI and FseI restriction sites at both ends, as shown, to allow fragments containing or not containing terminal CR / HL / CR sequences to be released from the bacterial vector backbone (pCCI-Brick) by enzymatic digestion. [Figure 2] This illustrates the reconstruction of sMVA. A schematic diagram of sMVA generation using the three sMVA fragments (F1-F3) shown in Figure 1 is illustrated. The three sMVA fragments are stably maintained in Escherichia coli (E. coli), then isolated from the bacteria, and transfected into MVA-tolerant BHK or CEF cells as circular or linear DNA molecules. Subsequently, the transfected cells are infected with fowlpox virus (FPV) as a helper virus to initiate transcription and replication of the MVA genome, and thus to initiate the reconstruction of the viral morphology of sMVA. [Figure 3]The construction of recombinant sMVA with one antigen is illustrated. A schematic diagram is shown for an example of the construction of recombinant sMVA (rsMVA) with one introduced antigen sequence, heterogeneous gene sequence, or other genomic modification. Using linear PCR-derived products or other forms of linear DNA constructs, the antigen or heterogeneous gene sequence (black circle) is inserted into the MVA genome sequence of F2 (or one of the other sMVA fragments (F1 or F3)) by bacterial recombination in E. coli. To initiate the reconstruction of rsMVA expressing one antigen or heterogeneous gene sequence, the modified sMVA fragment F1 and the unmodified sMVA fragments F2 and F3 are isolated from E. coli and cotransfected into FPV-infected BHK cells or CEF cells in circular or linear forms. Nucl. = cell nucleus. [Figure 4] The construction of recombinant sMVA with three antigens is illustrated. A schematic diagram is shown of an example of the generation of recombinant sMVA (rsMVA) into which three antigen sequences, heterogeneous gene sequences, or other genomic modifications have been introduced. Using linear PCR-derived products or other forms of linear DNA constructs, the antigens or heterogeneous gene sequences (indicated by squares, circles, or rectangles) are inserted into each of the three sMVA fragments (F1-F3) by bacterial recombination techniques in E. coli. The modified sMVA fragments F1, F2, and F3 are isolated from E. coli and cotransfected into tolerant BHK or CEF cells in circular or linear form, where, in the presence of FPV as a helper virus, rsMVA viruses expressing the three antigens or heterogeneous gene sequences are reconstituted. Nucl. = cell nucleus. [Figure 5]The sMVA rearrangement process is illustrated. A schematic diagram is shown of an example of the tissue culture rearrangement process of sMVA or rsMVA using three sMVA fragments (F1-F3) or modified versions thereof. BHK cells seeded in a 6-well tissue culture format are cotransfected with three sMVA fragments (unmodified, modified, or a combination thereof), and then infected with FPV as a helper virus to initiate transcription and replication of the sMVA fragments, and thus to initiate sMVA virus rearrangement. To promote sMVA rearrangement, the transfected / infected BHK cells are transferred to a larger tissue culture format every other day (on days 2, 4, and 6 (dpt / i) post-transfection / infection). Plaque development and progression may be detected after cell migration on days 2 and 4, and after migration on day 6, 90-100% infected BHK cells may be obtained at 7 or 8 dpt / i. [Figure 6] Figures 6A-6B show the sMVA infectivity analysis. Figure 6A is a plaque analysis showing representative bright-field microscopy images of five individual plaques in a BHK cell monolayer infected with sMVA or MVA NIH clone 1 on day 1 post-infection (dpi). Figure 6B shows the progression of infection. Representative bright-field microscopy images of a BHK cell monolayer infected with sMVA or MVA NIH clone 1 at 1 dpi, 2 dpi, and 3 dpi are shown. A pseudoinfected (uninfected) BHK cell monolayer was analyzed as a control. Plaques and infected areas were visualized by immunostaining for vaccinia virus B5R protein. [Figure 7]Figures 7A–7L show the PCR analysis of reconstituted sMVA. sMVA virus or MVA NIH clone 1, reconstituted from three sMVA fragments (F1–F3), was used to infect BHK cells at an MOI of 5. DNA extracted from infected BHK cells was evaluated by PCR. DNA (BHK) and H2O only (Ctrl) extracted from pseudoinfected (uninfected) BHK cells were analyzed as controls. PCR analysis includes terminal inversion sequences (ITRs) located within sMVA F1-derived DNA (Figure 7A); the transition region from the left ITR to the internal specific region (LITR / UR) (Figure 7B); and two MVA deletion sites (Del2) (Figure 7C); the intergenetic region (IGR) between MVA 69R and 70L located within sMVA F2-derived DNA (IGR69 / 70) (Figure 7D); three MVA deletion sites (Del3) located within sMVA F3-derived DNA (Figure 7E) and the transition region from the internal UR to the right ITR (UR / RITR) (Figure 7F); and sMVA This included recombination site reconstructions of F1 and F2 (F1 / F2) (Figure 7G) and F2 and F3 (F2 / F3) (Figure 7H); as well as sequence detection of MVA genome locations (Figures 7I-7L) containing five nucleotide polymorphisms (Polym. 1, 2, 3, 4 / 5) specific to the MVA Antoine strain. The expected PCR product is indicated by the arrow to the right of each panel. The expected size of the PCR product is given in parentheses below each panel. [Figure 8] This shows the reconstruction of sMVA containing a single fluorescent marker. Representative immunofluorescence and bright-field microscopy images of BHK cell monolayers at 6 and 7 days post-transfection / infection (dpt / i) are shown using unmodified sMVA fragments F1 and F3, as well as a modified sMVA fragment F2 (F2-RFP) with an inserted red fluorescent protein (RFP) marker. The method for sMVA reconstruction from these fragments is illustrated in Figure 5, with FPV used as the helper virus. Pseudotransfected / infected BHK cell monolayers were analyzed as a control. [Figure 9]This shows the reconstruction of sMVA containing dual fluorescent markers. Representative immunofluorescence and bright-field microscopy images of BHK cell monolayers at 6 and 7 days post-transfection / infection (dpt / i) are shown using unmodified sMVA fragment F1, modified sMVA fragment F2 (F2-RFP) with an inserted red fluorescent protein (RFP) marker, and modified sMVA fragment F3 (F3-BFP) with an inserted blue fluorescent protein (BFP) marker. The sMVA reconstruction method from these fragments is illustrated in Figure 5, with FPV used as the helper virus. Pseudotransfected / infected BHK cell monolayers were analyzed as a control. [Figure 10] Figures 10A–10H illustrate various examples of rsMVA HCMV vector construction. Examples are shown of using sMVA fragments F1–F3 to generate rsMVA vectors into which HCMV antigen sequences based on five subunits of the pentamer complex (PC; including UL128, UL130, UL131A, gH, and gL), glycoprotein B (gB), phosphorylated protein 65 (pp65), and / or pre-initial proteins 1 and 2 (IE1 and IE2) are inserted. The HCMV antigen sequences are inserted separately or combined as a multi-cistronic expression construct linked by 2A, as shown, into various MVA insertion sites, e.g., MVA deletion 3 (Del3) sites, or intergeneric regions between open reading frames 044 / 045 (44 / 45), 064 / 065 (64 / 65), or 069 / 070 (69 / 70). Examples are provided for rsMVA vectors expressing all PC subunits (sMVA-PC1 (Figure 10A) and sMVA-PC2 (Figure 10B)), rsMVA vectors expressing all PC subunits together with gB and pp65 (sMVA-7Ag1 (Figure 10C) and sMVA-7Ag2 (Figure 10D)), or rsMVA vectors expressing all PC subunits, gB, and pp65 together with IE1 / IE2 antigens (sMVA-8Ag1 / 2 (Figures 10E-10F) and sMVA-9Ag2 (Figures 10G-10H)). mH5 represents the vaccinia-modified H5 promoter, and ITR represents the terminal inversion sequence. [Figure 11]Figures 11A-11B show the characterization of sMVA. Figure 11A: PCR analysis. CEFs infected with sMVA (sMVA hp) obtained by FPV HP1.441 or sMVA (sMVA tv1 and sMVA tv2) obtained by TROVAC from two independent viral rearrangements were investigated by PCR for several MVA genomic locations (ITR sequences, transitions from left or right ITR to internal specific regions (left ITR / UR; UR / right ITR), Del2, IGR69 / 70, and Del3 insertion sites, as well as recombination sites for F1 / F2 and F2 / F3) and the absence of BAC vector sequences. PCR reactions with wtMVA-infected and uninfected cells, PCR reactions without samples (false), or PCR reactions with MVA BAC were performed as controls. Figure 11B: Restriction fragment length analysis. Viral DNA isolated from ultra-purified sMVA (sMVA tv1 and sMVA tv2) viruses or wtMVA viruses was compared by restriction enzyme digestion with KpnI and XhoI. [Figure 12]Figures 12A–12D show the replication characteristics of sMVA. The replication characteristics of sMVA (sMVA hp) obtained by FPV HP1.441 or sMVA (sMVA tv1 and sMVA tv2) obtained by TROVAC from two independent sMVA virus reconstitutions were compared with wtMVA. Figure 12A: Virus focus. CEF cells infected with reconstituted sMVA virus or wtMVA at a low MOI were immunostained using anti-vaccinia polyclonal antibody (αVAC). Figure 12B: Replication kinetics. BHK cells or CEF cells were infected with sMVA or wtMVA at 0.02 MOI, and viral titers in the inoculum and infected cells were determined in CEF cells at 24 and 48 hours post-infection. A mixed-effects model with Geisser-Greenhouse correction was applied. There were no significant differences between groups at 24 and 48 hours post-infection. Figure 12C: Viral focus size analysis. BHK or CEF cell monolayers were infected with sMVA or wtMVA at 0.002 MOI, and the area of ​​the viral focus was determined 24 hours post-infection by immunostaining with αVAC antibody. Figure 12D: Host cell range analysis. Various human cell lines (HEK293, A549, 143b, and HeLa), CEF cells, or BHK cells were infected with sMVA or wtMVA at 0.01 MOI, and viral titers were determined in CEF cells 48 hours post-infection. The dotted line shows the calculated viral titers of the inoculum based on 0.01 MOI. Differences between groups in Figures 12C-12D were calculated using Tukey (2C) or Dunnett (2D) multiple comparison tests after one-way ANOVA. ns = not significant. [Figure 13]Figures 13A-13D demonstrate the in vivo immunogenicity of sMVA. sMVA obtained by FPV HP1.441 (sMVA hp) or sMVA obtained by TROVAC from two independent viral rearrangements (sMVA tv1 and sMVA tv2) were compared to wtMVA by in vitro analysis. C57BL / 6 mice were immunized twice at 3-week intervals with either a low dose (1 × 10⁷ PFU) or a high dose (5 × 10⁷ PFU) of sMVA or wtMVA. Pseudoimmunized mice were used as controls. Figure 13A: Binding antibody. After the first and second immunizations, MVA-specific binding antibody (IgG titer) stimulated by sMVA or wtMVA was measured by ELISA. Figure 13B: NAb response. After additional immunization with recombinant wtMVA expressing a GFP marker, MVA-specific NAb titers induced by sMVA or wtMVA were measured. Figures 13C-13D: T cell responses. Following two immunizations, CD8+(13C) and CD4+(13D) T cell responses secreting MVA-specific IFNγ, TNFα, IL-4, and IL-10 were measured by flow cytometry after ex vivo antigen stimulation with B8R immunodominant peptide, induced by sMVA or wtMVA. Differences between groups were assessed using one-way ANOVA with Tukey's multiple comparison test. ns = not significant. [Figure 14]Figures 14A-14D demonstrate the in vivo immunogenicity of sMVA. sMVA obtained by the FPV HP1.441 strain (sMVA hp) or sMVA obtained by the FPV TROVAC strain from two independent viral rearrangements (sMVA tv1 and sMVA tv2) were compared to wtMVA by in vitro analysis. C57BL / 6 mice (N=4) were immunized twice at 3-week intervals with either a low dose (1 × 10⁷ PFU) or a high dose (5 × 10⁷ PFU) of sMVA or wtMVA. Pseudoimmunized mice were used as controls. Figure 14A: Binding antibody. Absorbance at 450 nm at different serum dilutions of MVA-specific binding antibodies, measured by ELISA after the first and second immunizations in mice receiving sMVA or wtMVA (IgG titer), is shown. Figure 14B: NAb response. MVA-specific NAb titers induced by sMVA or wtMVA were measured after additional immunization with wtMVA expressing the GFP marker. Measured GFP area of ​​infected cells at different serum dilutions is shown as square pixels (pix 2 × 10³). Figure 14C–14D: T cell response. MVA-specific CD8+ (14C) and CD4+ (14D) T cells expressing IFNγ, TNFα, IL-4, and IL-10 were measured by flow cytometry after ex vivo antigen stimulation with vaccinia A19L immunodominant peptide following two immunizations with sMVA or wtMVA. Differences between groups were assessed using one-way ANOVA with Tukey's multiple comparison test. ns = not significant. [Modes for carrying out the invention]

[0011] Detailed explanation Methods for generating poxvirus-based vectors and recombinant poxvirus vectors from circularized or linearized naturally-derived DNA or chemically synthesized DNA are disclosed herein. Specific examples for generating a fully synthetic version of MVA (sMVA) from circularized synthetic DNA fragments and for generating recombinant sMVA (rsMVA) that express one or more heterologous gene sequences, such as fluorescent markers or antigens for infectious diseases and cancer, are provided herein.

[0012] MVA has an excellent safety profile in addition to being a versatile expression system and having a large capacity (up to 30 kbp) for accommodating foreign DNA sequences 1 and is widely used for developing recombinant vaccine vectors against infectious diseases and cancer 7、12、13 . MVA has been pursued for developing various vaccine strategies for cancer treatment 14、15 and for developing various vaccine approaches to prevent human cytomegalovirus (HCMV) infection, a common cause of permanent congenital defects in newborns and complications in transplant recipients 16~18 . Some of these vaccines have completed Phase I or Phase II clinical evaluations 19 . As a member of the Poxviridae family, MVA provides its own enzymes for transcription and DNA replication and replicates entirely in the cytoplasm of infected cells 1、20 . Production of MVA virus fails in mammalian cells due to a late block in assembly, but MVA can efficiently infect most mammalian cells, including human cells, and initiate robust gene expression and DNA replication, and thus MVA is an ideal vehicle for efficiently delivering and expressing foreign antigens in vitro and in vivo 1、5 1、5、21 ​The most commonly used method for generating MVA recombinants is the so-called transfection / infection method, which uses a transfer plasmid that is co-delivered with MVA to accretachyon cells (CEF or BHK), thereby inserting the desired antigen, along with the upstream promoter sequence and downstream transcription termination signal, into the MVA genome via spontaneous homologous recombination. 1、5、21 .

[0013] Although this method is widely used, it can be cumbersome and hindered by the need for multiple selections to obtain a homogeneous population of recombinant MVA during antigen insertion and subsequent marker removal. 21 These drawbacks in conventional transfection / infection methods can be particularly problematic for the production of multi-antigenic MVA vectors with antigens inserted at two or more insertion sites, potentially reducing the stability of the MVA vaccine. 18、22 As an alternative to conventional transfection / infection methods, and to facilitate the production of multi-antigenic MVA vectors, methods for generating MVA recombinants using bacterial artificial chromosome (BAC) technology have been developed. 23~25 These methods enable repeatable operations to reconstruct a homogeneous viral population of recombinant MVA in BHK cells, using fowlpox virus (FPV) as a helper virus necessary to insert antigens into the MVA genome by a highly efficient and versatile mutagenesis technique, and to "jump-start" the transcription of the non-infectious MVA genome. 23 .

[0014] Construction of synthetic poxviruses Methods for constructing poxvirus-based vectors or recombinant poxvirus vectors from naturally occurring DNA or chemically synthesized DNA are disclosed herein. In certain embodiments, a single DNA fragment is obtained from viral DNA or chemically synthesized and contains the entire genome sequence of a poxvirus. This single DNA fragment can be used to transfect a host cell so that a poxvirus is reconstituted. In other embodiments, two or more naturally occurring DNA fragments or chemically synthesized DNA fragments or combinations thereof are used to cotransfect a host cell, where each DNA fragment contains a partial sequence of poxvirus genomic DNA having a duplicate sequence at the ends of two adjacent DNA fragments, so that when the two or more DNA fragments are cotransfected into a host cell, they are assembled together by homologous recombination to form a poxvirus containing the full-length sequence of a desired poxvirus genome. In certain embodiments, the duplicate sequence is about 100 bp to about 5000 bp long.

[0015] In certain embodiments, a shortened genome sequence that is not a complete genome sequence, or a modified genome sequence having deletions or alterations in non-essential genes or regions of the poxvirus genome sequence, or a hybrid derivative comprising genome sequences derived from two or more different poxviruses, may be used to construct the vectors disclosed herein.

[0016] In certain embodiments, one or more naturally occurring or chemically synthesized DNA fragments containing poxvirus genome or subgenomic DNA can be further modified to form an artificial hybrid fragment composed of natural and synthetic poxvirus genome DNA sequences. In other embodiments, one or more naturally occurring or chemically synthesized DNA fragments may be composed of sequences derived from two different poxviruses to form a poxvirus hybrid sequence. One or more DNA fragments are MVA (NCBI accession #U94848, #AY603355), vaccinia virus (#NC_006998, #LT966077), camelpox virus (#NC_003391), cowpox virus (#NC_003663), ectromelia virus (#NC_004105), monkeypox virus (#NC_003310), raccoonpox virus (#NC_027213), skunkpox virus (#NC_031038), Taterapox virus (#NC_008291), smallpox virus (#NC_001611, #L22579), velopox virus (#NC_0 The sequence may consist of poxvirus sequences derived from any other poxvirus or its variants, or from different poxvirus sequences.

[0017] In certain embodiments, host cells can be infected with a helper virus, such as FPV, before, during, or after transfection with one or more DNA fragments containing sequences of poxvirus genome or subgenomic DNA. The helper virus can be any suitable virus that infects the host cell and enables the initiation of poxvirus transcription and replication. Used as an example herein is an FPV in a host cell that does not undergo homologous recombination with poxvirus DNA. The helper virus cannot replicate in the host cell. Furthermore, for the purposes of this application, the helper virus, e.g., FPV, is not itself a component of the poxvirus being reconstituted. In certain embodiments, cowpox virus, Schoepp's fibroma virus, or other suitable poxviruses can be used as helper viruses. In certain embodiments, one or more DNA fragments for transfection may be linearized DNA fragments, cyclized DNA fragments, or a combination of linearized and cyclized DNA fragments. In a particular embodiment, one or more DNA fragments for transfection are cloned into a vector such as a plasmid or BAC and / or maintained in a host cell such as a bacterial cell, e. coli.

[0018] In certain embodiments, one or more DNA fragments further comprise one or both of the terminal inversion (ITR) regions of a poxvirus. In certain embodiments, the ITR sequence may contain one or more modifications or changes that do not affect the design scheme of the reconstructed poxvirus. In one embodiment, one or more DNA fragments reconstructing a poxvirus may contain only a portion of the ITR sequence. For example, sMVA fragments F1 and F3 and the reconstructed sMVA vector or recombinant sMVA vector (Figures 1-4) may contain only a portion of the MVA ITR sequence. In certain embodiments, one or more DNA fragments may be further modified to add a poxvirus terminal hairpin loop (HL) sequence to one or both ends as a double-stranded copy (double-stranded DNA) or to the 5' and / or 3' ends as a single-stranded nucleotide sequence. For example, the HL sequence may be added to one or both ends of the synthetic DNA fragment as a double-stranded or single-stranded DNA sequence in the sense (5'→3') direction or antisense (3'→5') direction. In certain embodiments, one or more DNA fragments may be further modified to add a poxvirus concatemer isolation (CR) sequence to one or both ends of a double-stranded DNA sequence, or to the 5' and / or 3' ends of a single-stranded nucleotide sequence. The CR sequence is the consensus sequence 5'-T6-N 7-9The sequence may be based on any sequence derived from -T / C-A3-T / A-3', where A is adenine, C is cytosine, G is guanine, T is thymine, and N is any nucleotide. In certain embodiments, two CR sequences may be attached to both ends of an HL sequence so that a CR-HL-CR sequence is formed. The CR-HL-CR sequence may be attached to one or more DNA fragments as a double-stranded DNA sequence at one or both ends, or as a single-stranded nucleotide sequence at the 5' end, 3' end, or both ends. In other embodiments, only the HL sequence or CR sequence may be attached to one or both ends of one or more DNA fragments as a double-stranded or single-stranded DNA sequence. In certain embodiments, all DNA fragments contain an HL sequence or a CR sequence, or a combination thereof, at one or both ends. In other embodiments, not all DNA fragments contain an HL sequence or a CR sequence. In certain embodiments, only a subset or subpopulation of DNA fragments contains an HL sequence or a CR sequence, or a combination thereof. ITR, HL, or CR sequences are MVA (NCBI accession #U94848, #AY603355), vaccinia virus (#NC_006998, #LT966077), camelpox virus (#NC_003391), cowpox virus (#NC_003663), ectromelia virus (#NC_004105), monkeypox virus (#NC_003310), raccoonpoxvirus (#NC_027213), skunkpoxvirus (#NC_031038), tatelapoxvirus (#NC_008291), and bariola virus (#NC_00161). 1. It may originate from any variant of the following poxviruses: 1. #L22579), velopoxvirus (#NC_031033), canary poxvirus (#NC_005309), porcine poxvirus (#NC_003389), FPV (#NC_002188, #MH734528), myxoma virus (#GQ409969), spongiform mites virus (NC_004002), goatpox virus (#NC_004003), (Aarf virus #NC_005336), rabbit fibroma virus (#NC_001266), or any other poxvirus or its variant.

[0019] In certain embodiments, the HL sequence or CR sequence used herein is disclosed as follows:

[0020] Sequence of a terminal CR-HL-CR sequence (215 bp long, 5'→3') (SEQ ID NO:1) in which the CR sequence is underlined and the HL sequence is italicized and double-underlined, and the MVA concatemer isolate sequence contains a double-stranded copy of the adjacent MVA terminal hairpin loop (HL): TIFF2026094336000002.tif18150.

[0021] Sequence of a terminal CR-HL-CR sequence (215 bp long, 5'→3') (SEQ ID NO:2) in which the concatemer isolate sequence contains a complementary double-strand copy of the adjacent MVA terminal hairpin loop (HL), with the CR sequence underlined and the HL sequence in italics and double underlined: TIFF2026094336000003.tif18150.

[0022] MVA terminal hairpin loop arrangement (HL, 165nt length, 5'→3') (SEQ ID NO:3): TIFF2026094336000004.tif11148.

[0023] Complementary sequence of MVA terminal hairpin loop (HL, 165nt length, 5'→3') (SEQ ID NO:4): TIFF2026094336000005.tif11148.

[0024] Sequence of a terminal CR-HL-CR sequence (154 bp long, 5'→3') (SEQ ID NO: 5) containing a double-stranded copy of a vaccinia-terminal hairpin loop (HL;S type) adjacent to a concatemer isolate sequence, where the CR sequence is underlined and the HL sequence is italicized and double-underlined: TIFF2026094336000006.tif11150.

[0025] Sequence of a terminal CR-HL-CR sequence (154 bp long, 5'→3') (SEQ ID NO: 6) containing a double-stranded copy of a vaccinia terminal hairpin loop (HL; F type) adjacent to a concatemer isolate sequence, where the CR sequence is underlined and the HL sequence is italicized and double-underlined: TIFF2026094336000007.tif11150.

[0026] Vaccinia virus terminal hairpin loop sequence 1 (HL, S type, 104 nt length, 5'→3') (SEQ ID NO: 7): TIFF2026094336000008.tif10149.

[0027] Vaccinia virus terminal hairpin loop sequence 2 (HL, type F, 104nt length, 5'→3') (SEQ ID NO:8): TIFF2026094336000009.tif10150.

[0028] Sequence 1 (CR, 20 bp length, 5'→3') (SEQ ID NO: 9, sense direction) of the MVA concatemer isolate sequence contained in the left end of the hairpin double-strand copy in the example: TIFF2026094336000010.tif4128.

[0029] Sequence 2 (CR, 20 bp length, 5'→3') (SEQ ID NO: 10, antisense direction) of the MVA concatemer isolate sequence contained in the right end of the hairpin double-strand copy in the example: TIFF2026094336000011.tif4128.

[0030] As demonstrated in the examples, the recombinant sMVA and rsMVA disclosed herein are derived from the MVA genome published by Antoine et al. (accession #U94848). 26It is generated based on the chemical synthesis of three approximately 60 kbp long DNA fragments, encompassing the entire approximately 178 kbp. This includes an internal intrinsic region (UR) and an adjacent approximately 9.6 kbp long terminal inversion (ITR) region, as illustrated in Figure 1. In this specification, the MVA genome sequence (accession #U94848) published by Antoine et al. is referred to as the MVA Antoine strain. 26 The sMVA fragment 1 (F1) contains approximately 5 base pairs of the internal UR of the National Institute of Health clone 1 (MVA NIH clone 1), which has been licensed and commercially available since 1974 and whose sequence is identical to the publicly released genome (accession #AY603355) of the MVA Acambis strain. sMVA fragment 1 (F1) contains approximately 50 kbp of the left ITR and the leftmost part of the internal UR of the MVA genome; sMVA fragment 2 (F2) contains approximately 60 kbp of the central part of the internal UR of the MVA genome; and sMVA fragment 3 (F3) contains approximately 50 kbp of the rightmost part of the internal UR and the right ITR of the MVA genome (Figure 1). sMVA F1 and F2, and sMVA F2 and F3 are designed to share approximately 3 kbp of overlapping sequences to enable the reconstruction of the complete MVA genome by homologous recombination (Figure 1). A 165-nucleotide-long double-stranded copy of the MVA terminal HL, adjacent to the MVA CR sequence, is attached to each of the three fragments to facilitate the separation and packaging of the MVA genome. 26 (Figure 1). Furthermore, the restriction sites of SfiI and FseI are present at both ends of each of the three fragments, so that fragments containing or not containing the HL and CR sequences may be released or linearized. The only exception is the HL and CR sequences in the ITRs of F1 and F3, which are fused to the ends in the same configuration as those present in the junction of the MVA concatemer replication intermediate. 26(Figure 1). Three sMVA fragments containing adjacent HL and CR sequences as well as overlapping homologous recombination sequences, as illustrated in Figure 1, were synthesized and generated using a yeast-based recombination system by Genscript Biotech. To ensure stable growth of the three fragments at low copy numbers in bacteria, all three sMVA fragments were cloned into a yeast shuttle vector called pCCI-Brick, which contains bacterial mini-F replicon elements that can be used as BAC vectors (Figure 1). The sMVA fragments were ultimately cloned into recombination-deficient E. coli cells DH10B or EPI300.

[0031] In a particular embodiment, in contrast to the terminals of the naturally occurring F1 and F3 ITRs in the concatemer replication intermediate, double-stranded copies of HL flanked by CR sequences are included at both ends of each of the three MVA fragments. This design is based on the inherent function of these sequence elements during poxvirus DNA replication. In the packaged poxvirus genome, the terminal HLs connect the two DNA strands to a continuous polynucleotide chain at the genome end, where they are present at both ends in an incompletely base-paired, AT-rich, inverted, complementary form. 26~29 The poxvirus HL sequence is crucial for the replication of the double-stranded DNA genome into multimeric head-to-tail or head-to-head concatemer replication intermediates, where the HL sequence resides at the concatemer junction as an exact double-stranded copy. 30~32 CR elements consist of highly conserved poxvirus isolation sequences and can be found in the packaged genome directly adjacent to the terminal HL, at both ends of a large ITR. 26、27、33、34 The concatemer replication intermediate poxvirus CR elements are located at any site in the HL double-strand copies of the genome junction, and these CR / HL / CR sequences are essential for the segregation of unit-length genomes and subsequent genome packaging. 33~36When a circular plasmid containing a poxvirus concatemer junction, in which the CR element consists of adjacent HL double-strand copies, is transfected into poxvirus-infected cells, they spontaneously separate into linear minichromosomes with terminal HL. 36、37 In certain embodiments, cyclized sMVA fragments can immediately replicate in host cells in an origin-independent manner when transfected into host cells. It has been reported that circular DNA molecules transfected into poxvirus-infected cells replicate origin-independently, and this non-specific sequence replication is not enhanced by insertion of viral DNA fragments. 38 In another embodiment, the cyclized sMVA fragment, when transfected into a host cell, can be replicated by a eukaryotic or viral replication origin inserted into the vector or MVA sequence.

[0032] Disclosed construction methods for synthetic poxviruses such as sMVA involve transfection of a circular plasmid or DNA molecule containing three synthetic MVA fragments having adjacent concatemer genome junctions (CR-HL-CR) and overlapping genome sequences into FPV-infected BHK or CEF cells, thereby promoting (1) transcription and replication of the three sMVA fragments, (2) isolation of the three sMVA fragments from the plasmid vector sequence, and (3) recombination of the three sMVA fragments into a full-length genome, ultimately resulting in the packaging of a vector-free genome with terminal HL into a pre-formed viral particle (Figures 1 and 2). These events may be initiated immediately as a result of FPV infection or may occur as a stepwise process after transcription initiation and replication of the three sMVA fragments. In another embodiment, the sMVA reconstruction process from the three sMVA fragments may be initiated without FPV or other helper viruses. Instead of this strategy of reconstituted sMVA from three MVA fragments using a circular plasmid or DNA molecule, sMVA reconstitution can also be facilitated by an approach based on the linearized morphology of the three sMVA fragments using added FseI and SfiI restriction sites, for example, as shown in Figures 1 and 2.

[0033] Whether using the circular form of the three MVA fragments, the linear form, or a combination of the circular and linear forms, the ends of the fragments may or may not contain HL and CR sequences. In certain embodiments, only a subset of the fragments may contain HL and CR sequences, or they may be appended to one or both ends of the fragments as single-stranded or double-stranded DNA sequences in the sense or antisense direction, for example, only in the naturally occurring F1 and F3 MVA subsequences of the putative concatemer replication intermediate. In another example, not all F1 fragments contain HL and CR sequences; F1 fragments containing HL and CR sequences and F1 fragments not containing them may be mixed during the construction process. Similarly, it is not required that all F2 or F3 fragments contain HL and CR sequences; a subset or subpopulation of F2 or F3 fragments may contain HL and CR sequences. In certain embodiments, the HL sequence and / or CR sequence may be chemically ligated as a single-stranded or double-stranded DNA sequence in a linearized form of three fragments.

[0034] In another embodiment, sMVA viruses reconstituted from sMVA fragments may be modified by insertion, deletion, or introduction of point mutations, or by insertion of one or more heterologous DNA sequences encoding one or more antigens, their subunits, or fragments. These modifications or antigen sequences may be introduced into the sMVA DNA fragment by conventional transfection / infection methods using transfer plasmids having homology flanks that mediate homologous recombination. In certain embodiments, one or more nucleotide sequences encoding one or more antigens, their subunits, or fragments may be codon-optimized for expression in eukaryotes or vaccinia. For example, antigens, their subunits, or fragments may be optimized for transcriptional or expression stability in host cells. Various codon optimization techniques may be used, but are not limited to, those involving changes to four identical consecutive nucleotides (e.g., GGGG, CCCC, TTTT, AAAA) by introducing silent point mutations that do not result in amino acid changes in the encoded protein, or adaptation of codon usage frequencies to a particular host species.

[0035] In another embodiment, an sMVA virus reconstituted from an sMVA fragment in a host cell, e.g., a BHK cell or a CEF cell, may be used to generate an sMVA bacterial artificial chromosome (BAC) containing the full-length sMVA genome. The BAC vector sequence may be inserted into the sMVA genome by a transfer construct containing a BAC sequence with adjacent homologous sequences that mediate homologous recombination. The circular replication intermediate with the inserted BAC sequence may be isolated from a host cell, e.g., a BHK cell or a CEF cell, and transferred to an E. coli cell, e.g., DH10B or EPI300, by electroporation or chemical transformation, allowing for stable growth of the large DNA construct. In another embodiment, the sMVA BAC may be transferred to an E. coli cell, e.g., a GS1783 cell, and manipulated by a Red recombination technique, e.g., en passant mutagenesis.

[0036] In another embodiment, sMVA fragments may be used to reconstruct a complete or full-length sMVA genome by in vitro ligation or other in vitro DNA assembly methods, such as Gibson assembly or Golden Gate assembly.

[0037] Expression of antigens or other heterogeneous gene sequences The techniques disclosed herein have the flexibility to insert various antigens, subunits or fragments, or other heterologous DNA sequences into one or more naturally derived or chemosynthetic DNA fragments prior to transfection, so that when reconstituted, a synthetic poxvirus expressing an antigen, its subunit or fragment is obtained. These antigens, subunits or fragments may be derived from or based on cytomegalovirus (CMV), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), other herpesviruses, Zika virus, Lassa virus, hepatitis C virus (HCV), hepatitis (HBV), coronaviruses (e.g., 2019-nCoV, SARS, MERS), influenza, or other viral, bacterial, or other forms of infectious pathogens. Antigen sequences may be derived from or based on cancer-related proteins (e.g., p53, retinoblastioma, neoantigens). Similarly, other heterogeneous gene sequences may be inserted into naturally derived or chemosynthetic poxvirus DNA fragments. Such heterogeneous gene sequences include, but are not limited to, fluorescent markers, RNA, cDNA copies such as RNAi, shRNA, LNCRNA, miRNA, interferons, cytokines, antibodies or fragments thereof, or other proteins expressed in prokaryotic or eukaryotic cells. In certain embodiments, an antigen sequence or heterogeneous gene sequence may be inserted into an sMVA fragment containing an upstream natural or synthetic poxvirus promoter (pSyn, P11, H5, mH5, P28, ATI, pHyb, p7.5) and a downstream transcription termination signal (TTTTTAT) so that the antigen sequence or heterogeneous gene sequence is expressed when the poxvirus fragment is transfected into a host cell.

[0038] The DNA sequences of an antigen, its subunit or fragment, or other heterologous genetic sequences can be inserted into one or more poxvirus insertion sites within one or more DNA fragments. For example, using sMVA, the DNA sequence of one antigen or fragment can be inserted into a single MVA insertion site located in one sMVA DNA fragment, e.g., sMVA F2, prior to transfection of host cells (Figure 3). In another example, the DNA sequences of two or more antigens or fragments thereof can be inserted into a single MVA insertion site located in one DNA fragment, e.g., sMVA F2, prior to transfection of host cells. These antigen DNA sequences may be under the control of a single promoter or two or more promoters. Similarly, the antigen DNA sequences may share the same transcription termination signal or have different transcription termination signals. Nucleotide sequences encoding two or more antigens may be linked by picornavirus 2A sequences (P2A, T2A, F2A, etc.) that mediate ribosome skipping, or by intra-sequence ribosome entry sites (IRESs), so that the antigens are processed post-translation and form multi-component antigen complexes by self-assembly.

[0039] In a particular embodiment, when one or more naturally derived poxvirus DNA fragments or chemosynthetic poxvirus DNA fragments are transfected into host cells, one or more fusion proteins are expressed, each consisting of a poxvirus protein and one or more antigens, subunits or fragments thereof, or other heterogeneous protein sequences attached to the C-terminus, N-terminus, or any internal position of the poxvirus protein, by inserting one or more DNA sequences encoding one or more antigens, subunits or fragments thereof, or other heterogeneous DNA sequences in-frame at the 5' end, 3' end, or any internal position of one or more essential or non-essential poxvirus open reading frames (ORFs). In some embodiments, one or more expressed fusion proteins, each comprising a poxvirus protein and one or more antigens, subunits or fragments thereof, or other heterologous protein sequences attached to the C-terminus, N-terminus, or any internal position of the poxvirus protein, are processed ("cleaved") into individual components at the 2A linker sequence by a ribosome skipping mechanism, such that one or more DNA sequences encoding one or more antigens, subunits or fragments thereof, or other heterologous DNA sequences are linked to one or more poxvirus ORFs by a sequence encoding picornavirus 2A (P2A, F2A, T2A, etc.). In another embodiment, when one or more naturally derived poxvirus DNA fragments or chemosynthetic poxvirus DNA fragments are transfected into host cells, one or more poxvirus proteins and one or more antigens, their subunits or fragments, or other heterogeneous protein sequences are simultaneously expressed through a chimeric polycistronic expression construct having multiple translation initiation sites at the 5' end of each ORF in the expression construct, such that one or more DNA sequences encoding one or more antigens, their subunits or fragments, or other heterogeneous DNA sequences are ligated to one or more essential or non-essential poxvirus ORFs by an intracellular ribosome entry site sequence.

[0040] In certain embodiments, two or more antigens, their subunits or fragments, or other heterogeneous sequences may be inserted into two or more MVA insertion sites, which may be located on the same sMVA fragment or on different sMVA fragments. For example, two or more antigens, their subunits or fragments, may be inserted into two different MVA insertion sites, both located on sMVA F1. In another example, two or more antigens, their subunits or fragments, may be inserted into two different MVA insertion sites, one located on sMVA F1 and the other on sMVA F2 (Figure 4). It is understood that the same antigen, its subunit or fragment DNA sequence may be inserted into different DNA fragments. Alternatively, different antigens, their subunits or fragments, or fragment DNA sequences may be inserted into different sMVA fragments such that, upon reconstitution after transfection, the different antigens, their subunits or fragments expressed by the synthetic MVA self-process and self-assemble into a complete antigen containing a viral particle, a viral complex, or its subunit or fragment.

[0041] As demonstrated in the examples, a previously established method for generating recombinant MVA vectors using BAC technology. 16、18、24、25 This was adapted to generate an rsMVA recombinant using three sMVA fragments. Since the three sMVA fragments are cloned into a BAC vector containing a mini-F replicon, they can grow stably in bacteria and therefore can be manipulated by highly efficient recombination methods, such as en passant mutagenesis or other E. coli-based manipulation methods. 39、40Using these methods, naturally derived or synthetic heterologous antigen sequences can be inserted, along with upstream vaccinia virus promoters and downstream transcription termination signals, into one, two, or all three sMVA fragments, in parallel, sequentially, or repeatedly, at virtually all MVA genomic locations (Figures 3 and 4). These insertion sites may include commonly used insertion sites, such as the MVA deletion 2 (Del2) site, the intergenetic region (IGR) between open reading frames (ORF) 44L and 45L (IGR44 / 45), the IGR between ORF 69R and 70L (IGR69 / 70), the IGR between 64L and 65L (IGR64 / 65), the thymidine kinase (TK) gene insertion site, or the MVA deletion 3 (Del3) site (Figure 1), or any other MVA deletion site, intergenetic region, or gene insertion site (ORF numbers are based on the MVA Antoine strain (accession #U94848)). These methods may also be used to generate one or more point mutations, insertions, or deletions at one or more locations on one or more of the sMVA fragment. Next, to initiate the rearrangement of rsMVA containing single, double, or multiple antigen insertions or genomic modifications, the modified sMVA fragment can be isolated from E. coli and co-transfected as a circular or linear DNA molecule into helper virus-infected BHK or CEF cells by various transfection methods (Figures 3 and 4). In certain embodiments, rsMVA rearrangement may be initiated without the addition of helper virus. In certain embodiments, three sMVA fragments may also be used to generate sMVA with single or multiple antigen insertions or genomic modifications by in vitro ligation. In certain embodiments, one or more antigens, their subunits, or fragments can be inserted into one or more MVA insertion sites of the sMVA fragment. In certain embodiments, one or more antigens or fragments are inserted into only one sMVA fragment (Figure 3). In certain embodiments, one or more antigens or fragments are inserted into two or more sMVA fragments (Figure 4).In certain embodiments, all sMVA fragments may have one or more antigens, subunits, or fragments inserted (Figure 4). When antigens, subunits, or fragments are inserted into two or more sMVA fragments, the antigens, subunits, or fragments may be the same or different for different sMVA fragments. For example, sMVA F1 may have one type of antigen or fragment inserted, and sMVA F2 may have a different type of antigen or fragment inserted. Alternatively, sMVA F1 and F2 may have the same type of antigen or fragment inserted, and sMVA F3 may have the same type of antigen or fragment, or a different type of antigen, subunit, or fragment inserted.

[0042] Poxvirus vectors produced by the techniques disclosed herein can be used, for example, to generate multi-antigenic vaccine vectors for stimulating multifunctional humoral and cellular immune responses against various conditions, such as viral infections and cancer. As an example, the disclosed technique based on three sMVA fragments F1-F3 can be used to generate a multi-antigenic rsMVA vaccine vector for stimulating multifunctional humoral and cellular immune responses against human cytomegalovirus (HCMV). This may include immunodominant antigen sequences based on the five subunits of the HCMV pentamer complex (PC), glycoprotein B (gB), phosphorylated protein 65 (pp65), and pre-initial 1 and 2 proteins (IE1 and IE2). Antigen sequences may be inserted separately into various commonly used MVA insertion sites (Del2, Del3, IGR44 / 45, IGR69 / 70, IGR64 / 65) of the sMVA fragment, or combined as a multi-cistronic expression construct linked by 2A, to generate rsMVA vaccine vectors expressing five, six, seven, eight, or nine HCMV antigens, as illustrated in Figures 6 and 10. rsMVA vectors expressing only a single HCMV antigen, or any other number or combination of the aforementioned nine HCMV antigens, may also be generated. All antigen sequences or expression constructs may be inserted along with an upstream mH5 promoter and a downstream transcription termination signal. These vectors may be used to stimulate an immune response to HCMV in animal models or in humans.

[0043] Expression systems and vaccines An MVA expression system is provided herein by embodiments described herein. In certain embodiments, the expression system may express one or more desired antigens, their subunits or fragments, or other heterologous protein sequences.

[0044] As described above, DNA sequences encoding one or more antigens, subunits, or fragments can be inserted into one or more sMVA fragments so that the reconstituted sMVA simultaneously expresses the antigen, its subunits, and fragments.

[0045] In certain embodiments, the antigen DNA sequence inserted into the sMVA fragment may be based on a native DNA sequence or may be derived from chemical synthesis. In other embodiments, the antigen DNA sequence may be optimized for expression and stability in the expression system.

[0046] The sMVA described herein may be part of a vaccine composition that can be used in methods for treating or preventing viral infections or in methods for treating cancer, depending on the antigen expressed by the sMVA. The vaccine composition described herein may contain the sMVA described herein in a therapeutically effective amount and may further include a pharmaceutically acceptable carrier according to standard methods. Examples of acceptable carriers include physiologically acceptable solutions, such as sterile saline and sterile buffered saline.

[0047] In some embodiments, a vaccine or pharmaceutical composition may be used in combination with a pharmaceutically effective amount of an adjuvant to enhance its prophylactic or therapeutic effect. Any immunological adjuvant that can stimulate the immune system and increase the response to the vaccine without having a specific antigenic effect itself may be used as an adjuvant. Many immunological adjuvants mimic evolutionarily conserved molecules known as pathogen-associated molecular patterns (PAMPs) and are recognized by a set of immune receptors known as Toll-like receptors (TLRs). Examples of adjuvants that may be used according to the embodiments described herein include Freund's complete adjuvant, Freund's incomplete adjuvant, double-stranded RNA (TLR3 ligand), LPS, LPS analogs, e.g., monophosphoryl lipid A (MPL) (TLR4 ligand), flagellin (TLR5 ligand), lipoproteins, lipopeptides, single-stranded RNA, single-stranded DNA, imidazoquinoline analogs (ligands for TLR7 and TLR8), and CpG. This includes DNA (TLR9 ​​ligand), Ribi adjuvant (monophosphoryl lipid A / trehalose dicorynoycolate), glycolipid (α-GalCer analog), unmethylated CpG islands, oil emulsion, liposomes, visomes, saponins (active fraction of saponins, e.g., QS21), muramyl dipeptide, alum, aluminum hydroxide, squalene, BCG, cytokines, e.g., GM-CSF and IL-12, chemokines, e.g., MIP1α and RANTES, activated cell surface ligands, e.g., CD40L, N-acetylmuramin-L-alanyl-D-isoglutamine (MDP), and thymosin α1. The amount of adjuvant used may be appropriately selected depending on the degree of symptoms that may occur in humans or animals as part of the immune response after administration of this type of vaccine, such as skin softening, pain, erythema, fever, headache, and muscle pain.

[0048] In further embodiments, as described above, the therapeutic effect achieved by the administration of the vaccine or pharmaceutical composition can be enhanced by using various other adjuvants, drugs, or additives in conjunction with the vaccine of the present invention. A pharmaceutically acceptable carrier may contain trace amounts of additives, such as substances that enhance isotonicity and chemical stability. Such additives should be nontoxic to human or other mammalian subjects at the dosage and concentration used, and examples include buffers, e.g., phosphoric acid, citrate, succinic acid, acetic acid, and other organic acids, and their salts; antioxidants, e.g., ascorbic acid; low molecular weight (e.g., less than about 10 residues) polypeptides (e.g., polyarginine and tripeptides), proteins (e.g., serum albumin, gelatin, and immunoglobulins); amino acids (e.g., glycine, glutamic acid, aspartic acid, and arginine); monosaccharides, disaccharides, and other carbohydrates (e.g., cellulose and its derivatives, glucose, mannose, and dextrin); chelating agents (e.g., EDTA); sugar alcohols (e.g., mannitol and sorbitol); counterions (e.g., sodium); nonionic surfactants (e.g., polysorbate and poloxamer); antibiotics; and PEG.

[0049] The vaccines or pharmaceutical compositions containing sMVA described herein may be stored in single-dose or multi-dose containers, such as sealed ampoules or vials, as aqueous solutions or lyophilized products.

[0050] Prevention or treatment of viral infections or cancer Poxviruses reconstituted from one or more naturally derived or chemosynthetic DNA fragments, with or without inserted antigens, their subunits or fragments, or other heterologous sequences, can be used as vaccine compositions for the prevention or treatment of various viral infections or cancers. Methods for selecting specific viral or cancer antigens for conditions or diseases to be prevented or treated will be known to those skilled in the art. These may include, but are not limited to, antigens of any infectious disease or cancer that can induce an immune response, such as viral envelope glycoproteins or glycoprotein complexes, immunodominant T cell antigens, or variant cancer neoantigens. These antigen sequences or portions thereof may be derived from or based on viruses, such as CMV, EBV, KSHV, other herpesviruses, Zika virus, Lassa virus, HCV, HBV, coronavirus, influenza, or other viral, bacterial, or other forms of infectious pathogens.

[0051] The following embodiments are for illustrative purposes only, to illustrate various aspects of the present invention. Therefore, the specific embodiments described should not be constructed to limit the scope of the invention. It will be apparent to those skilled in the art that various equivalents, variations, and modifications can be made without departing from the scope of the invention, and it will be understood that such equivalent embodiments are included herein. Furthermore, all references cited herein are incorporated herein by reference in their entirety as if they were fully shown herein. [Examples]

[0052] Example 1: Reconstruction of sMVA using the cyclic morphology of synthetic MVA fragments As an initial test to validate the sMVA platform, the procedure illustrated in Figure 2 was used to evaluate the reconstruction of sMVA from the circular morphology of three sMVA fragments. From E. coli, EPI300 or DH10B, which allows for stable growth of large DNA fragments cloned as BACs, an alkali-dissolved sMVA was used. 41Three sMVA fragments (F1-F3) were isolated and co-transfected into a monolayer of BHK cells (6-well plate format) as circular plasmids by lipofection (approximately 1-2 μg / fragment) (Figure 5). Four hours post-infection (hpi), BHK cells were infected with helper FPV at an infection multiplicity (MOI) of 0.1 to initiate MVA transcription and DNA replication. FPV-infected BHK cells transfected with one or two of the MVA fragments, pseudo-infected (uninfected) BHK cells transfected with all three MVA fragments, and pseudo-transfected FPV-infected BHK cells were used as controls. Transfected / infected BHK cells were grown under appropriate conditions and dispersed (passaged) into larger tissue culture formats every two days at a ratio of approximately 1:2-1:3 using the procedure illustrated in Figure 5. Signs of cytopathic effects (CPE) and viral plaque formation and progression, indicating sMVA reconstitution, were detected in FPV-infected BHK cell monolayers transfected with all three synthetic MVA fragments at 3–6 days post-transfection (dpt / i). After dispersion of FPV-infected BHK cells transfected with all three MVA fragments, BHK cell monolayers obtained at 7–8 dpt / i showed 90–100% infection. In contrast, no signs of CPE or characteristic MVA viral plaque formation were observed in any of the controls. sMVA was prepared from BHK cell monolayers with 100% CPE using a conventional freeze / thaw method, titrated in BHK cells, and used to infect BHK cell monolayers at a low MOI. MVA infection and spread were evaluated at 16–72 hpi by immunostaining for vaccinia virus glycoprotein B5R. 16、42 .

[0053] This analysis showed that the reconstituted sMVA had a similar ability to form characteristic MVA viral foci and diffuse into the BHK cell monolayer compared to MVA NIH clone 1 (Figure 6), thus confirming the formation of sMVA from the three sMVA fragments. At day 1 post-infection (dpi), characteristic viral foci were detected in the BHK cell monolayer infected with sMVA and MVA NIH, at 2dpi MVA viral infection was visible throughout the entire BHK cell monolayer infected with sMVA or MVA NIH, and at 3dpi most of the cells in the BHK cell monolayer infected with sMVA or MVA NIH detached. In contrast, no characteristic MVA viral infection or CPE was detected in the pseudo-infection control. Other research groups have definitively demonstrated that FPV, used as a helper virus, either does not provide genetic material or is recombined with the MVA genome when introduced into the transfection / infection incubation of BHK or CEF cells. 43、44 .

[0054] To characterize the reconstituted sMVA genome, viral DNA prepared from sMVA-infected BHK cells was evaluated by PCR using primers specific to different genomic locations within the three synthetic MVA fragments. DNA prepared from pseudoinfected (uninfected) BHK cells and BHK cells infected with NIH MVA clone 1 were used as controls. This PCR analysis revealed that DNA derived from all three sMVA fragments was present within the reconstituted sMVA genomic DNA (Figure 7). All PCR products derived from sMVA-infected BHK cells were similar (in molecular weight) to those derived from MVA NIH-infected BHK cells. This included (1) PCR products derived from ITR sequences located in sMVA F1 and F3 (Figure 7A); (2) PCR products corresponding to sMVA F1-derived DNA, e.g., the transition from the left ITR to the left end of the internal UR (Figure 7B) and the Del2 insertion site (Figure 7C); (3) PCR products corresponding to sMVA F2-derived DNA, e.g., the G1L / I8R insertion site (IGR69 / 70) (Figure 7D); and sMVA F3-derived DNA, e.g., the Del3 insertion site (Figure 7E) and the transition from the right end of the internal UR to the right ITR (Figure 7F). Furthermore, PCR analysis using primers containing binding sites adjacent to the duplicate homologous sequences of sMVA F1 / F2 or sMVA F2 / F3 (Figure 1) showed similar product sizes for MVA-infected BHK cells and MVA NIH-infected BHK cells (Figures 7G-7H), indicating successful recombination of the three sMVA fragments and removal of the vector sequence within the sMVA.

[0055] Sanger sequencing analysis of all PCR products derived from sMVA-infected BHK cells revealed that the MVA Antoine strain contains recombination sites for F1 / F2 and F2 / F3 cells. 26 The sequences of these genomic locations were identified as being identical to the publicly released sequences. The only exception to this was the open reading frame (ORF) 021L. 26The PCR product originated from the Del2 site of sMVA, showing a one-nucleotide change from T to A located three base pairs downstream of the IGR. BLAST analysis showed that this sequence change in sMVA DNA is not present in published MVA or vaccinia virus genome sequences. Sanger sequencing of sMVA F1 purified from E. coli showed that this specific nucleotide change at the Del2 site was already present before sMVA rearrangement in BHK cells, suggesting that it originates from the chemosynthesis of F1 or is a result of cloning or proliferation of sMVA F1 in E. coli. Additional sequencing analysis of PCR products derived from sMVA-infected BHK cells (Figures 7I-7L) showed that the sMVA genome sequence contains all five Antoine-specific nucleotide polymorphisms in the internal UR that distinguish it from the MVA NIH clone 1 genome sequence.

[0056] The following DNA sequence is shown as the nucleotide sequence (5' to 3') of the sense strand of the DNA molecule. Sequence of sMVA fragment 1 (F1, 60021 bp long) (SEQ ID NO: 11): TIFF2026094336000012.tif128150TIFF2026094336000013.tif231150TIFF2026094336000014.t if231150TIFF2026094336000015.tif231150TIFF2026094336000016.tif231150TIFF20260943360 00017.tif231150TIFF2026094336000018.tif231150TIFF2026094336000019.tif231150TIFF202 6094336000020.tif231150TIFF2026094336000021.tif231150TIFF2026094336000022.tif231150 TIFF2026094336000023.tif231150TIFF2026094336000024.tif231150TIFF2026094336000025.t if231150TIFF2026094336000026.tif231150TIFF2026094336000027.tif231150TIFF20260943360 00028.tif231150TIFF2026094336000029.tif231150TIFF2026094336000030.tif231150TIFF2026 094336000031.tif231150TIFF2026094336000032.tif231150TIFF2026094336000033.tif176150.

[0057] Any of the sequences of sMVA fragments F1, F2, and F3 may contain one or more modifications or changes. For example, the sequence of sMVA fragment F1 (deposited with NCBI under accession number MW023923 (www.ncbi.nlm.nih.gov / nuccore / MW023923.1 / )) contains a one-nucleotide modification in the non-coding determination region downstream of open reading frame 021 (SEQ ID NO: 12): TIFF2026094336000034.tif231150TIFF2026094336000035.tif231150TIFF2026094336000036.t if231150TIFF2026094336000037.tif231150TIFF2026094336000038.tif231150TIFF20260943360 00039.tif231150TIFF2026094336000040.tif231150TIFF2026094336000041.tif231150TIFF202 6094336000042.tif231150TIFF2026094336000043.tif231150TIFF2026094336000044.tif231150 TIFF2026094336000045.tif231150TIFF2026094336000046.tif231150TIFF2026094336000047.t if231150TIFF2026094336000048.tif231150TIFF2026094336000049.tif231150TIFF20260943360 00050.tif231150TIFF2026094336000051.tif231150TIFF2026094336000052.tif231150TIFF202 6094336000053.tif231150TIFF2026094336000054.tif231150TIFF2026094336000055.tif73150.

[0058] Sequence of sMVA fragment 2 (F2, 63035 bp long) (deposited with NCBI under accession number MW023924 (www.ncbi.nlm.nih.gov / nuccore / MW023924.1 / )) (SEQ ID NO: 13): TIFF2026094336000056.tif 128150 TIFF2026094336000057.tif 231150 TIFF2026094336000058.tif 231150 TIFF2026094336000059.tif 231150 TIFF2026094336000060.tif 231150 TIFF2026094336000061.tif 231150 TIFF2026094336000062.tif 231150 TIFF2026094336000063.tif 231150 TIFF2026094336000064.tif 231150 TIFF2026094336000065.tif 231150 TIFF2026094336000066.tif 231150 TIFF2026094336000067.tif 231150 TIFF2026094336000068.tif 231150 TIFF2026094336000069.tif 231150 TIFF2026094336000070.tif 231150 TIFF2026094336000071.tif 231150 TIFF2026094336000072.tif 231150 TIFF2026094336000073.tif 231150 TIFF2026094336000074.tif 231150 TIFF2026094336000075.tif 231150 TIFF2026094336000076.tif 231150 TIFF2026094336000077.tif 231150 TIFF2026094336000078.tif 224150 TIFF2026094336000079.tif 11150。

[0059] Sequence of sMVA fragment 3 (F3, 62068 bp long) (SEQ ID NO:14): TIFF2026094336000080.tif196150TIFF2026094336000081.tif231150TIFF2026094336000082.tif23 1150TIFF2026094336000083.tif231150TIFF2026094336000084.tif231150TIFF2026094336000085.t if231150TIFF2026094336000086.tif231150TIFF2026094336000087.tif231150TIFF20260943360000 88.tif231150TIFF2026094336000089.tif231150TIFF2026094336000090.tif231150TIFF20260943360 00091.tif231150TIFF2026094336000092.tif231150TIFF2026094336000093.tif231150TIFF2026094 336000094.tif231150TIFF2026094336000095.tif231150TIFF2026094336000096.tif231150TIFF202 6094336000097.tif231150TIFF2026094336000098.tif231150TIFF2026094336000099.tif231150TIF F2026094336000100.tif231150TIFF2026094336000101.tif231150TIFF2026094336000102.tif93150.

[0060] Alternatively, the sMVA F3 sequence (deposited with NCBI under accession number MW030459 (www.ncbi.nlm.nih.gov / nuccore / MW030459.1 / )) may contain a single nucleotide change in the non-coding region at 88 bp near the end of the ITR sequence (SEQ ID NO: 15): TIFF2026094336000103.tif100150TIFF2026094336000104.tif231150TIFF2026094336000105.tif23 1150TIFF2026094336000106.tif231150TIFF2026094336000107.tif231150TIFF2026094336000108.t if231150TIFF2026094336000109.tif231150TIFF2026094336000110.tif231150TIFF20260943360001 11.tif231150TIFF2026094336000112.tif231150TIFF2026094336000113.tif231150TIFF20260943360 00114.tif231150TIFF2026094336000115.tif231150TIFF2026094336000116.tif231150TIFF2026094 336000117.tif231150TIFF2026094336000118.tif231150TIFF2026094336000119.tif231150TIFF2026 094336000120.tif231150TIFF2026094336000121.tif231150TIFF2026094336000122.tif231150TIFF 2026094336000123.tif231150TIFF2026094336000124.tif231150TIFF2026094336000125.tif190150.

[0061] An example of a synthetic vaccinia virus (sVAC) for reconstructing a complete vaccinia virus genome is also disclosed. An example of the sequence of sVAC fragment 1 (F1, 66679 bp long) (SEQ ID NO: 16): TIFF2026094336000126.tif11150TIFF2026094336000127.tif231150TIFF2026094336000128.tif231150TIFF2026094336000129.tif231150TIFF2026094336000130.tif231150TIFF2026094336000131.tif231150TIFF2026094336000132.tif231150TIFF2026094336000133.tif231150TIFF2026094336000134.tif231150TIFF2026094336000135.tif231150TIFF2026094336000136.tif231150TIFF2026094336000137.tif231150TIFF2026094336000138.tif231150TIFF2026094336000139.tif231150TIFF2026094336000140.tif231150TIFF2026094336000141.tif231150TIFF2026094336000142.tif231150TIFF2026094336000143.tif231150TIFF2026094336000144.tif231150TIFF2026094336000145.tif231150TIFF2026094336000146.tif231150TIFF2026094336000147.tif231150TIFF2026094336000148.tif231150TIFF2026094336000149.tif231150TIFF2026094336000150.tif128150。

[0062] An example of sVAC fragment 2 (66,679 bp long) (SEQ ID NO:17): TIFF2026094336000151.tif80150TIFF2026094336000152.tif231150TIFF2026094336000153.tif231150TIFF2026 094336000154.tif231150TIFF2026094336000155.tif231150TIFF2026094336000156.tif231150TIFF20260943360 00157.tif231150TIFF2026094336000158.tif231150TIFF2026094336000159.tif231150TIFF2026094336000160.t if231150TIFF2026094336000161.tif231150TIFF2026094336000162.tif231150TIFF2026094336000163.tif231150 TIFF2026094336000164.tif231150TIFF2026094336000165.tif231150TIFF2026094336000166.tif231150TIFF202 6094336000167.tif231150TIFF2026094336000168.tif231150TIFF2026094336000169.tif231150TIFF20260943360 00170.tif231150TIFF2026094336000171.tif231150TIFF2026094336000172.tif231150TIFF2026094336000173.t if231150TIFF2026094336000174.tif231150TIFF2026094336000175.tif231150TIFF2026094336000176.tif38150.

[0063] An example of the sequence of sVAC fragment 3 (F3, 66679 bp long) (SEQ ID NO: 18): TIFF2026094336000177.tif169150TIFF2026094336000178.tif231150TIFF2026094336000179.tif231150TIFF2026094336000180.tif231150TIFF2026094336000181.tif231150TIFF2026094336000182.tif231150TIFF2026094336000183.tif231150TIFF2026094336000184.tif231150TIFF2026094336000185.tif231150TIFF2026094336000186.tif231150TIFF2026094336000187.tif231150TIFF2026094336000188.tif231150TIFF2026094336000189.tif231150TIFF2026094336000190.tif231150TIFF2026094336000191.tif231150TIFF2026094336000192.tif231150TIFF2026094336000193.tif231150TIFF2026094336000194.tif231150TIFF2026094336000195.tif231150TIFF2026094336000196.tif231150TIFF2026094336000197.tif231150TIFF2026094336000198.tif231150TIFF2026094336000199.tif231150TIFF2026094336000200.tif231150TIFF2026094336000201.tif32150。

[0064] To evaluate the removal of bacterial vector sequences after sMVA reconstitution, DNA from sMVA-infected BHK cells during successive viral passages of sMVA in BHK cells was evaluated by PCR using primers specific to the sopA and cat chloramphenicol resistance genes of the bacterial vector. This PCR analysis showed that residual vector sequences could be detected in sMVA DNA for 7 or 8 dpt / i, but could no longer be detected in sMVA DNA after two further viral passages of sMVA. Taken together, these results indicate that cotransfection of three synthetic MVA fragments as circular plasmid molecules into FPV-infected BHK cells with MVA Antoine strain 26 This provides evidence that it leads to the formation of a vector-free, full-length genome sMVA, which is thought to have a sequence composition similar to the publicly released genome sequence.

[0065] Example 2: Generation of recombinant sMVA expressing a single heterologous gene sequence To evaluate the use of three sMVA fragments to generate rsMVA, we performed en passant mutagenesis. 39、40 GS1783 E. coli cells used to manipulate large DNA molecules cloned into BAC 40 Three fragments were introduced. This highly efficient method allows for the introduction of point mutations or large or small sequence insertions or deletions into DNA molecules cloned in BAC by a two-step mutagenesis-based Red recombination method, without preserving bacterial marker sequences. 39、40 As the first test of an sMVA platform for generating rsMVA expressing heterologous gene sequences, the generation of rsMVA expressing a single fluorescent marker was evaluated. MVABAC-TK 18、24As previously described for the insertion of antigen sequences into , an expression cassette consisting of a red fluorescent protein (RFP) marker, an upstream mH5 promoter, and a downstream TTTTTAT vaccinia virus transcription termination signal was inserted into the IGR69 / 70 insertion site (also known as G1L / I8R) within sMVA fragment F2 (Figure 3) using en passant mutagenesis to obtain F2-RFP. Sequence integrity of the manipulated sMVA fragment was determined by restriction fragment length polymorphism (RFLP) analysis and PCR and Sanger sequencing analysis of the gene insertion site. Modified sMVA fragment F2 with the inserted RFP marker, as well as unmodified sMVA fragments F1 and F3, were isolated from E. coli and co-transfected into BHK cells in the presence of FPV as a helper virus to evaluate rsMVA rearrangement using the procedure illustrated in Figure 5. As shown in Figure 8, immunofluorescence imaging revealed the formation and progression of red fluorescent viral plaques in BHK cell monolayers transfected with the F1 / F2-RFP / F3 plasmid combination at 3–6 dpt / i, and RFP expression was visible throughout the BHK cell monolayer at 7 dpt / i. In contrast, no RFP expression was observed in pseudotransfected / infected BHK cell monolayers. Taken together, these results demonstrate that the sMVA platform can be used to rapidly generate rsMVAs expressing a single heterologous gene sequence.

[0066] Example 3: Generation of recombinant sMVA expressing two heterologous gene sequences To evaluate the sMVA platform for generating rsMVA with multiple heterologous gene sequences inserted at different genomic locations, the construction of rsMVA expressing two fluorescent markers was assessed. Using en passant mutagenesis in GS1783 cells, gene expression cassettes consisting of the P11 promoter, an RFP marker or a blue fluorescent protein (BFP) marker, and a TTTTTAT transcription termination signal were separately inserted at IGR69 / 70 in sMVA F2 and the Del3 site in sMVA F3. Modified sMVA F2 with the RFP marker inserted (F2-RFP) and modified sMVA F3 with the BFP marker inserted (F3-BFP) were isolated from GS1783 E. coli cells and co-transfected into BHK cells along with unmodified sMVA F1 in the presence of a helper virus to evaluate the reconstitution of rsMVA with the RFP and BFP markers using the procedure shown in Figure 5. As shown in Figure 9, immunofluorescence imaging demonstrated the formation of bifluorescent (red and blue) viral plaques in a monolayer of BHK cells cotransfected with the F1 / F2-RFP / F3-BFP plasmid combination at 3–6 dpt / i, and at 7 dpt / i, the majority or all of the BHK cells appeared to exhibit bifluorescent protein expression of RFP and BFP. These results provide evidence that the sMVA platform can be used to rapidly generate rsMVAs expressing two heterologous gene sequences inserted at different insertion sites.

[0067] Example 4: Generation of recombinant sMVA expressing three heterologous gene sequences To further evaluate the sMVA platform for generating rsMVA with multiple heterologous gene sequences inserted, we investigated the generation of rsMVA expressing three fluorescent markers. Using en passant mutagenesis in E. coli, the green fluorescent protein (GFP) marker was inserted into IGR44 / 45 of MVA F1, along with the upstream P11 promoter and downstream transcription termination signal. Then, using the procedure shown in Figure 5, the modified sMVA fragment F1 with the inserted GFP marker (F1-GFP) was tested in various combinations with the fluorescently tagged forms of F2 and F3 (F2-RFP and F3-BFP), as well as the unmodified sMVA fragments of F1, F2, and F3, to evaluate single-fluorescence, double-fluorescence, or triple-fluorescence sMVA expression vectors. The fragment combinations used for cotransfection included F1-GFP / F2-RFP / F3-BFP, F1-GFP / F2-RFP / F3, F1-GFP / F2 / F3-BFP, F1 / F2-RFP / F3-BFP, F1-GFP / F2 / F3, F1 / F2-RFP / F3, and F1 / F2 / F3-BFP. Cotransfection of three unmodified sMVA fragments (F1 / F2 / F3) into FPV-infected BHK cells was evaluated as a negative control. In BHK cell monolayers transfected with different fragment combinations (excluding the negative control), fluorescent gene expression was observed at 3–6 dpt / i by immunofluorescence imaging. Fluorescent marker expression was observed in the majority of cells in the BHK monolayer at 7–8 dpt / i, indicating the reconstitution of the fluorescent recombinant sMVA expression vector. Recombinant sMVA expression vectors were prepared using a freeze / thaw method, titrated in BHK cells, and used to infect BHK cell monolayers at a low MOI. At 16–24 hpi, the infected BHK cell monolayers were evaluated by IF imaging to confirm the formation and reconstitution of sMVA expressing different fluorescent marker sequences.This analysis revealed the formation of viral foci, showing triple fluorescence expression in BHK cell monolayers infected with sMVA derived from F1-GFP / F2-RFP / F3-BFP; double fluorescence expression in BHK cell monolayers infected with sMVA derived from F1-GFP / F2-RFP / F3, F1-GFP / F2 / F3-BFP, or F1 / F2-RFP / F3-BFP; and single fluorescence expression in BHK cell monolayers infected with sMVA derived from F1-GFP / F2 / F3, F1 / F2-RFP / F3, or F1 / F2 / F3-BFP. Taken together, these results indicate that the three sMVA fragments can be manipulated in E. coli and used as circular DNA molecules to reconstitute single, double, or triple recombinant sMVA expression vectors, highlighting the potential of the three sMVA fragments for generating recombinant vaccine vectors.

[0068] Example 5: Construction and evaluation of a case of infectious disease antigen-modified MVA To demonstrate the usability of the sMVA platform for generating multi-antigenic vaccine vectors for infectious diseases, we evaluate the construction of rsMVA expressing a five-member HCMV envelope pentamer complex (PC) composed of gH, gL, UL128, UL130, and UL131A. Antigen expression cassettes consisting of a gH / gL subunit and a UL128 / UL130 / UL131A subunit linked by 2A are separately inserted into the IGR69 / 70 site located at sMVA F2 and the Del3 site located at sMVA F3 using en passant mutagenesis in E. coli. To reconstitute sMVA expressing all five PC subunits (rsMVA-PC), modified sMVA fragments from F2 and F3 are transfected into FPV-infected BHK cells along with the unmodified sMVA fragment from F1. As controls, we generate rsMVA expressing only the gH / gL subunit or the UL128 / 130 / 131A subunit, and sMVA without an inserted antigen sequence. Antigen sequence expression is confirmed by Western blotting, and the formation of multiprotein complexes by PC subunits expressed from rsMVA-PC is verified by flow cytometry (FC) using a neutralizing antibody (NAb) targeting the structural epitope formed by two or more PC subunits. To investigate the immunogenicity of the rsMVA-PC vector for NAb stimulation, Balb / c mice are immunized three times with either rsMVA-PC or a control vector via the intraperitoneal route at 4-week intervals. HCMV-specific NAb responses are measured in MRC-5 fibroblasts (FB) and ARPE-19 epithelial cells (EC) at various postimmunization time points. This analysis reveals potent stimulation of FB and EC-specific NAb responses in mice immunized with rsMVA-PC, while no response or minimal response is measured in mice immunized with the control vector. These results demonstrate that an sMVA vaccine platform could be used to generate a multi-antigenic MVA vaccine vector expressing all five PC subunits that assemble together to form a NAb epitope, thereby stimulating a potent NAb response in mice.

[0069] Example 6: Construction and evaluation of 2019-nCoV expressed from sMVA An example of the potential use of the sMVA platform for generating a vaccine against the recently emerged coronavirus 2019-nCoV strain (Wuhan), a highly transmissible and pathogenic novel coronavirus, is illustrated. The coronavirus S (spike) protein is involved in receptor recognition, as well as viral attachment and entry, and is a major target of protective humoral and cellular immunity, thus representing a potential target for the development of vaccines and therapeutics against coronaviruses (CoV). The full-length S protein (approximately 1273 amino acids) of the 2019-nCoV strain, the immunogenic S1 domain of the S protein, or the receptor-binding domain (RBD) of the S1 domain are separately inserted into the MVA Del3 site of sMVA fragment F3. Next, using a procedure illustrated in Figure 5, the modified sMVA F3 fragment obtained is cotransfected into BHK cells along with the unmodified versions of sMVA fragments F1 and F2 to initiate the reconstitution of sMVA expressing the novel coronavirus S protein (sMVA-2019-nCoV-S), S1 domain (sMVA-2019-nCoV-S1), or RBD domain (sMVA-2019-nCoV-RBD). The expression of the S protein, S1 domain, or RBD domain from the recombinant sMVA is confirmed by immunoblotting and flow cytometry. Convalescent serum derived from infected humans, mice, or rabbits is used as a detection reagent for immunoblotting. In some cases, tags such as HA are covalently attached to the carboxyl terminus, and antibodies against them are used as detection reagents for either immunoblotting or flow cytometry. Similarly, to develop a multi-antigen vaccine, additional immunogenic proteins, such as N or M antigens derived from 2019-nCoV, are inserted into the unique gene insertion regions of the sMVA. For example, one or more of these 2019-nCoV antigens are inserted into one or more insertion sites, such as the Del3 site of sMVA fragment F3 and IGR69 / 70 of sMVA fragment F2.

[0070] Two animal models can be used to test the immunogenicity and protective efficacy of the sMVA construct: (1) 12-month-old Balb / c mice, 1 × 10⁶, as more severe symptoms are observed in older individuals. 7 ~1 × 10 8 (2) Immunization is performed twice, at 4-week intervals, via subcutaneous and intramuscular pathways, with sMVA plaque-forming units (Pfu). (3) Considering the possible involvement of human angiotensin-converting enzyme 2 (hACE-2) in the entry of nCoV into lung cells, transgenic mice expressing the human hACE2 receptor are used to test the efficacy of the vaccine.

[0071] To test the immunogenicity and protective efficacy of the sMVA construct, transgenic Balb / c mice were used to test for angiotensin-converting enzyme 2 (ACE2) at a rate of 1 × 10⁻¹⁴. 7 ~1 × 10 8 The mice are immunized twice, 4 weeks apart, via subcutaneous and intramuscular pathways, with sMVA plaque-forming units (Pfu). Neutralizing antibody responses and T-cell responses specific to the 2019-nCoV-S protein and derivatives are measured one week after each immunization, as described above. On day 45 after booster immunization, the mice are immunized with 7 × 10⁶ 2019-nCoV homologous to the vaccine antigen, heterologous strains (SARS-CoV and MERS-CoV). 4Animals were infected with a 50% tissue culture infectious dose (TICD50), sacrificed 4 days after challenge, and lungs were collected for viral load measurement and histopathological analysis. Large viral loads were found in both the pseudoimmune control and non-recombinant MVA immunoimmune control groups, and in animals challenged with heterologous CoV strains, with an average of over 11,000–20,000 2019-nCoV genomic equivalents per ng of total RNA. In stark contrast, lung tissue targeted for the protein (sMVA-nCoV-S) or S1 domain (sMVA-nCoV-S1) contained significantly lower levels of 2019-nCoV RNA (viral load), indicating efficient inhibition of 2019-nCoV replication by the vaccine-induced immune response. All placebo-controlled animals (MVA-GFP) succumbed to the infection 4–8 days after infection, while MVA-S, MVA-N, and MVA-N / S showed no signs of disease at all or only minimal signs, such as minimal weight loss. Mice immunized with MVA-N / S were completely protected.

[0072] Histopathology focuses on the substantially reduced lung injury observed in animals immunized with 2019-nCoV subunit antigens. The possibility of antibody enhancement of the disease is also evaluated in these subunit vaccine models and is found to be absent. NAb responses to the S protein antigen and its S1 and RBD derivatives are assessed using a pseudotyping strategy for nCoV robust to recognition by antigen-specific NAb, but the use of pathogenic CoV strains is avoided. High titer neutralizing antibodies are generated in serum from animals immunized with the S protein and derivatives against homologous 2019-nCoV strains, but not against heterologous CoV strains, such as the MERS and SARS pathogens. IC90 neutralizing titers are calculated for each mouse serum sample. Based on the success of vaccine studies in the transgenic ACE2 Balb / c mouse strain, we will investigate progressively larger animal species known to be susceptible to 2019-nCoV challenge and respond to MVA-based vaccines, namely rabbits, ferrets, and rhesus monkeys.

[0073] Example 7: Construction and evaluation of a case of cancer antigen-modified sMVA This embodiment is ATCC or NIAID 46This study demonstrates the insertion of the mouse p53 gene previously used in research using the MVA skeleton obtained from [source]. The mouse p53 cDNA is inserted into the deletion (DEL3) locus using en passant to ensure that no additional nucleotides are added or deleted at the precise insertion site. The insertion is verified by sequence analysis, and all relevant regions are intact and 100% sequence-verifiable. After modification of plasmid F3, all three plasmids are combined into a single aliquot, and a modified version of the sMVA is generated using the same method as described in Example 2, yielding live virus two days after transfection / infection with FPV. The live virus is enriched and amplified in BHK cells to create a stock, and sequence-verified using Sanger to establish that the virus has the correct sequence of the p53 gene insertion at the DEL3 locus. Furthermore, the virus stock is prepared, frozen, and stored long-term, while simultaneously producing 1 × 10⁶ modified sMVA stocks. 9 To expand the titer to above pfu / mL, a working stock is prepared using a published method. Validation of the expanded stock is performed using a combination of qPCR, Sanger sequencing, infectivity titer, and Western blot analysis. All four of these methods are performed to ensure the nucleotide sequence is of appropriate size and the infectivity titer is 1 × 10⁻⁶. 9 To confirm that the result is within the range of pfu / mL or higher, appropriate results are obtained and a protein band of approximately 53Kd is confirmed by Western blot analysis. Those skilled in the art can apply this approach using a human version of the p53 gene as a vaccine to protect humans from cancer progression.

[0074] Mouse (mu)p53-sMVA Female 6-8 week old BALB / C mice are obtained from Jackson Laboratory (Bar Harbor, ME) and maintained in a pathogen-free environment. All studies are approved by the Research Animal Care Committee of City of Hope National Medical Center and conducted under the American Association for the Accreditation of Laboratory Animal Care guidelines. Meth A sarcoma cells (Meth A) are generously donated by Dr. LJ Old (Memorial Sloan-Kettering Cancer Center (New York, NY)). Meth A cells are passaged as ascites tumors. Anti-CTLA-4 (9H10) is donated by Dr. James Allison (MD Anderson Cancer Center (Houston, TX)). Antibodies are produced using a CELLine device (BD Biosciences, Mountain View, CA). IgG antibodies are purified by absorption with Protein G Sepharose (Amersham Biosciences, Uppsala, Sweden) followed by elution with 0.1 M glycine HCl (pH 2.7). The product is dialyzed against phosphate-buffered ordinary saline and concentrated using a Sentry Plus centrifugal filter (Millipore, Bedford, MA). Control Syrian hamster IgG is obtained from Jackson ImmunoResearch (West Grove, PA). rsMVA titers are determined by immunostaining of infected cultures using the VECTASTAIN Elite ABC kit (Vector Laboratories, Burlingame, CA). Antigen-specific detection of mouse p53 protein expressed from sMVA is performed using the anti-p53 antibody pab122, followed by incubation with the peroxidase-labeled goat anti-mouse secondary antibody provided in the kit. Control sMVA expressing CMV-pp65 is also constructed using the same method as used in the construction of mouse sMVAp53.

[0075] In vivo tumor challenge experiment In the left flank of a 6-week-old female BALB / C mouse, 5 × 10¹⁴ cells were found subcutaneously. 5 Inject 5 x 10⁶ METH A cells. On the third day, administer 5 x 10⁶ cells via intraperitoneal injection. 7 Mice were treated with pfu sMVAp53. Negative control mice were treated with 5 × 10⁶ 7 Inject pfu-containing sMVApp65 or phosphate-buffered saline. An additional positive control is used, identical to the mouse MVAp53 described in the initial report. Evaluate the subcutaneous tumor weekly by IVIS imaging using the luciferase method, or by caliper if the luciferase gene is not inserted into METH A sarcoma cells. An additional approach is to inject 1 × 10⁶ smears into the left flank of BALB / C mice of the same age as sMVA infection. 6 The procedure involves injecting 5 × 10⁶ METH A cells. This tumor has been shown to rapidly produce lethal tumors in the majority of mice, despite CTLA-4(9H10) antibody treatment. Seven days after tumor inoculation, 5 × 10⁶ cells are administered. 7 Pfu-sMVAp53 was intraperitoneally injected into mice. The control was the same as described above. Anti-CTLA-4(9H10) antibody or control hamster antibody was intraperitoneally injected at doses of 100 micrograms, 50 micrograms, and 50 micrograms, respectively, on days 6, 9, and 12 after tumor injection.

[0076] Example 8: Construction of sMVA and in vitro and in vivo feature determination The three sMVA fragments, designed as shown in Figure 1, are cotransfected into helper virus-infected cells as circular DNA plasmids. These fragments then separate into linear minichromosomes, recombinate with each other via shared homologous sequences, and ultimately package into a full-length MVA genome. All three sMVA fragments were cloned into E. coli as bacterial artificial chromosome (BAC) clones.

[0077] Methods previously used to rescue MVA from BAC 16、24、25Using this method, sMVA virus was reconstituted by cotransfecting BHK cells, which are intolerant to FPV, with three DNA plasmids along with fowlpox (FPV) as a helper virus (Figure 2). Two different FPV strains (HP1.441 and TROVAC) were used to facilitate sMVA virus reconstitution (Figure 12A). After viral replication in CEF, which is commonly used for MVA vaccine production, highly purified sMVA virus was produced. The viral titers achieved by the reconstituted sMVA virus were similar to those achieved by wild-type MVA (wtMVA) (Table 1 below).

[0078] TIFF2026094336000202.tif41128 * The stock was prepared in CEF after infection (MOI 0.02) of a 30 x 15 cm dish.

[0079] To characterize the viral DNA of sMVA, DNA extracts from sMVA-infected CEFs and wtMVA-infected CEFs were compared at several MVA genomic locations by PCR. Similar PCR results were obtained for sMVA and wtMVA at all evaluated genomic locations, including the F1 / F2 and F2 / F3 recombination sites (Figure 11A), indicating efficient recombination of the three sMVA fragments. Additional PCR analysis showed the absence of BAC vector sequences within the sMVA viral DNA (Figure 11A), suggesting that bacterial vector elements are spontaneously and efficiently removed by sMVA viral recombination. Comparison of viral DNA from highly purified sMVA and wtMVA viruses by restriction enzyme digestion revealed similar genomic patterns between sMVA and wtMVA (Figure 11B). Sequencing analysis of sMVA viral DNA confirmed the MVA genomic sequence at several locations, including the F1 / F2 and F2 / F3 recombination sites. Furthermore, whole-genome sequencing of one of the sMVA virus isolates reconstructed by FPV TROVAC confirmed the assembly of the reference MVA genome sequence and the absence of vector-specific sequences in the viral DNA resulting from the reconstructed sMVA virus.

[0080] To characterize the replication characteristics of sMVA, the proliferation kinetics of sMVA and wtMVA were compared in two cell types known to support productive MVA replication: BHK cells and CEF cells. 4 This analysis revealed similar proliferation kinetics of sMVA and wtMVA in both BHK and CEF cells (Figure 12B). Furthermore, similar areas of viral focus were determined in monolayers of BHK and CEF cells infected with sMVA or wtMVA (Figure 12C), suggesting similar diffusive capabilities of sMVA and wtMVA in MVA-tolerant cells compared to the productive replication of sMVA and wtMVA in BHK and CEF cells. 4Following infection of various human cell lines, only limited viral production was observed with sMVA or wtMVA (Figure 12D). These results are consistent with the strictly limited replication characteristics of MVA and indicate that sMVA viruses can efficiently replicate in BHK and CEF cells, but not in human cells.

[0081] To characterize sMVA in vivo, the immunogenicity of sMVA and wtMVA was compared in C57BL / 6 mice after two immunizations with high or low doses. MVA-specific binding antibodies stimulated by sMVA and wtMVA after the first and second immunizations were comparable (Figures 13A, 14A). After the first immunization, antibody levels in the high-dose vaccine group were higher than those in the low-dose vaccine group, but after the second immunization, similar antibody levels were observed in both groups. Furthermore, no significant difference was detected in the level of MVA-specific NAb response induced by sMVA and wtMVA after the second immunization (Figures 13B, 14B). Immunodominant peptide 47 MVA-specific T cell responses determined after additional immunization induced by ex vivo antigen stimulation using revealed similar MVA-specific T cell levels in mice receiving sMVA or wtMVA (Figures 13C-13D and 14C-14D). These results indicate that sMVA viruses have a similar ability to wtMVA in inducing MVA-specific humoral and cellular immunity in mice.

[0082] References The references, patents, and published patent applications listed below, as well as all references cited in the above specification, are incorporated herein by reference in their entirety as if they were fully presented herein. TIFF2026094336000203.tif190150TIFF2026094336000204.tif228150TIFF2026094336000205.tif225150TIFF2026094336000206.tif225150TIFF2026094336000207.tif225150TIFF2026094336000208.tif104150

Claims

1. A step of transfecting one or more DNA fragments into a host cell, wherein the one or more DNA fragments include the entire genomic DNA sequence of the poxvirus so that a desired poxvirus is reconstituted in the host cell. A method for producing a poxvirus vector or recombinant poxvirus vector containing [the specified substance].

2. The method according to claim 1, wherein two or more DNA fragments are cotransfected into the host cell, and each DNA fragment comprises a partial sequence of the poxvirus genome such that the two or more DNA fragments are assembled by homologous recombination and, when reconstituted in the host cell, comprise the full sequence of the poxvirus genome.

3. The method according to claim 1 or 2, further comprising the step of infecting the host cell with a helper virus before, during, or after transfection of the one or more DNA fragments in order to initiate transcription of the one or more DNA fragments.

4. The method according to claim 3, wherein the helper virus is fowlpox virus (FPV), sheep fibroma virus, vaccinia virus, or cowpox virus.

5. The method according to any one of claims 1 to 4, wherein one or more DNA fragments are cyclized before transfection or transfected into the host cell in a circular form.

6. The method according to claim 5, wherein one or more DNA fragments are cloned into a plasmid vector or a bacterial artificial chromosome (BAC) vector.

7. The method according to any one of claims 1 to 4, wherein one or more DNA fragments are linearized before cotransfection or transfected into the host cell in a linearized form.

8. The method according to any one of claims 1 to 7, wherein the one or more DNA fragments are a naturally derived DNA fragment, a chemically synthesized DNA fragment, or a combination of a naturally derived DNA fragment and a chemically synthesized DNA fragment.

9. The method according to any one of claims 1 to 8, wherein the poxvirus genome sequence comprises the sequence of modified vaccinia ankara (MVA) accession number #U94848 or #AY603355.

10. The method according to any one of claims 1 to 9, wherein two adjacent DNA fragments have overlapping sequences to facilitate homologous recombination.

11. The method according to claim 10, wherein the overlapping sequence is approximately 100 bp to approximately 5000 bp in length.

12. The method according to any one of claims 1 to 11, wherein the one or more DNA fragments further comprises an ITR region.

13. The method according to any one of claims 1 to 12, wherein the one or more DNA fragments further comprises a poxvirus terminal hairpin loop (HL) sequence, a poxvirus genome resolution (CR) sequence, or both, and the HL sequence or the CR sequence is appended to one or both ends of the DNA fragment as a sense-directed or antisense-directed single-stranded or double-stranded DNA sequence.

14. The method according to claim 13, wherein the one or more DNA fragments further comprises one or more HL sequences and one or more CR sequences.

15. The method according to claim 14, wherein each HL sequence is adjacent to two CR sequences at both ends of the HL sequence.

16. The method according to any one of claims 13 to 15, wherein only a subset of the one or more DNA fragments comprises the HL sequence or the CR sequence.

17. The method according to any one of claims 1 to 16, wherein the one or more DNA fragments further comprises one or more DNA sequences encoding one or more antigens, subunits or fragments thereof, or other heterogeneous DNA sequences.

18. The method according to claim 17, wherein two or more DNA fragments comprise the same antigen, the DNA sequence of its subunits or fragments, or the same heterologous DNA sequence.

19. The method according to claim 17, wherein two or more DNA fragments comprise different antigens, DNA sequences of their subunits or fragments, or other heterogeneous DNA sequences.

20. The method according to any one of claims 17 to 19, wherein the DNA sequence of the antigen, its subunit or fragment, or other heterologous DNA sequence is codon-optimized for expression in the host cell.

21. The method according to any one of claims 17 to 20, wherein the one or more DNA fragments further comprises a viral promoter upstream of the DNA sequence of the antigen, its subunit or fragment, or other heterologous DNA sequence, or further comprises a transcription termination signal downstream of the DNA sequence of the antigen, its subunit or fragment, or other heterologous DNA sequence, or both.

22. The method according to any one of claims 17 to 21, wherein a DNA sequence encoding the antigen, a subunit or fragment thereof, or other heterologous DNA sequence is inserted into one or more poxvirus insertion sites or intergenetic regions.

23. (i) A single DNA fragment containing the entire genome of the desired poxvirus, When expressed in host cells by cotransfection, two or more DNA fragments are sequentially assembled and contain the full sequence of the poxvirus genome, and each of the two or more DNA fragments contains a partial sequence of the desired poxvirus genome, enabling the reconstruction of the desired recombinant poxvirus, and (ii) One or more DNA sequences or other heterologous DNA sequences that encode one or more antigens, subunits or fragments thereof, inserted into one or more insertion sites of the poxvirus, wherein the antigens, subunits or other heterologous DNA sequences are expressed in the host cell by transfection of one or more poxvirus DNA fragments and rearrangement of the poxvirus. An expression system that includes this.

24. The expression system according to claim 23, wherein one or more of the DNA fragments are cyclized before transfection or transfected into the host cell in a circular form.

25. The expression system according to claim 24, wherein one or more of the DNA fragments are cloned into a plasmid vector or a BAC vector.

26. The expression system according to claim 23, wherein one or more of the DNA fragments are linearized before transfection, or are transfected into the host cell in a linearized form.

27. The expression system according to any one of claims 23 to 26, wherein one or more of the DNA fragments are naturally derived DNA fragments, chemically synthesized DNA fragments, or a combination of naturally derived DNA fragments and chemically synthesized DNA fragments.

28. The expression system according to any one of claims 23 to 27, wherein the genome sequence of the poxvirus includes the sequence of MVA accession number #U94848 or #AY603355.

29. The expression system according to any one of claims 23 to 28, wherein the two adjacent DNA fragments have overlapping sequences to facilitate homologous recombination.

30. The expression system according to claim 29, wherein the overlapping sequence is approximately 100 bp to approximately 5000 bp in length.

31. The expression system according to any one of claims 23 to 30, wherein one or more of the DNA fragments further comprises a terminal inversion (ITR) region.

32. The expression system according to any one of claims 23 to 31, wherein one or more of the DNA fragments further comprises a poxvirus terminal hairpin loop (HL) sequence, a poxvirus genome isolation (CR) sequence, or both, and the HL sequence or the CR sequence is appended to one or both ends of the DNA fragment as a sense-directed or antisense-directed single-stranded or double-stranded DNA sequence.

33. The expression system according to claim 32, wherein one or more DNA fragments further comprise one or more HL sequences and one or more CR sequences.

34. The expression system according to claim 33, wherein each HL sequence is adjacent to two CR sequences at both ends of the HL sequence.

35. The expression system according to any one of claims 32 to 34, wherein only one or more subsets of the DNA fragments contain the HL sequence or CR sequence.

36. An expression system according to any one of claims 23 to 35, wherein one or more of the DNA fragments further comprises a viral promoter upstream of the DNA sequence encoding the antigen, its subunits or fragments, or upstream of another heterogeneous DNA sequence, or further comprises a transcription termination signal downstream of the DNA sequence encoding the antigen, its subunits or fragments, or downstream of another heterogeneous DNA sequence, or both.

37. An expression system according to any one of claims 23 to 36, wherein one or more antigens, DNA sequences encoding subunits or fragments thereof, or other heterologous DNA sequences are inserted into one or more poxvirus insertion sites.

38. (i) A single DNA fragment containing the entire genome of the desired poxvirus, When transferred to a host cell by cotransfection, two or more DNA fragments are sequentially assembled and contain the full sequence of the poxvirus genome, such that each of the two or more DNA fragments contains a partial sequence of the desired poxvirus genome, and (ii) One or more DNA sequences or other heterologous DNA sequences that encode one or more antigens, subunits or fragments thereof, inserted into one or more insertion sites of the poxvirus, wherein the antigens, subunits or fragments thereof, or other heterologous DNA sequences are expressed in the host cell by transfection of one or more poxvirus DNA fragments; A vaccine composition for the prevention or treatment of cancer or viral infection, comprising [the specified element].

39. The vaccine composition according to claim 38, wherein the antigen, its subunit or fragment, or other heterologous DNA sequence is inserted into one or more poxvirus insertion sites.

40. The vaccine composition according to claim 38 or 39, further comprising a pharmaceutically acceptable carrier, adjuvant, additive, or combination thereof.

41. A method for preventing or treating cancer or viral infection in a subject, The process includes administering a preventive or therapeutically effective amount of the vaccine composition to the subject, The aforementioned vaccine, (i) A single DNA fragment containing the entire genome of the desired poxvirus, When transferred to a host cell by cotransfection, two or more DNA fragments are sequentially assembled and contain the full sequence of the poxvirus genome, such that each of the two or more DNA fragments contains a partial sequence of the desired poxvirus genome, and (ii) One or more DNA sequences or other heterologous DNA sequences that encode one or more antigens, subunits or fragments thereof, inserted into one or more insertion sites of the poxvirus, wherein the antigens, subunits or fragments thereof, or other heterologous DNA sequences are expressed in the host cell by transfection of one or more poxvirus DNA fragments; including, The aforementioned method.

42. The method according to claim 41, wherein the antigen, its subunit or fragment, or other heterologous DNA sequence is inserted into one or more poxvirus insertion sites.

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