KR-102963059-B1 - Novel vaccinia virus strain with increased extracellular enveloped virus production

Abstract

The present invention relates to a vaccinia virus with increased extraenveloped virus production. The vaccinia virus of the present invention is expected to exhibit a significantly higher effect than conventional vaccinia viruses by increasing the production of extraenveloped viruses associated with viral transmissibility, thereby enhancing viral spread within the tumor and maximizing the potential for tumor lysis of the vaccinia virus.

Inventors

• 손우찬
• 이동현
• 이지영
• 손호선
• 김태희
• 고수민
• 이장미
• 강지연
• 정다솜
• 임희선

Assignees

• 재단법인 아산사회복지재단
• 울산대학교 산학협력단

Dates

Publication Date

20260511

Application Date

20221228

Priority Date

20211229

Claims (7)

1. A novel vaccinia virus deposited under accession number KCTC 15195BP, characterized by increased extracellular enveloped virus (EEV) production compared to wild-type vaccinia virus, The above wild-type vaccinia virus is a vaccinia virus that is the VR-156 IHD strain of the American Type Culture Collection (ATCC).
2. In paragraph 1, A vaccinia virus characterized by being an IHD strain, wherein the novel vaccinia virus deposited under the above accession number KCTC 15195BP.
3. In paragraph 1, The novel vaccinia virus deposited under the above accession number KCTC 15195BP is a vaccinia virus characterized by having deleted genes C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, F4L, F5L, A52R, A53R, A55R, and A56R.
4. In paragraph 1, The novel vaccinia virus deposited under the above accession number KCTC 15195BP is a vaccinia virus characterized by forming a comet plaque.
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Description

Novel vaccinia virus strain with increased extracellular enveloped virus production The present invention relates to a novel vaccinia virus variant with increased extraenveloped viral productivity. Vaccinia virus is a membrane-bound virus with a linear DNA genome of approximately 190 kb, and it has been used as an ideal expression vector since the early 1980s. This is because it can stably insert more than 25 kb of external DNA, replicates in the cytoplasm allowing for gene expression without interference from the host genome, possesses a relatively high level of protein synthesis, and undergoes complete post-translational modification. Furthermore, due to these utility properties of vaccinia virus, research is actively underway to use it as a recombinant vaccine or oncolytic virus. Vaccinia viruses infect and spread to cells through four types of particles, each playing a different role from infection to transmission based on the characteristics of its respective form. The four types of viral particles are Intracellular Mature Virus (IMV), Intracellular Enveloped Virus (IEV), Cell Associated Virus (CEV), and Extracellular Enveloped Virus (EEV). After infecting a cell, the vaccinia virus replicates and assembles in the cytoplasm to produce Intracellular Mature Viruses (IMVs) enclosed by a single membrane. While many Intracellular Mature Viruses spread out of the cell through cell lysis, some are packaged with components derived from the endoplasmic reticulum or Golgi apparatus to form Intracellular Enveloped Viruses (IEVs) with a double membrane. After intracellular enveloped viruses (IEVs) move to the cell surface via microtubules, the outer membrane of the intracellular enveloped virus fuses with the cell membrane to form a cellular virus (CEV); the extraenveloped virus (EEV) is formed when this cellular virus detaches from the cell surface through the polymerization of actin and is secreted into the culture medium. Among the four forms mentioned above, cellular viruses (CEVs) play an important role in intercellular infection, while extraenveloped viruses (EEVs) play an important role in systemic transmission of the virus. Meanwhile, extraenveloped viruses (EEVs) of the vaccinia virus typically account for a low proportion of the total viruses produced (1-2%), but they are responsible for systemic transmission and possess resistance to the complement-mediated immune system. Furthermore, extraenveloped viruses play a biologically important role in the survival and spread of the vaccinia virus itself, such as possessing relative resistance to neutralizing antibodies compared to intracellular mature viruses. Therefore, it is known that the production of extraenveloped viruses of the vaccinia virus is related to the ability to form comet plaques, which are considered important for the spread of the vaccinia virus from cell to cell. Figure 1 is a diagram showing the results of performing a plaque assay for each passage after serially culturing to isolate a novel vaccinia virus according to one embodiment of the present invention. FIGS. 2a to 5b show the results of next-generation sequencing analysis performed on a wild-type vaccinia virus to verify genomic mutations of a vaccinia virus according to an embodiment of the present invention. FIGS. 2a to 3c show the next-generation sequencing results of the left arm region of a vaccinia virus isolate according to an embodiment of the present invention, and FIGS. 4a to 5b show the next-generation sequencing results of the A52R to A56R gene region. FIGS. 3a to 3c represent a single figure as a whole, in which FIG. 3a is located on the right, FIG. 3b in the middle, and FIG. 3c on the left. FIGS. 5a and 5b represent a single figure as a whole, in which FIG. 5a is located on the left and FIG. 5b on the right. Figure 6 is a diagram showing the results of measuring the titers of vaccinia virus and wild-type vaccinia virus according to one embodiment of the present invention. The inventors of the present invention conducted research to secure a vaccinia virus that maintains infectivity while having excellent extraenveloped virus (EEV) productivity, which is related to the virus's transmissibility within a tumor, and is also advantageous for use as an anticancer virus vector. As a result, they completed the present invention by isolating a vaccinia virus variant that forms comet plaques by repeating passages in which only the extraenveloped virus in the supernatant of an IHD strain (ATCC VR-156) that did not form comet plaques related to extraenveloped virus production infects cells. The present invention will be described in detail below. The present invention provides a novel vaccinia virus variant characterized by increased production of extracellular enveloped virus (EEV) deposited under accession number KCTC 15195BP. A novel vaccinia virus according to the present invention can be obtained through the following steps: Before infecting cells with wild-type vaccinia viru