• BMB Reports
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• 제28권3호
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• pp.216-220
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• 1995

cDNA for human cholesteryl ester transfer protein (CETP), a potent atherogenic plasma protein that redistributes the neutral lipids among lipoproteins, was expressed in recombinant vaccinia virus-infected cells (CV-1). Two insertion vectors regulated by different promoters were constructed. The vectors were introduced into human thymidine kinase-negative ($TK^-$) 1438 cells infected with wild-type vaccinia virus (WR strain). Recombinant viruses were selected with 5-bromodeoxyuridine (BUdR) and X-gal and identified with DNA dot blot analysis (vSC11-CETP and vTM1-CETP). The CETP cDNA insert in the recombinant vaccinia virus genome was identified by Southern blot analysis. Transcription of CETP cDNA in CV-1 cells infected with recombinant vaccinia virus was monitored by Northern blot analysis using the CETP cDNA as a probe. Positive signals were detected at 1.8 kb in cells infected with vSC11-CETP and at 2.3 kb in cells infected with vTM1-CETP. The recombinant vaccinia virus-infected CV-1 cells were shown to produce functional CETP when the culture medium was subjected to the CETP assay.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.82-82
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• 1997

The hepatitis B virus(HBV) is a small, enveloped virus with a circular, double-stranded DNA genome. HBV causes active and chronic hepatitis worldwide, including Korea, and is considered to be a major factor for liver cirrhosis and hepatocellular carcinoma. In contrast to the wealth of knowledge on the gene structure and expressional regulation, immunological and pathological mechanisms for HBV-induced hepatocellular injury are not well known. In the present study, vaccinia virus which has been demonstrated to be a useful eukaryotic expression vector was used to clone the gene for HBV surface antigen, L(S+preS2+preS1). The recombinant vaccinia virus vector, pMJ-L, which contains L surface antigen gene of adr-type HBV was constructed, and subseouently used for making recombinant vaccinia virus VV-$\textrm{HBV}_{L}$. Expression of the HBV antigen was examined by immunofluorescent antibody (IFA) test using mouse monoclonal anti-hepatitis B surface antigen. HBsAg was detected in the recombinant virus indicating that the VV-$\textrm{HBV}_{L}$ expressed S antigen successfully. The HBV-Vaccinia Virus recombinant obtained in this study is currently being used for studying the immunological aspects of HBV infection.

• 대한바이러스학회지
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• 제28권1호
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• pp.71-78
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• 1998

Vaccinia virus is the prototype orthopoxvirus that has been used as a vaccine strain for small pox. This virus has been used to express a variety of cellular and viral genes in mammalian cells at high levels. Interleukin-4 (IL-4) has been found to stimulate the proliferation of T cells and enhance the cytolytic activity of cytotoxic T lymphocytes. To test the immunotherapeutic potential of IL-4 delivered in vivo by poxvirus, a recombinant vaccinia virus expressing the murine IL-4 gene (RVVmIL-4) was constructed. A high level of IL-4 production was confirmed by infecting HeLa cells and measuring IL-4 in cell culture supernatant by ELISA. As a tumor model, two cell lines were used; the murine T leukemic line P388 and the murine breast cancer line TS/A. CDF1 mice were intraperitoneally inoculated with $1\;{\times}\;10^5$ cells of P388. Mice were injected at the same site with $5\;{\times}\;10^5\;PFU$ of recombinant vaccinia virus; first, 3 days after the injection of tumor cells and thereafter once every week for 3 weeks. Intraperitoneal injections of RVVmIL-4 significantly prolonged the survival time of mice inoculated with tumor cells. All mice injected with RVVmIL-4 remained alive for 30 days after the postinoculation of tumor cells, while 100% and 70% of the animals injected with saline or wild type vaccinia virus died, respectively. In another tumor model using TS/A, tumor was established by subcutaneously inoculating $2{\times}10^5$ tumor cells to BALB/c mice. After tumor formation was confirmed on day 4 in all mice, $5\;{\times}\;10^6\;PFU$ of RVVmIL-4 was inoculated subcutaneously three times, once every week for 3 weeks. The TS/A tumor was eradicated in two of the nine mice. Seven of the nine mice treated with RVVmIL-4 developed a tumor, but tumor growth was significantly delayed compared to those treated with saline or wild type vaccinia virus. These results indicate that recombinant vaccinia viruses may be used as a convenient tool for delivering immunomodulator genes to a variety of tumors.

• KSBB Journal
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• 제11권4호
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• pp.438-444
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• 1996

모델 재조합 단백질인 $\beta$galactosidase를 발현하 는 Vaccinia virus를 생산하기 위하여 숙주 동물세 포의 배양조건을 관찰한 결과 감염비 5일 경우 H HeLa는 감염후 60시간 후, HeLa 83는 감염후 40 시간 후에 회수할 때 최 대 의 ${\beta}$-galactosidase 수율 을 얻을 수 있으며, 세포가 대수증식기 일 때 감염하 고 배양온도는 $37^{\circ}C$ 로 하는 것이 최적 배양조건으로 나타났다. 감염후 혈청의 농도는 단백질 수율에 크 게 영향을 미치지는 않으나 3~5% 에서 가장 높은 단백질 수율을 보였으며, 낮은 이온 농도의 용액으 로 세포층을 세척하는 것과 virus 감염시 온도를 20~∼$30^{\circ}C$ 로 낮추는 것은 Vaccinia-HeLa system에서 감염능 증대 효과를 나타내 였다. Dexamethasone 전처리는 HeLa 83에서 ViruS 복제 증대를 HeLa에 서는 virus 복제 감소를 가져왔다.

• 대한수의학회지
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• 제29권3호
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• pp.407-416
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• 1989

The prospect of live vaccines consisting of genetically modified vaccinia virus expressing foreign genes is exciting, but important issues concerning safety and efficacy need to resolved. Vaccinia virus (VV) is an efficient expression vector with broad host range infectivity and large DNA capacity. This vector has been particularly useful for identifying target antigens for humoral and cell-mediated immunity. The WHO smallpox eradication program, involving the extensive use of VV vaccines, resulted in the late 1970s in the elimination of one of the world's most feared diseases. This achievement is a triumph for preventive medicine and for international collaboration in public health. In 1980, WHO recommended that the routine use of smallpox vaccine should be stopped. Against this background, the prospect of li ve vaccines consisting of genetically modified VV expressing foreign antigens arising from the work of Moss, and Paoletti and their colleagues in 1982 has been greeted with enthusiasm. These investigators have shown that genes coding for immunogenic proteins can be inserted into VV DNA without impairing the ability of the virus to grow in cell culture. Moreover experimental animals infected with VV recombinants containing genes coding for a variety of immunizing proteins have been shown to be protected against challenge infection with the corresponding infectious agent. In this communication, I describe current progress in the construction of a novel plasmid vector that facilitate the insertion and expression of foreign genes in VV as well as the selection of recombinants.

• IMMUNE NETWORK
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• 제5권2호
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• pp.89-98
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• 2005

Background: DNA vaccination represents an anticipated approach for the control of numerous infectious diseases. Used alone, however, DNA vaccine is weak immunogen inferior to viral vectors. In recent, heterologous prime-boost vaccination leads DNA vaccines to practical reality. Methods: We assessed prime-boost immunization strategies with a DNA vaccine (minigene, $gB_{498-505}$ DNA) and recombinant vaccinia virus $(vvgB_{498-505})$ expressing epitope $gB_{498-505}$ (SSIEF ARL) of CD8+ T cells specific for glycoprotein B (gB) of herpes simplex virus (HSV). Animals were immunized primarily with $gB_{498-505}$ epitope-expressing DNA vaccine/recombinant vaccinia virus and boosted with alternative vaccine type expressing entire Ag. Results: In prime-boost protocols using vvgBw (recombinant vaccinia virus expressing entire Ag) and $vvgB_{498-505}$, CD8+ T cell-mediated immunity was induced maximally at both acute and memory stages if primed with vvgBw and boosted with $vvgB_{498-505}$ as evaluated by CTL activity, intracellular IFN-staining, and MHC class I tetramer staining. Similarly $gB_{498-505}$ DNA prime-gBw DNA (DNA vaccine expressing entire Ag) boost immunization elicited the strongest CD8+ T cell responses in protocols based on DNA vaccine. However, the level of CD8+ T cell-mediated immunity induced with prime-boost vaccination using DNA vaccine expressing epitope or entire Ag was inferior to those based on vvgBw and $vvgB_{498-505}$. Of particular interest CD8+ T cell-mediated immunity was optimally induced when $vvgB_{498-505}$ was used to prime and gB DNA was used as alternative boost. Especially CD7+ T cell responses induced by such protocol was longer lasted than other protocols. Conclusion: These facts direct to search for the effective strategy to induce optimal CD8+ T cell-mediated immunity against cancer and viral infection.

• 대한의생명과학회지
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• 제18권3호
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• pp.210-217
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• 2012

Oncolytic viral vectors have shown good candidates for cancer treatment but have many limitations. To improve the therapeutic potential of oncolytic vaccinia virus, we developed a recombinant vaccinia virus expressing the 4-1BBL co-stimulatory molecule or CCL21. 4-1BBL and CCL21 expression was identified by FACS analysis and immunoblotting. rV-4-1BBL vaccination shows significant tumor regression compared to rV-LacZ, but rV-CCL21 shows rapid tumor growth compared to rV-LacZ in the poorly immunogenic B16 murine melanoma model. 4-1BBL expression resulted in the increase of the number of CD8+ T cells and especially the increase of effector (CD62L-CD44+) CD8+ T cells. These data suggest 4-1BBL may be the potential target for enhancement of tumor immunotherapy.

• IMMUNE NETWORK
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• 제11권5호
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• pp.268-280
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• 2011

Background: Dengue virus, which belongs to the Flavivirus genus of the Flaviviridae family, causes fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) with infection risk of 2.5 billion people worldwide. However, approved vaccines are still not available. Here, we explored the immune responses induced by alternating prime-boost vaccination using DNA vaccine, adenovirus, and vaccinia virus expressing E protein of dengue virus type 2 (DenV2). Methods: Following immunization with DNA vaccine (pDE), adenovirus (rAd-E), and/or vaccinia virus (VV-E) expressing E protein, E protein-specific IgG and its isotypes were determined by conventional ELISA. Intracellular CD154 and cytokine staining was used for enumerating CD4+ T cells specific for E protein. E protein-specific CD8+ T cell responses were evaluated by in vivo CTL killing activity and intracellular IFN-${\gamma}$ staining. Results: Among three constructs, VV-E induced the most potent IgG responses, Th1-type cytokine production by stimulated CD4+ T cells, and the CD8+ T cell response. Furthermore, when the three constructs were used for alternating prime-boost vaccination, the results revealed a different pattern of CD4+ and CD8+ T cell responses. i) Priming with VV-E induced higher E-specific IgG level but it was decreased rapidly. ii) Strong CD8+ T cell responses specific for E protein were induced when VV-E was used for the priming step, and such CD8+ T cell responses were significantly boosted with pDE. iii) Priming with rAd-E induced stronger CD4+ T cell responses which subsequently boosted with pDE to a greater extent than VV-E and rAd-E. Conclusion: These results indicate that priming with live viral vector vaccines could induce different patterns of E protein-specific CD4+ and CD8+ T cell responses which were significantly enhanced by booster vaccination with the DNA vaccine. Therefore, our observation will provide valuable information for the establishment of optimal prime-boost vaccination against DenV.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.153-163
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• 1994

P450s are a superfamily of heme-containing monooxygenases and important in the metabolism of numerous physiological substrates and foreign compounds. It has been established that tilers are at least 30 distinct human isoforms of P450. Four families containing numerous individual P450s are mainly responsible for metabolizing foreign compounds, A cDNA expression system in which individual human P450s are synthesized in cultured human hepatoma (Hep G2) cells infected with a recombinant vaccinia virus containing human P450 cDNA has been constructed.

• KSBB Journal
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• 제13권1호
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• pp.83-89
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• 1998

Cellulose로 만들어진 다공성 미립담체를 이용하여 HeLa cell을 working volume 100mL의 spinner flask에서 배양하였으며, 세포가 완전히 자란 미립담제를 계대배양하기 위하여 담체간 세포 전이 배양 방법을 시도하였다. 부유세포의 농도는 다공성 미립담체-HeLa 시스템의 경우에 담체간 세포 전이 배양에 영향을 미치는 중요한 인자로 작용하였으며, 낮은 칼슘농도의 배지인 RPMI-1640과 빠른 교반 속도를 이용하여 활성을 유지한 많은 세포가 떨어지도록 유도하였으며, 담체간 세포 전이 배양을 효과적으로 3회 이상 실시할 수 있었다. 이렇게 배양한 세포에 재 조합 Vaccinia virus를 감염하여 그 수율을 비교한 결과 T-flask에서 떼어낸 세포로 접종한 미립담체 배양과 거의 비슷한 재조합 단백질($\beta$-galactosidase) 수율을 나타내었다. Trypsin 처리 방법에 의한 미립담체 계대 배양도 경우에 따라서는 유용한 미립담체 계대 배양 방법이 될 수 있지만 실제 생산 규모에의 적용에는 공정이 복잡해지고 정확한 제어가 필요하다는 등의 문제가 있다. 따라서, 추가적인 비용이나 공정이 필요 없는 간편한 방법인 담체간 세포 전이 배양은 동물세포 배양을 이용한 유용 단백질 및 바이러스 생산 공정의 규모 증대에 매우 유용한 수단이다.