Preclinical and clinical trials of oncolytic vaccinia virus in cancer immunotherapy:a comprehensive review

Oncolytic virotherapy has emerged as a promising treatment for human cancers owing to an ability to elicit curative effects via systemic administration.Tumor cells often create an unfavorable immunosuppressive microenvironment that degrade viral structures and impede viral replication;however,recent studies have established that viruses altered via genetic modifications can serve as effective oncolytic agents to combat hostile tumor environments.Specifically,oncolytic vaccinia virus(OVV)has gained popularity owing to its safety,potential for systemic delivery,and large gene insertion capacity.This review highlights current research on the use of engineered mutated viruses and gene-armed OVVs to reverse the tumor microenvironment and enhance antitumor activity in vitro and in vivo,and provides an overview of ongoing clinical trials and combination therapies.In addition,we discuss the potential benefits and drawbacks of OVV as a cancer therapy,and explore different perspectives in this field.

Extracts from rabbit skin inflamed by the vaccinia virus attenuate bupivacaine-induced spinal neurotoxicity in pregnant rats

Extracts from rabbit skin inflamed by the vaccinia virus can relieve pain and promote repair of nerve injury. The present study intraperitoneally injected extracts from rabbit skin inflamed by the vaccinia virus for 3 and 4 days prior to and following intrathecal injection of bupivacaine into pregnant rats. The pain threshold test after bupivacaine injection showed that the maximum possible effect of tail-flick latency peaked 1 day after intrathecal injection of bupivacaine in the extract-pretreatment group, and gradually decreased, while the maximum possible effect in the bupivacaine group continued to increase after intrathecal injection of bupivacaine. Histological observation showed that after 4 days of intrathecal injection of bupivacaine, the number of shrunken, vacuolated, apoptotic and caspase-9-positive cells in the dorsal root ganglion in the extract-pretreatment group was significantly reduced compared with the bupivacaine group. These findings indicate that extracts from rabbit skin inflamed by the vaccinia virus can attenuate neurotoxicity induced by intrathecal injection of bupivacaine in pregnant rats, possibly by inhibiting caspase-9 protein expression and suppressing nerve cell apoptosis.

Lethality in mice infected with recombinant vaccinia virus expressing hepatitis C virus core protein

OBJECTIVE: To establish a mouse model of HCV core expression and investigate the toxicity of HCV core protein or the possible pathogenic effects. METHODS: A series of vaccinia viral expression vectors were engineered to express 5' portion of HCV genes including 5' non-translated region (NTR), core protein, and portion of the E1 gene. These HCV sequences were fused to a luciferase reporter gene and inserted into a vaccinia virus expression vector (pSC11) adjacent to the vaccinia virus promoter, p7.5. The recombinant DNA constructs were packed into infectious recombinant chimeric viruses. The expression of HCV core protein was examined in cultured cells after infection with these viruses. Death of the infected mice was investigated by specific correlation to the expression of HCV core protein and its expression levels. RESULTS: The recombinant virus (VNCE-LUA) expressed HCV core protein and an envelope-luciferase fusion protein in cultured cells. When Balb/c mice were inoculated intraperitoneally with more than 10~7 pfu per mouse of VNCE-LUA, death occurred immediately. The mortality was dependent on the amount of VNCE-LUA virus inoculated. All mice inoculated with 3×10~8 pfu of VNCE-LUA died within 4 days of infection and 50% of mice inoculated with 3×10~7 pfu of VNCE-LUA died within 7 days of infection. No death occurred in mice inoculated with 3×10~8 pfu of a control recombinant vaccinia virus, which expressed luciferase but not the HCV core and envelope proteins. Deletion of core sequences from VNCE-LUA rapidly reduced the mortality of infected mice whereas deletion of envelope sequence did not. SCID mice infected with VNCE-LUA died 2-3 days after infection, suggesting that the HCV-core induced mortality is not dependent on host T-or B-cell responses to core protein. CONCLUSIONS: HCV core protein can be lethal to mice when expressed in vivo and this specific lethality is independent of T-cells or B-cells. The findings and model itself provide a useful tool for further investigation on potential pathological effects as well as the potential toxicity of the HCV core protein.

Recombinant Vaccinia Virus is an Effective and Non-perturbing Vector for Human Dendritic Cells Transfected with Epstein-Barr Virus Latent Membrane Protein 2A

ObjectiveTo study the effects of dendritic cells (DC) transfected with recombinant vaccinia virus encoding Epstein Barr virus (EBV) latent membrane protein 2A(LMP2A) gene,and to provide evidence for further investigation on the therapeutic vaccines against EBV associated malignancies. MethodsMature DC were transfected with EBV LMP2A recombinant vaccinia virus (rVV LMP2A). Before and after the transfection,the expression of surface antigens on mature DC including CD1a,CD83,CD40,CD80,HLA DR was measured by fluorescence activated cell sorter (FACS) and the function of DC to stimulate allogeneic T cells proliferation was measured by mixed leukocyte reactions (MLR). ResultsLMP2A protein was highly expressed (66.1 %) in DC after the transfection of rVV LMP2A. No significant changes in the primary surface antigens expression and in the MLR were detected during the transfection. Transfected DC still had strong potential in stimulating the proliferation of allogeneic T cells. ConclusionRecombinant vaccinia virus was an effective and non perturbing vector to mediate the transfection of LMP2A into DC. The functions of mature DC were not affected significantly by the transfection of Vac LMP2A. This study could provide evidence for the further immunotherapy of EBV associated malignancies,e.g. nasopharyngeal carcinoma (NPC).

Brazilian vaccinia virus strains show a classical orthopoxvirus infection course and cross-protection

Objectives:The purpose of this work was to study the infection course and cross-protection in mice after intradermal injection of Vaccinia virus(VACV) strain Western Reserve and three Brazilian VACV strains: Aracatuba,Muriae and BeAn58058 isolated from cow,human and rodent,respectively.Methods:Balb/c mice were inoculated by footpad and back scarification and daily monitored regarding lesion development and weight loss.To check cross protection after intradermal VACV inoculation,mice were subsequendy infected with different VACV strains and monitored to check lesion development.Serum neutralization assays were performed to check for the presence of antibodies against Orthopoxvirus.Results:After VACV intradermal inoculation the lesion development pattern was similar in mice infected with the different virus strains.By using the footpad scarification model,cross-protection among VACV strains was observed.Moreover,neutralizing antibodies against Orthopoxvirus were detected in sera from mice infected with all VACV strains.Conclusion:Although it was not possible to observe virulence differences among VACV strains isolated from cow,rodent and human using the murine model,this inoculation route showed to be an appropriated model to study lesions development since it mimics natural infections by VACV in nature.

ANTI-METASTATIC EFFECT OF ONCOLYSATES FROM MURINE MELANOMA CELLS TRANSFECTED WITH RECOMBINANT VACCINIA VIRUS ENCODING HUMAN IL-2

Oncolysates, debris of tumor cells, have been proven to be effective in active immunotherapy of cancer. In this experiment, the oncolysates from murine melanoma cells B16F10 transfected by recombinant vaccinia viruses encoding human IL2(IL2VBO) were used as vaccine. After treatment of tumor bearing mice with pulmonary metastases by intravenous injection of IL2VBO or rVVIL2, higher level IL2 activity was detected in the serum of IL2VBO or rVVIL2 treated mice at 8h. Further experiment results demonstrated that IL2VBO significantly reduced the number of pulmonary metastases and prolonged the survival time of tumorbearing mice when compared with other preparations. Fresh peripheral blood lymphocytes from IL2VBO treated mice showed potent cytotoxicity to B16F10 cells and YAC1 cells. But only cytotoxicity to B16F10 cells is more marked than that in rVVIL2 group, indicating that the IL2VBO could induce specific and non specific antitumor immunity. Because IL2 expression was at the same level in the serum of IL2VBO or rVVIL2 treated mice, the results suggested that the specific antitumor immunity induced by IL2VBO might contribute to the enhanced therapeutic effect of IL2VBO.

Construction of a vaccinia virus vectored multi--epitope live vaccine candidate for Plasmodium falciparum

To construct live recombinant vaccinia virus, The HGFSP gene encoding CSP, MSA1, MSA2, RESA,IL-1 and TT epitopes was inserted into the Eec RI and Burn HI sites of pSK plasmid. After digested with Eco RI,bluntly ended by Klenow enzyme and digested with Sac I, the HGFSP gene was cloned into the Sma I and Sac I sites of the vaccinia virus insertion vector (pJ2--16). Recombinant plasmids were identified by gel electrophoresis,restriction enzyme and enzyme map. Results evidenced that HGFSP gene fragment was correctly inserted into the cloning site of hemagglutinin (HA) gene of the pJ2--16 vector. The recombinant plasmids were trans feeted into Cos--7 cells, which were infected with wild type of vaccinia virus Tiantan strain, by means of lipofectamine. Two recombinant vaccinia viruses (HA) were screened and cloned by chicken hemadorption test in BHK21 cells. Indirect immunofluorescence assay (IFA), Dot--ELISA and Western blot with the antibodies against HGFSP protein expressed by E. colt showed that one of the 2 recombinant vaccinia virus expressed desired proteins in infected BHK21 cells. Western blot also showed that the molecular weight of 2 of expressed protein bands was about 23 kDa, according to the theoretical molecular weight of HGFSP protein. Further identification of immunological characters of recombinant virus is under way.

EFFECTS OF GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR GENE ENCODED VACCINIA VIRUS VECTOR ON MURINE PULMONARY METASTATIC MELANOMA

A recombinant vaccinia virus expressing murine granulocyte-macrophage colony-stimulating factor (VVGM-CSF) was tested for its antitumor activity.Murine pulmonary metastasis was established by injecting 20×10~5 B16F10 melanoma cells into the tail vein of C57BL/6 mice. Three days after B16F10 inoculation,WGM-CSF or VVTK, a thymidine kinase gene deficient control vaccinia virus, were injected intraperitoneally twice weekly for 2 weeks. Two weeks later, the mice were sacrificed and pulmonary metastasis fool counted.The results demonstrated that VVGM-CSF treatment significantly decreased the number of pulmonary metastasis and prolonged the survival time of tumorbearing mice. Cytotoxic and phagocytic activities of the peritoncal macrophages were found to be markedly elevated in mice treated with WGM-CSF. Nitric oxide released from the macrophages was also found to be increased. These data, together with our other results,strongly demonstrated that continuous secretion of GMCSF and activation of macrophages might pal-tially explain the therapeutic effects of VVGM-CSF on murine pulmonary metastasis.

The Expression of Sperm Membrane Peptide-Hepatitis B SurfaceAntigen Fusion Protein with Recombinant Vaccinia Virus

A synthetic oligonucleotide, HSD-2a, encoding a peptide segment of the extracel-lular domain of a human sperm membrane protein, YWK-II, was fused with hepatitisB surface antigen gene (HBs gene ). The fused gene was then cloned to pUC18plasmid. The fused gene was prepared from the recombinant pUC18 plasmid byBamH I and Eco R I digestion, and then cloned into the transfer vector pGJP- 5 underthe control of P;., promoter, designated as pGJP-HSD/HBs. CV-1 cells were co-transfected with vaccinia virus (Tian Tan strain) and pGJP-HSD/IIBs and homolo-gous re combination occurred between the vaccinia virus TK gene of the plasmid flank-ing the foreign gene and the same sequences within the virus genome. TK phen0tyPerecombinant virus, vv-HSD/HBs, were selected from trandected HuTK' cells byplaque purthcation technique. The eopressi0n of HSD-b in spent medium and cellu-lar protein of HuTK cells infected with vv-HSD/HBs was determined by ELISAand Western-blot analysis using anti-rwK-II antiserum. The present study indicatesthat the vv-HSD/HBs seems promising as an antdertility vaccine.

STRUCTURE AND EXPRESSION OF EPSTEIN-BARR VIRUS MEMBRANE ANTIGEN IN RECOMBINANT VACCINIA VIRUS

The Epstein-Barr virus membrane antigen was constructed and inserted into vaccinia virus, Tian-tan strain in order to study the effect of this virus on EB infection and tumorogenesis. The EBV-derived membrane antigen was expressed under the control of a 7.5 K promoter of vaccinia virus. The antibody against the membrane antigen of EB virus was produced on rabbits vaccinated with recombinant vaccinia virus.

Construction of non-replicating recombinant vaccinia virus expressing HPV16 L1,L2E7 proteins

Objective： To construct a non-replicating vaccinia virus expressing human papillomavirus 16 （HPV16） L1, L2E7 proteins as a candidate vaccine for cervical cancer. Methods： Using vaccinia virus vector, we generated a strain of non-replicating recombinant vaccinia virus vaccine expressing HPV16 L1, L2E7 proteins by homologous recombination and identified by PCR and Westernloting. Results： We demonstrated that the L1, L2E7 gene of HPV16 were integrated into vaccinia genosome and could express L1, L2E7 protein stably when infected the CEF using PCR and Western-blot assay. Conclusion： NTVJL1/L2E7 can express L1, L2E7 protein of HPV16 and can be taken as a candidate vaccine for HPV16-associated diseases.

SARS-CoV-2重组痘苗病毒疫苗rVTT△TK-RBD的构建、筛选及免疫原性研究

目的 构建重组痘苗病毒载体疫苗rVTT△TK-RBD,并评价其安全性和免疫原性。方法 参考严重急性呼吸综合征冠状病毒2(SARS-CoV-2)基因序列合成受体结合域(RBD)基因,并将其插入自主构建的重组质粒pSTKE的多克隆位点上,构建重组痘苗病毒穿梭载体pSTKE-RBD,并转染到预先感染天坛株痘苗病毒(VTT)的BHK-21细胞内,经多轮的荧光噬斑筛选成功获得重组痘苗病毒rVTT△TK-RBD;通过滴鼻方式免疫小鼠后,检测rVTT△TK-RBD对BALB/c小鼠体质量的影响;通过肌肉免疫小鼠后,分析rVTT△TK-RBD对BALB/c小鼠产生的特异性抗体和中和抗体的水平;通过流式细胞术检测rVTT△TK-RBD对BALB/c小鼠T细胞亚群的影响。结果 利用同源重组、增强型绿色荧光蛋白(EGFP)筛选标记和多次荧光噬斑筛选,成功筛选获得了表达RBD的胸腺激酶(TK)基因缺失型重组痘苗病毒rVTT△TK-RBD,且PCR验证成功。BALB/c小鼠体内实验表明rVTT△TK-RBD具有较好的抗SARS-CoV-2的免疫原性且相比于亲本株VTT明显降低了对机体的毒性作用。结论 成功构建并获得SARS-CoV-2重组痘苗病毒疫苗rVTT△TK-RBD并通过各项试验证明其安全性和免疫原性。

Vaccinia virus viability under different environmental conditions and different disinfectants treatment

Monkeypox(mpox)outbreak in 2022 has caused more than 91,000 cases,has spread to 115 countries,regions,and territories,and has thus attracted much attention.The stability of poxvirus particles in the environment is recognized as an important factor in determining their transmission.However,few studies have investigated the persistence of poxviruses on material surfaces under various environmental conditions,and their sensitivity to biocides.Here,we systematically measured the stability of vaccinia virus(VACV)under different environmental conditions and sensitivity to inactivation methods via plaque assay,quantitative real‐time polymerase chain reaction(qPCR),and Gaussia luciferase(G‐luciferase)reporter system.The results show that VACV is stable on the surface of stainless steel,glass,clothing,plastic,towel,A4 paper,and tissue and persists much longer at 4℃ and?20℃,but is effectively inactivated by ultraviolet(UV)irradiation,heat treatment,and chemical reagents.Our study raises the awareness of long persistence of poxviruses in the environment and provides a simple solution to inactivate poxviruses using common disinfectants,which is expected to help the control and prevention of mpox virus and future poxvirus outbreaks.

Immune therapy for human papillomaviruses-related cancers

Human papillomaviruses(HPVs) are a large family of double strand DNA viruses comprising more than 180 types. Infection with HPV is very common and it is associated with benign and malignant proliferation of skin and squamous mucosae. Many HPVs, considered lowrisk such as HPV 6 and 11, produce warts; while highrisk viruses, such as HPVs 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, and 58, induce tumors. About 5% of all cancers in men and women are associated with HPV infection. Because there are not antiviral drugs for HPV infection, current therapies for low-risk HPV infections involve physical removal of the lesion by cryotherapy, trichloracetic acid, laser, or surgical removal. Surgical procedures are effective in the treatment of precancerous lesions, however after these procedures, many recurrences appear due to new re-infections, or to failure of the procedure to eliminate the HPV. In addition, HPV can inhibit recognition of malignant cellsby the immune system, leading to the development of cancer lesions. When this occurs, radiotherapy and chemotherapy are then used. Unfortunately, about 50% of the HPV-cancer patients still die. In the past decade, a better knowledge of the natural history of the virushost interaction and of the immune response against this viral infection has brought new therapeutic strategies geared to modulate the immune system to generate an efficient virus-specific cytotoxic response. Novel HPV protein-expressing vaccines have shown some significant clinical efficacy and systemic HPV-specific cytotoxic T cell responses. This review will describe the current status of the several therapeutic strategies used to treat HPV-induced lesions, and discuss the various new therapies now being tested.

Interleukin-2 expression and glioma cell proliferation following Vaccinia vector gene transfection in vivo

BACKGROUND： The effectiveness of gene therapy is closely related to the efficiency of vector transfection and expression. OBJECTIVE： This study was designed to transfect a human brain glioma cell line with recombinant Vaccinia virus expressing the interleukin-2 （rVV-IL-2） gene, and to observe IL-2 expression and glioma cell proliferation potential after transfection. DESIGN： Experimental observation. SETTING： Department of Neurosurgery, Shenyang Military Area Command of Chinese PLA. MATERIALS： The rVV-IL-2 vectors were obtained through homologous recombination and screening in the Second Military Medical University of Chinese PLA. The human brain glioma cell line and IL-2-dependent cells were produced by the Second Military Medical University of Chinese PLA. Human IL-2 was produced by Genzyme Corporation. METHODS： At passage day l, Veto cells were amplified l ; 1 for virus and cells. A human brain glioma cell line was transfected using amplified Vaccinia viral vectors at varying multiplicities of infection （MOI）. At 2, 4, 6, 8, 12, and 24 hours post-transfection, superuatant was collected to determine by MTT assay IL-2 expression levels in IL-2 dependent cells. The transfected and non-transfected cells were divided into 4 groups, namely MOI1 ： 1, MOI 5 ： 1, MOI 10 ： 1, and control groups. MAIN OUTCOME MEASURES： IL-2 expression at different time points after transfection of human brain glioma cells with varying MOI of Vaccinia viral vectors; in vitro proliferation capacity of human brain glioma cells among the 4 groups. RESULTS： IL-2 expression was detectable 4 hours after Vaccinia viral vector transfection and reached 300 kU/L by 8 hours. There was no significant difference in the proliferating rate of human brain glioma cells among the 4 groups （P 〉 0.05）. CONCLUSION： Vaccinia viral vectors can transfect human brain glioma cells in vitro and express high levels of IL-2. Vaccinia virus and high IL-2 expression do not influence the proliferation rate of human brain glioma cells in vitro.

ANTITUMOR EFFECT OF GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR(GM-CSF)-GENE ENCODED VACCINIA MELANOMA ONCOLYSATE AND ITS IMMUNOLOGICAL MECHANISMS

Vaccinia melanoma oncolysate (VMO) prepared by infecting B16F10 melanoma cells with recombinant vaccinia virus encoding murine GMCSF gene was tested for its therapeutic effect on the preestablished melanoma. C57BL/6 mice were inoculated s.c. with 1×105 B16F10 melanoma cells and received s.c. administration with VMO prepared with GMCSF gene encoded vaccinia virus(GMCSFVMO), VMO prepared with thymidine kinase genedeficient vaccinia virus(TKVMO), B16F10 melanoma oncolysate(BMO), or PBS 3 days after tumor inoculation. The same treatment was bolstered one week later. The results demonstrated that GMCSFVMO treatment significantly inhibited the growth of subcutaneous tumor and prolonged the survival period of tumorbearing mice. Further study elucidated that cytotoxicity of PBL and splenocytes towards B16F10 increased obviously after treatment with GMCSFVMO, but NK activity remained unchanged. These results suggest that the tumor oncolysate vaccine prepared with GMCSF geneencoded vaccinia virus might exert potent therapeutic effect on the preestablished tumor through the efficient induction of specific antitumor immune response of the host.

Near obstructing painful anorectal mass and facial rash in a man with monkeypox: A case report

BACKGROUND Monkeypox(MPX)is a zoonotic infection that is endemic in Western and Central Africa along the Congo River basin.It has a high case fatality rate especially in younger age groups.It belongs to the virus family orthopoxvirus like smallpox.It is transmitted from wild animals to humans but human to human transmission has been established.It is often a self-limited infection in endemic regions.Recently,attention has been given to MPX with the spread of infection to Europe and the United States of America(USA).There is currently sporadic infection of MPX in the USA especially amongst men who have sex with men(MSM).It is a serious life-threatening infection in human immunodeficiency virus/acquired immunodeficiency syndrome co-infected individuals especially those who are treatment naïve with severe immunosuppression.CASE SUMMARY We report a 38-year old man who presented with rectal pain,and anal,torso,and facial rash.Abdominal computed tomography scan showed a near obstructive rectal mass with peri-anal fistula.MPX was positive.He was started on tecovirimat(TPOXX)and HAART therapy.Additional treatment provided included vaccinia immunoglobulin following his clinical deterioration.CONCLUSION This case highlights a rare presentation of MPX with peri-anal fistula and near obstructive rectal mass,and the significance of MPX as a differential diagnosis in proctitis in MSM in addition to other sexually transmitted infection like gonorrhea and chlamydia.

An MPXV mRNA-LNP vaccine candidate elicits protective immune responses against monkeypox virus

The monkeypox virus(MPXV)outbreak,declared a Public Health Emergency of International Concern(PHEIC)by the World Health Organization(WHO)in 2022,continues to pose a significant threat due to the absence of vaccines or drugs for MPXV infection.In this study,we developed an mRNA vaccine that expressing the A29L antigen,a specific protein of the intracellular mature virus.Our vaccine utilizes a thermostable ionizable lipid nanoparticle(iLNP)platform and has been administered to mice.Our find-ings demonstrate that the MPXV A29L mRNA vaccine candidate induces robust cross-neutralizing immune responses against both vaccinia virus(VACV)and MPXV live virus.Furthermore,immunization with the vaccine candidate provided protection against the VACV challenge in mice.These findings underscore the potential of mRNA-LNP vaccines as safe and effective candidates against monkeypox epidemics.Given the current absence of specific interventions for MPXV infection,our study represents a significant step forward in developing a viable solution to combat this ongoing public health threat.

痘苗病毒基因组密码子使用频率分析

密码子使用的差别是普遍存在的现象 ,每一个密码子被某些生物偏爱 ,而在另一些生物中则很少使用。以往这方面的研究多集中在自养生物中 ,而对纯寄生的病毒本身及其与宿主细胞基因密码子使用频率关系的研究则很少。分析痘苗病毒哥本哈根株 189个基因的密码子使用频率发现 :总体上痘苗病毒偏爱使用以A/U为结尾的密码子 ;基因的异质性不强 ,没有影响密码子使用的主要趋势 ;在不同转录方向上和表达时相上 ,基因密码子使用略有不同 ;不同功能的基因其密码子使用上差别较大 ;晚期基因比早期基因与宿主密码子使用频率的差别大。上述结果表明 :密码子是影响病毒和细胞相互作用。

人癌胚抗原-重组痘苗病毒的构建和制备

痘苗病毒的基因组庞大，结构复杂而特殊，不可能将外源基因直接插入它的基因组，必须利用一种特殊的痘苗病毒质粒，才能构建成功重组痘苗病毒．在分析了痘苗病毒质粒ｐＪ１２０〔含有我国天花疫苗－痘苗病毒天坛株７６１的启动子和胸苷激酶（ｔｈｙｍｉｄｉｎｅｋｉｎａｓｅ，简称ＴＫ基因），及含有人癌胚抗原（ｃａｒｃｉｎｏｅｍｂｒｙｎｉｃａｎｔｉｇｅｎ，简称ＣＥＡ）ｃＤＮＡ全序列的质粒ｐ９１０２３Ｂ－ｃｅａ－１７结构的基础上，设计出三步法构建了重组疫苗病毒质粒ｐＪ－ＣＥＡ．经酶切及ＰＣＲ鉴定ｐＪ－ＣＥＡ中ＣＥＡｃＤ－ＮＡ的存在，进一步用同源重组方法构建了表达人ＣＥＡ的重组痘苗病毒，并以人体成纤维细胞作为宿主细胞，对ＣＥＡ－重组痘苗病毒进行了大量培养．再次证实痘苗病毒是良好的真核表达载体，可以高效而准确地表达细胞膜糖蛋白ＣＥＡ．