非复制重组痘苗病毒天坛株C-K缺失区表达载体的构建及重组病毒生物学性状的研究

将反向串联的痘苗病毒启动p11k和p7.5k表达结构引入非复制痘苗病毒质粒载体,得到非复制痘苗病毒表达载体pNEOCK11β75、pNEOCK7511β、pNEOCK1175和pNEOCK7511。利用载体pNEOCK11β75、pNEOCK7511β及pNEOCK11β75IL6和RVJ123重组病毒进行同源重组,分别得到非复制重组痘苗病毒RVJ123△11β75、RVJ123△7511β和RVJ123△11β75IL6。Southern-blot证实：重组病毒RVJ123△11β75 C－K片段间大片段基因的稳定缺失,同时β－半乳糖苷酸基因插入到缺失区内并稳定表达,不影响另外非必要区外源基因的表达,缺失区内两外源基因的表达夫相互干扰,并且缺失区内外源基因的转录方向对其DNA复制及蛋白表达以及另外非必要区基因的表达无影响。     

甲3型流感病毒M2基因在痘苗病毒天坛株中的表达

为获得表达甲3型流感病毒(H3N2)M2蛋白的重组天坛株痘苗病毒RVJ1175M2,使用PCR方法扩增流感病毒全长M2基因,将其克隆到天坛株痘苗病毒同源重组质粒pJSC1175中,获得重组质粒pJSC1175M2,通过与痘苗病毒载体同源重组,构建了含流感病毒M2基因的重组痘苗病毒株RVJ1175M2。PCR检测结果证明,流感病毒(H3N2)M2蛋白基因准确插入到天坛株痘苗病毒TK区;Western blot、免疫荧光和流式细胞计数表明重组病毒RVJ1175M2可以有效地表达M2蛋白,表达的M2蛋白有两条带,分别为15kD和13kD,与相关文献报道一致;M2蛋白可有效分布在感染细胞的细胞膜上。这些结果表明重组痘苗病毒株RVJ1175M2可以有效地表达流感病毒M2蛋白,为使用表达M2蛋白的不同类型疫苗进行广谱流感疫苗效果的比较研究奠定了基础。     

表达绿色荧光蛋白的重组鸡痘病毒的构建

[目的]旨在构建表达绿色荧光蛋白(GFP)的重组鸡痘病毒。[方法]使用overlap PCR进行表达载体质粒的构建,使用菌液PCR和测序技术进行了鉴定;使用鸡胚成纤维细胞作为重组鸡痘病毒同源重组的介质,尝试了可能影响重组鸡痘病毒构建的不同条件;最后使用PCR和Western Blot方法对重组病毒进行了鉴定。[结果]将人工合成的痘苗病毒早晚期启动子SP与绿色荧光蛋白基因一同插入到鸡痘病毒载体质粒的多克隆位点,挑取LA平板上的重组子,菌液PCR及测序验证鉴定结果为阳性;利用重组质粒转染被野生鸡痘病毒感染的鸡胚成纤维细胞制备重组病毒,利用倒置荧光显微镜观察到含有重组病毒的在激发光下呈绿色的细胞;改进重组质粒转染的条件以达到更好的重组病毒包装成效,研究得出被野生痘病毒感染的细胞进行重组质粒传染制备重组病毒的效率是用野生痘病毒感染已转染重组质粒的细胞的6倍,且感染用野生病毒滴度高时重组病毒的制备效果更好;最后,用PCR方法和Western Blot方法对重组病毒进行鉴定,鉴定结果为阳性。[结论]成功构建了表达GFP的重组鸡痘病毒。     

表达中东呼吸综合征冠状病毒S蛋白的重组痘苗病毒的构建及免疫效果初步评价

目的:以痘苗病毒天坛株为载体,构建表达中东呼吸综合征冠状病毒(MERS-Co V)S蛋白的重组病毒疫苗,并进行免疫效果评价。方法:通过PCR扩增获得去除跨膜区的S蛋白基因片段SQ,并构建重组痘苗病毒载体质粒p JSC11Lac Z7.5SQ,将重组质粒与痘苗病毒同源重组并单斑纯化获得重组病毒毒株RVVMERS-SQ,重组病毒免疫小鼠后用酶联免疫吸附实验(ELISA)和假病毒微量中和实验检测其诱发的S蛋白体液免疫反应水平。结果:构建了表达MERS-Co V去跨膜区S蛋白的重组病毒RVVMERS-SQ,其免疫小鼠后诱发了强的S蛋白体液免疫反应,血清Ig G抗体滴度为1∶3200,中和抗体滴度达到1∶1000。结论:重组痘苗病毒RVVMERS-SQ可在BALB/c小鼠体内诱发强的免疫反应,为MERS-Co V疫苗的研发提供了实验基础。     

IL-5在非复制重组痘苗病毒中的表达及其对重组病毒免疫效果的影响

利用非复制型痘苗病毒表达载体 pNEOCK11β75IL5和重组病毒RVJ12 3,通过两步重组构建了能同时表达IL 5和乙型肝炎病毒HBsAg的非复制型重组痘苗病毒RVJ12 3Δ11β75IL5。Southern blot证实 ,痘苗病毒C K片段间基因缺失的同时伴有IL 5基因的插入。鼻腔吸入分别免疫Balb/c小鼠和新西兰白兔 ,ELISPOT实验证实 ,免疫后两周小鼠肺淋巴细胞的抗HBsAgIgA抗体分泌细胞 (ASC)数比对照组 (RVJ12 3Δ11β75 )增加约 2倍 ,而同时小鼠肺淋巴细胞的抗HBsAgIgG抗体分泌细胞 (ASC)数与对照组无差别。可在小鼠血液、肺浸出液以及新西兰白兔血液、肺浸出液、其它分泌液样品中检测到抗乙型肝炎病毒HBsAg的特异性的IgA、IgG抗体 ,与对照组相比 ,IgA抗体阳转率及抗体滴度提高 ,而IgG则无差异。本实验说明 :IL 5可在体内选择性地增强机体的粘膜IgA反应。提示非复制载体疫苗中 ,表达的该细胞因子可有效的增强疫苗的粘膜免疫反应 ,为粘膜疫苗的发展策略提供了新的途径     

IL—5在非复制重组痘苗病毒中的表达及其对重组病毒免疫效效果的影响

利用非复制痘苗病毒表达载体pNEOCKβ751IL5和重组病毒RVJ123,通过两步重组构建了能同时表达IL－5和乙型肝炎病毒HBsAg的非复制型重组痘苗病毒RVJ123△11β75IL5。Southern-blot证实,痘苗病毒C－K片段间基因缺失的同时伴有IL－5基因的插入,鼻腔吸入分别免疫Balb/c小鼠和新西兰白兔。ELISPOT实验证实,免疫后两周小鼠肺淋巴细胞的抗HBsAgIgA抗分分泌细胞（ASC）数比对照组（RVJ123△11β75）增加约2倍,而同时小鼠肺淋巴细胞的抗HBsAgIgG抗体分泌细胞（ASC）数与对照组无差别。可在小鼠血液、肺浸出液以及新西兰白兔血液、肺浸出液、其它分泌液样品中检测到抗乙型肝炎病毒HBsAg的特异性的IgA,IgG抗体,与对照组相比,IgA抗体阳性转率及抗体滴度提高,而IgG则无差异。本实验说明：IL－5可在体内选择性地增强机体的粘膜IgA反应。提示非复制载体疫苗中,表达的该细胞因子有效的增强疫苗的的粘膜免疫反应,为粘膜疫苗的发展策略提供了新的途径。     

三个HIV-1广谱中和表位与HBV S抗原融合表达可诱导小鼠特异性抗体应答

为了增强HIV-1交叉中和表位的免疫原性,本研究使用PCR克隆技术将HIV-1三个具有一定广谱中和活性的线性抗原表位ELDKWA(简称2F5)、NWFDIT(简称4E10)和GPGRAFY(简称447-52D)基因分别融合到HBV S基因的3味端,构建了分别表达这三种融合基因的天坛株重组痘苗病毒疫苗RVJ1175S-2F5、RVJ1175S-4E10和RVJ1175S-447-52D,使用这三种重组痘苗病毒感染的细胞培养上清液经分离纯化制备了三种相应的蛋白亚单位疫苗PS-2F5、PS-4E10和PS-447-52D,对重组痘苗病毒和亚单位疫苗中三种融合抗原的生物学及免疫学特性进行了比较研究.PCR和测序结果表明,三种融合基因序列正确重组到痘苗病毒TK区,HBsAg的ELISA检测表明三种融合蛋白有效表达并分泌到细胞培养上清液中,SDS-PAGE凝胶电泳显示三种纯化后的融合蛋白均含分子量为23kD和27kD两种典型HBsAg条带,Western blot证明这两个条带均能与HBsAg抗体反应,并分别能与三种表位相应的HIV-1单抗2F5、4E10和447-52D反应.小鼠免疫结果显示,三种重组痘苗病毒疫苗和三种蛋白亚单位疫苗均能诱发较高水平的HBsAg抗体和相应HIV-1交叉中和表位抗体,蛋白亚单位疫苗诱生的这两类抗体均明显高于对应的重组痘苗病毒疫苗.这些结果为进一步研究三种表位抗体的中和活性和通过不同类型疫苗联合免疫进一步增强其免疫效果研究奠定了基础.     

表达乙型肝炎病毒表面抗原的非复制型重组痘苗病毒疫苗株RVJ123ΔCK的构建及其生物学性状鉴定

利用重组质粒 pNeo-CK与 pNeo-CKLacZ和表达乙型肝炎 (乙肝 )病毒S抗原的重组痘苗病毒RVJ12 3[1] ,构建了非复制型重组痘苗病毒RVJ12 3ΔCK。Southernblot证实 ,非复制型重组病毒RVJ12 3ΔCK基因组C和K片段间与宿主范围和毒力相关的基因稳定缺失 ,同时 ,J片段中插入的乙肝S抗原基因稳定存在。重组病毒RVJ12 3ΔCK在鸡胚成纤维母细胞中可良好繁殖 ,而在人源细胞系中不繁殖或仅低度繁殖 ,但都能表达HBsAg ,并且在病毒一个复制周期内 ,复制型和非复制型病毒HBsAg表达水平无明显差别。     

一种基于EGFP的、安全的抗HIV药物评价系统

目的：建立基于EGFP的、安全的抗人免疫缺陷病毒（HIV）药物评价系统。方法：用增强型绿色荧光蛋白（EGFP）基因替代HIV感染性克隆质粒pUC18-HIV-NL4-3中的部分包膜基因（env）,构建重组假病毒质粒pUC18-NL4-3-EGFP,将其与水疱性口炎病毒糖蛋白（VSV-G）真核表达载体共转染人胚肾293FT细胞,观察绿色荧光蛋白的表达,同时用该细胞培养上清进一步感染其他293FT细胞培养物。为了检验该假病毒系统能否用于抗病毒药物的评价,在假病毒复制和感染过程中加入不同浓度的抗HIV药物AZT（Zidovudine）,采用荧光显微镜检测和流式细胞仪定量检测,分析AZT对假病毒的抑制作用。结果：假病毒质粒pUC18-NL4-3-EGFP能够在转染细胞和再感染细胞中有效地表达绿色荧光蛋白基因,不同浓度的AZT能以剂量依赖方式抑制假病毒的感染和报告基因的表达。结论：建立了一种基于EGFP表达和检测的、安全的HIV假病毒复制和感染系统,该系统可以用于抗HIV药物的筛选和评价。     

表达绿色荧光蛋白的重组耻垢分枝杆菌的构建

目的 建立可表达绿色荧光蛋白的耻垢分枝杆菌,便于对耻垢分枝杆菌进行直观检测和快速定量。方法利用PCR技术从真核表达质粒pLVTH扩增获得绿色荧光蛋白的编码基因,克隆人大肠埃希菌一分枝杆菌穿梭载体pMV261,建立重组质粒pMVGFP,并经酶切鉴定证实。利用电穿孔技术将pMVGFP转化入耻垢分枝杆菌,利用卡那霉素抗性筛选重组耻垢分枝杆菌克隆,扩大培养后直接涂片,荧光显微镜镜检。结果重组质粒pMVGFP构建正确;将重组耻垢分枝杆菌在荧光显微镜下观察,证实绿色荧光蛋白在重组耻垢分枝杆菌中的表达。结论自发释放荧光的重组耻垢分枝杆菌的成功建立,为研究结核病致病机制和快速筛选化学药物等奠定了基础。     

Use of single-cycle infectious lymphocytic choriomeningitis virus to study hemorrhagic fever arenaviruses

Several arenaviruses, chiefly Lassa virus (LASV) and Junin virus in West Africa and Argentina, respectively, cause hemorrhagic fever (HF) disease in humans that is associated with high morbidity and significant mortality. The investigation of antiviral strategies to combat HF arenaviruses is hampered by the requirement of biosafety level 4 (BSL-4) facilities to work with these viruses. These biosafety hurdles could be overcome by the use of recombinant single-cycle infectious arenaviruses. To explore this concept, we have developed a recombinant lymphocytic choriomeningitis virus (LCMV) (rLCMVΔGP/GFP) where we replaced the viral glycoprotein (GP) with the green fluorescent protein (GFP). We generated high titers of GP-pseudotyped rLCMVΔGP/GFP via genetic trans complementation using stable cell lines that constitutively express LCMV or LASV GPs. Replication of these GP-pseudotyped rLCMVΔGP/GFP viruses was restricted to GP-expressing cell lines. This system allowed us to rapidly and reliably characterize and quantify the neutralization activities of serum antibodies against LCMV and LASV within a BSL-2 facility. The sensitivity of the GFP-based microneutralization assay we developed was similar to that obtained with a conventionally used focus reduction neutralization (FRNT) assay. Using GP-pseudotyped rLCMVΔGP/GFP, we have also obtained evidence supporting the feasibility of this approach to identify and evaluate candidate antiviral drugs against HF arenaviruses without the need of BSL-4 laboratories.     

Construction of recombinant herpes simplex virus type I expressing green fluorescent protein without loss of any viral genes

For use in various applications in research on herpes simplex virus type 1, we attempted to generate recombinant HSV-1 expressing green fluorescent protein (GFP) without any loss of viral genes. Our results were as follows. (i) A recombinant HSV-1 (YK333), in which a GFP expression cassette driven by the Egr-1 promoter was inserted into the intergenic region between UL3 and UL4, was constructed. (ii) YK333 replicated as well as wild-type HSV-1 F strain in Vero cells. (iii) As one application of the recombinant YK333 for research on HSV-1, we developed a system to detect anti-herpetic activity, termed a fluorescence-based anti-viral assay. The 50% inhibitory concentration of ganciclovir for YK333 determined using our newly developed assay was comparable to that determined using a plaque reduction assay. YK333 will be a convenient tool for herpes simplex virus research, including such applications as monitoring of viral replication in vitro and in vivo, and rapid screening of potential anti-herpetic agents.     

Multiple-cloning-site plasmids for the rapid construction of recombinant poxviruses

Plasmid vectors containing multiple cloning sites suitable for the rapid insertion of protein-coding sequences into poxviruses have been constructed. They are based on pUC plasmids and carry the thymidine kinase (TK) gene of vaccinia virus interrupted by a vaccinia virus promoter. Six unique restriction enzyme sites (BamHI, SalI/HincII, PstI, HindIII, EcoRI), located within 40 bp of vaccinia virus promoters transposed from the HindIII-F or HindIII-C fragment of the vaccinia virus genome, allow rapid insertion of foreign-protein-coding sequences into these plasmids. Such plasmids can be used to construct recombinant poxviruses expressing foreign proteins using marker-rescue recombination techniques and selection for TK negative viruses. Vaccinia viruses expressing the haemagglutinin (HA) gene of swine influenza virus, A/NJ/11/76 (H1N1), have been constructed.     

Vaccinia virus recombinants expressing the SA11 rotavirus VP7 glycoprotein gene induce serotype-specific neutralizing antibodies.

A cDNA copy of the gene coding for the major outer neutralizing protein (VP7) of simian 11 rotavirus was incorporated into the vaccinia virus genome under the control of the vaccinia promoter (molecular weight, 7,500). A deletion mutant of this gene which codes for a secreted form of VP7 when expressed under the control of the simian virus 40 late promoter (M. S. Poruschynsky, C. Tyndall, G. W. Both, F. Sato, A. R. Bellamy, and P. H. Atkinson, J. Cell Biol. 101:2199-2209, 1985) was also inserted. Each recombinant vaccinia virus directed the synthesis of a rotavirus protein in infected cells, and the product encoded by the mutated gene was secreted. Rabbits immunized with the two types of recombinant vaccinia virus generated antibodies that were able both to recognize simian 11 rotavirus in an enzyme-linked immunosorbent assay and to neutralize the virus in a plaque-reduction test. Antibodies induced by the recombinant vaccinia viruses expressing either form of VP7 were serotype specific.     

A New Method for the Detection of Neutralizing Antibodies against Mumps Virus

Neutralization test is the most reliable method of evaluating immunity against viral diseases but there is no standard procedure for mumps virus, with tests differing in the infectivity of the challenge virus, 50% plaque reduction or complete inhibition of cytopathic effects (CPE), and usage of complement. A reliable, easy, and simple neutralization test for mumps virus was developed in this study. A recombinant mumps virus expressing GFP was generated as a challenge virus. Complement was added to the neutralizing mixture at 1∶200 when stocked serum samples were used. Neutralizing antibody titers were expressed as the reciprocal of the highest dilution that did not exceed two-fold of FU values (GFP expression) of the cell control wells. A total of 1,452 serum samples were assayed by inhibition of GFP expression in comparison with those examined by conventional 100% inhibition of CPE. 1,367 (94.1%) showed similar neutralizing antibody titers when examined by both methods. The GFP expression inhibition assay, using a recombinant mumps virus expressing GFP, is a simple and time- saving method.     

Protein of macaques against infection with simian type D retrovirus (SRV-1) by immunization with recombinant vaccinia virus expressing the envelope glycoproteins of either SRV-1 or Mason-Pfizer monkey virus (SRV-3).

Rhesus macaques were immunized with live vaccinia virus recombinants expressing the envelope glycoproteins (gp70 and gp22) of simian type D retrovirus (SRV), serotype 1 or 3. All of the animals immunized with either the SRV-1 env or the SRV-3 env vaccinia virus recombinant developed neutralizing antibodies against the homologous SRV. In addition, both groups developed cross-reactive antibodies and were protected against an intravenous live-virus challenge with SRV-1. The four control animals immunized with a vaccinia virus recombinant expressing the G protein of respiratory syncytial virus were not protected against the same SRV-1 challenge. Although SRV-1 and SRV-3 immune sera showed cross-neutralization, they failed to neutralize a separate, more distantly related serotype, SRV-2, in an in vitro assay. These findings are consistent with the known degree of serologic and genetic relatedness of these three SRV strains.     

Immunization with a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: long-term protection and effect of revaccination.

Previously we showed that mice immunized with a vaccinia virus vector expressing the herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) gene (vaccinia/gD) were protected against both lethal and latent infections with HSV-1 for at least 6 weeks after immunization (K. J. Cremer, M. Mackett, C. Wohlenberg, A. L. Notkins, and B. Moss, Science 228:737-740, 1985). In the experiments described here, we examined long-term immunity to HSV following vaccinia/gD vaccination, the effect of revaccination with vaccinia/gD, and the impact of previous immunity to vaccinia virus on immunization with the gD recombinant. Mice immunized with vaccinia/gD showed 100, 100, and 80% protection against lethal infection with HSV-1 at 18, 44, and 60 weeks postimmunization, respectively. Protection against latent trigeminal ganglionic infection was 70, 50, and 31% at 6, 41, and 60 weeks postvaccination, respectively. To study the effect of reimmunization on antibody levels, mice vaccinated with vaccinia/gD were given a second immunization (booster dose) 3 months after the first. These mice developed a 10-fold increase in neutralizing-antibody titer (221 to 2,934) and demonstrated a significant increase in protection against lethal HSV-1 challenge compared with animals that received only one dose of vaccinia/gD. To determine whether preexisting immunity to vaccinia virus inhibited the response to vaccination with vaccinia/gD virus, mice were immunized with a recombinant vaccinia virus vector expressing antigens from either influenza A or hepatitis B virus and were then immunized (2 to 3 months later) with vaccinia/gD. These mice showed reduced titers of neutralizing antibody to HSV-1 and decreased protection against both lethal and latent infections with HSV-1 compared with animals vaccinated only with vaccinia/gD. We conclude that vaccination with vaccinia/gD produces immunity against HSV-1 that lasts over 1 year and that this immunity can be increased by a booster but that prior immunization with a vaccinia recombinant virus expressing a non-HSV gene reduces the levels of neutralizing antibody and protective immunity against HSV-1 challenge.     

Multigene DNA priming-boosting vaccines protect macaques from acute CD4+-T-cell depletion after simian-human immunodeficiency virus SHIV89.6P mucosal challenge

We evaluated four priming-boosting vaccine regimens for the highly pathogenic simian human immunodeficiency virus SHIV89.6P in Macaca nemestrina. Each regimen included gene gun delivery of a DNA vaccine expressing all SHIV89.6 genes plus Env gp160 of SHIV89.6P. Additional components were two recombinant vaccinia viruses, expressing SHIV89.6 Gag-Pol or Env gp160, and inactivated SHIV89.6 virus. We compared (i) DNA priming/DNA boosting, (ii) DNA priming/inactivated virus boosting, (iii) DNA priming/vaccinia virus boosting, and (iv) vaccinia virus priming/DNA boosting versus sham vaccines in groups of 6 macaques. Prechallenge antibody responses to Env and Gag were strongest in the groups that received vaccinia virus priming or boosting. Cellular immunity to SHIV89.6 peptides was measured by enzyme-linked immunospot assay; strong responses to Gag and Env were found in 9 of 12 vaccinia virus vaccinees and 1 of 6 DNA-primed/inactivated-virus-boosted animals. Vaccinated macaques were challenged intrarectally with 50 50% animal infectious doses of SHIV89.6P 3 weeks after the last immunization. All animals became infected. Five of six DNA-vaccinated and 5 of 6 DNA-primed/particle-boosted animals, as well as all 6 controls, experienced severe CD4(+)-T-cell loss in the first 3 weeks after infection. In contrast, DNA priming/vaccinia virus boosting and vaccinia virus priming/DNA boosting vaccines both protected animals from disease: 11 of 12 macaques had no loss of CD4(+) T cells or moderate declines. Virus loads in plasma at the set point were significantly lower in vaccinia virus-primed/DNA-boosted animals versus controls (P = 0.03). We conclude that multigene vaccines delivered by a combination of vaccinia virus and gene gun-delivered DNA were effective against SHIV89.6P viral challenge in M. nemestrina.     

In vivo and in vitro protein solubility assays using split GFP

The rapid assessment of protein solubility is essential for evaluating expressed proteins and protein variants for use as reagents for downstream studies. Solubility screens based on antibody blots are complex and have limited screening capacity. Protein solubility screens using split beta-galactosidase in vivo and in vitro can perturb protein folding. Split GFP used for monitoring protein interactions folds poorly, and to overcome this limitation, we recently developed a protein-tagging system based on self-complementing split GFP derived from an exceptionally well folded variant of GFP termed 'superfolder GFP'. Here we present the step-by-step procedure of the solubility assay using split GFP. A 15-amino-acid GFP fragment, GFP 11, is fused to a test protein. The GFP 1-10 detector fragment is expressed separately. These fragments associate spontaneously to form fluorescent GFP. The fragments are soluble, and the GFP 11 tag has minimal effect on protein solubility and folding. We describe high-throughput protein solubility screens amenable both for in vivo and in vitro formats. The split-GFP system is composed of two vectors used in the same strain: pTET GFP 11 and pET GFP 1-10 (Fig. 1 and Supplementary Note online). The gene encoding the protein of interest is cloned into the pTET GFP 11 vector (resulting in an N-terminal fusion) and transformed into Escherichia coli BL21 (DE3) cells containing the pET GFP 1-10 plasmid. We also describe how this system can be used for selecting soluble proteins from a library of variants (Box 1). The large screening power of the in vivo assay combined with the high accuracy of the in vitro assay point to the efficiency of this two-step split-GFP tool for identifying soluble clones suitable for purification and downstream applications.     

Remodeling of the tumor microenvironment using an engineered oncolytic vaccinia virus improves PD-L1 inhibition outcomes

Immune checkpoint inhibitor (ICI) immunotherapies have vastly improved therapeutic outcomes for patients with certain cancer types, but these responses only manifest in a small percentage of all cancer patients. The goal of the present study was to improve checkpoint therapy efficacy by utilizing an engineered vaccinia virus to improve the trafficking of lymphocytes to the tumor, given that such lymphocyte trafficking is positively correlated with patient checkpoint inhibitor response rates. We developed an oncolytic vaccinia virus (OVV) platform expressing manganese superoxide dismutase (MnSOD) for use as both a monotherapy and together with anti-PD-L1. Intratumoral OVV-MnSOD injection in immunocompetent mice resulted in inflammation within poorly immunogenic tumors, thereby facilitating marked tumor regression. OVV-MnSOD administration together with anti-PD-L1 further improved antitumor therapy outcomes in models in which these monotherapy approaches were ineffective. Overall, our results emphasize the value of further studying these therapeutic approaches in patients with minimally or non-inflammatory tumors.