Yuki Sato, Ramjay Vatsan, Bharat H Joshi, Syed R Husain, Raj K Puri
{"title":"一种表达白细胞介素-13 受体 α2抗原的新型重组改良安卡拉疫苗病毒，可用于潜在的癌症免疫疗法。","authors":"Yuki Sato, Ramjay Vatsan, Bharat H Joshi, Syed R Husain, Raj K Puri","doi":"10.2174/1566524023666230331085007","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Genetically altered recombinant poxviruses hold great therapeutic promise in animal models of cancer. Poxviruses can induce effective cellmediated immune responses against tumor-associated antigens. Preventive and therapeutic vaccination with a DNA vaccine expressing IL-13Rα2 can mediate partial regression of established tumors <i>in vivo</i>, indicating that host immune responses against IL-13Rα2 need further augmentation.</p><p><strong>Objective: </strong>The aim of the study is developing a recombinant modified vaccinia Ankara (MVA) expressing <i>IL-13Rα2</i> (rMVA-IL13Rα2) virus and study <i>in vitro</i> infectivity and efficacy against IL-13Rα2 positive cell lines.</p><p><strong>Methods: </strong>We constructed a recombinant MVA expressing <i>IL-13Rα2</i> and a green fluorescent protein (<i>GFP</i>) reporter gene. Purified virus titration by infection of target cells and immunostaining using anti-vaccinia and anti-IL-13Rα2 antibodies was used to confirm the identity and purity of the rMVA-IL13Rα2.</p><p><strong>Results: </strong>Western Blot analysis confirmed the presence of IL-13Rα2 protein (~52 kDa). Flow cytometric analysis of IL-13Rα2 negative T98G glioma cells when infected with rMVA-IL13Rα2 virus demonstrated cell-surface expression of IL-13Rα2, indicating the infectivity of the recombinant virus. Incubation of T98G-IL13Rα2 cells with varying concentrations (0.1-100 ng/ml) of interleukin-13 fused to truncated Pseudomonas exotoxin (IL13-PE) resulted in depletion of GFP<sup>+</sup> fluorescence in T98G-IL13Rα2 cells. IL13-PE (10-1000 ng/ml) at higher concentrations also inhibited the protein synthesis in T98G-IL13Rα2 cells compared to cells infected with the control pLW44-MVA virus. IL13- PE treatment of rMVA-IL13Rα2 infected chicken embryonic fibroblast and DF-1 cell line reduced virus titer compared to untreated cells.</p><p><strong>Conclusion: </strong>rMVA-IL13Rα2 virus can successfully infect mammalian cells to express IL-13Rα2 in a biologically active form on the surface of infected cells. To evaluate the efficacy of rMVA-IL13Rα2, immunization studies are planned in murine tumor models.</p>","PeriodicalId":10873,"journal":{"name":"Current molecular medicine","volume":" ","pages":"758-770"},"PeriodicalIF":2.5000,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"A Novel Recombinant Modified Vaccinia Ankara Virus expressing Interleukin-13 Receptor α2 Antigen for Potential Cancer Immunotherapy.\",\"authors\":\"Yuki Sato, Ramjay Vatsan, Bharat H Joshi, Syed R Husain, Raj K Puri\",\"doi\":\"10.2174/1566524023666230331085007\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Genetically altered recombinant poxviruses hold great therapeutic promise in animal models of cancer. Poxviruses can induce effective cellmediated immune responses against tumor-associated antigens. Preventive and therapeutic vaccination with a DNA vaccine expressing IL-13Rα2 can mediate partial regression of established tumors <i>in vivo</i>, indicating that host immune responses against IL-13Rα2 need further augmentation.</p><p><strong>Objective: </strong>The aim of the study is developing a recombinant modified vaccinia Ankara (MVA) expressing <i>IL-13Rα2</i> (rMVA-IL13Rα2) virus and study <i>in vitro</i> infectivity and efficacy against IL-13Rα2 positive cell lines.</p><p><strong>Methods: </strong>We constructed a recombinant MVA expressing <i>IL-13Rα2</i> and a green fluorescent protein (<i>GFP</i>) reporter gene. Purified virus titration by infection of target cells and immunostaining using anti-vaccinia and anti-IL-13Rα2 antibodies was used to confirm the identity and purity of the rMVA-IL13Rα2.</p><p><strong>Results: </strong>Western Blot analysis confirmed the presence of IL-13Rα2 protein (~52 kDa). Flow cytometric analysis of IL-13Rα2 negative T98G glioma cells when infected with rMVA-IL13Rα2 virus demonstrated cell-surface expression of IL-13Rα2, indicating the infectivity of the recombinant virus. Incubation of T98G-IL13Rα2 cells with varying concentrations (0.1-100 ng/ml) of interleukin-13 fused to truncated Pseudomonas exotoxin (IL13-PE) resulted in depletion of GFP<sup>+</sup> fluorescence in T98G-IL13Rα2 cells. IL13-PE (10-1000 ng/ml) at higher concentrations also inhibited the protein synthesis in T98G-IL13Rα2 cells compared to cells infected with the control pLW44-MVA virus. IL13- PE treatment of rMVA-IL13Rα2 infected chicken embryonic fibroblast and DF-1 cell line reduced virus titer compared to untreated cells.</p><p><strong>Conclusion: </strong>rMVA-IL13Rα2 virus can successfully infect mammalian cells to express IL-13Rα2 in a biologically active form on the surface of infected cells. To evaluate the efficacy of rMVA-IL13Rα2, immunization studies are planned in murine tumor models.</p>\",\"PeriodicalId\":10873,\"journal\":{\"name\":\"Current molecular medicine\",\"volume\":\" \",\"pages\":\"758-770\"},\"PeriodicalIF\":2.5000,\"publicationDate\":\"2024-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Current molecular medicine\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.2174/1566524023666230331085007\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"MEDICINE, RESEARCH & EXPERIMENTAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current molecular medicine","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.2174/1566524023666230331085007","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"MEDICINE, RESEARCH & EXPERIMENTAL","Score":null,"Total":0}
A Novel Recombinant Modified Vaccinia Ankara Virus expressing Interleukin-13 Receptor α2 Antigen for Potential Cancer Immunotherapy.
Background: Genetically altered recombinant poxviruses hold great therapeutic promise in animal models of cancer. Poxviruses can induce effective cellmediated immune responses against tumor-associated antigens. Preventive and therapeutic vaccination with a DNA vaccine expressing IL-13Rα2 can mediate partial regression of established tumors in vivo, indicating that host immune responses against IL-13Rα2 need further augmentation.
Objective: The aim of the study is developing a recombinant modified vaccinia Ankara (MVA) expressing IL-13Rα2 (rMVA-IL13Rα2) virus and study in vitro infectivity and efficacy against IL-13Rα2 positive cell lines.
Methods: We constructed a recombinant MVA expressing IL-13Rα2 and a green fluorescent protein (GFP) reporter gene. Purified virus titration by infection of target cells and immunostaining using anti-vaccinia and anti-IL-13Rα2 antibodies was used to confirm the identity and purity of the rMVA-IL13Rα2.
Results: Western Blot analysis confirmed the presence of IL-13Rα2 protein (~52 kDa). Flow cytometric analysis of IL-13Rα2 negative T98G glioma cells when infected with rMVA-IL13Rα2 virus demonstrated cell-surface expression of IL-13Rα2, indicating the infectivity of the recombinant virus. Incubation of T98G-IL13Rα2 cells with varying concentrations (0.1-100 ng/ml) of interleukin-13 fused to truncated Pseudomonas exotoxin (IL13-PE) resulted in depletion of GFP+ fluorescence in T98G-IL13Rα2 cells. IL13-PE (10-1000 ng/ml) at higher concentrations also inhibited the protein synthesis in T98G-IL13Rα2 cells compared to cells infected with the control pLW44-MVA virus. IL13- PE treatment of rMVA-IL13Rα2 infected chicken embryonic fibroblast and DF-1 cell line reduced virus titer compared to untreated cells.
Conclusion: rMVA-IL13Rα2 virus can successfully infect mammalian cells to express IL-13Rα2 in a biologically active form on the surface of infected cells. To evaluate the efficacy of rMVA-IL13Rα2, immunization studies are planned in murine tumor models.
期刊介绍：
Current Molecular Medicine is an interdisciplinary journal focused on providing the readership with current and comprehensive reviews/ mini-reviews, original research articles, short communications/letters and drug clinical trial studies on fundamental molecular mechanisms of disease pathogenesis, the development of molecular-diagnosis and/or novel approaches to rational treatment. The reviews should be of significant interest to basic researchers and clinical investigators in molecular medicine. Periodically the journal invites guest editors to devote an issue on a basic research area that shows promise to advance our understanding of the molecular mechanism(s) of a disease or has potential for clinical applications.
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