HIGH EFFICIENCY RETROVIRUS-MEDIATED GENE TRANSFERTO LEUKEMIA CELLS

Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (NeoR) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1. The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells. Results: The titer of DOSPER directly transfected GP+envAm12 cells determined on NIH3T3 cells was 8.0×105 CFU/ml, while that of producer infected with retrovirus was 1.6×107 CFU/ml. Integration of NeoR gene into all leukemia cells was confirmed by polymerase chain reaction (PCR). Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated NeoR gene on colonies after 7 days’ culture. Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.

No transmission of porcine endogenous retrovirus in an acute liver failure model treated by a novel hybrid bioartificial liver containing porcine hepatocytes

BACKGROUND： A novel hybrid bioartificial liver（HBAL） was constructed using an anionic resin adsorption column and a multi-layer flat-plate bioreactor containing porcine hepatocytes co-cultured with bone marrow mesenchymal stem cells（MSCs）. This study aimed to evaluate the microbiological safety of the HBAL by detecting the transmission of porcine endogenous retroviruses（PERVs） into canines with acute liver failure（ALF） undergoing HBAL.METHODS： Eight dogs with ALF received a 6-hour HBAL treatment on the first day after the modeling by D-galactosamine administration. The plasma in the HBAL and the whole blood in the dogs were collected for PERV detection at regular intervals until one year later when the dogs were sacrificed to retrieve the tissues of several organs for immunohistochemistry and Western blotting for the investigation of PERV capsid protein gag p30 in the tissue. Furthermore, HEK293 cells were incubated to determine the in vitro infectivity.RESULTS： PERV RNA and reverse transcriptase activity were observed in the plasma of circuit 3, suggesting that PERV particles released in circuit 3. No positive PERV RNA and reverse transcriptase activity were detected in other plasma. No HEK293 cells were infected by the plasma in vitro. In addition, all PERV-related analyses in peripheral blood mononuclear cells and tissues were negative.CONCLUSION： No transmission of PERVs into ALF canines suggested a reliable microbiological safety of HBAL based on porcine hepatocytes.

Multifunctional facets of retrovirus integrase

The retrovirus integrase(IN) is responsible for integration of the reverse transcribed linear c DNA into the host DNA genome. First, IN cleaves a dinucleotide from the 3' OH blunt ends of the viral DNA exposing the highly conserved CA sequence in the recessed ends. IN utilizes the 3' OH ends to catalyze the concerted integration of the two ends into opposite strands of the cellular DNA producing 4 to 6 bp staggered insertions, depending on the retrovirus species. The staggered ends are repaired by host cell machinery that results in a permanent copy of the viral DNA in the cellular genome. Besides integration, IN performs other functions in the replication cycle of several studied retroviruses. The proper organization of IN within the viral internal core is essential for the correct maturation of the virus. IN plays a major role in reverse transcription by interacting directly with the reverse transcriptase and by binding to the viral capsid protein and a cellular protein. Recruitment of several other host proteins into the viral particle are also promoted by IN. IN assists with the nuclear transport of the preintegration complex across the nuclear membrane. With several retroviruses, IN specifically interacts with different host protein factors that guide the preintegration complex to preferentially integrate the viral genome into specific regions of the host chromosomal target. Human gene therapy using retrovirus vectors is directly affected by the interactions of IN with these host factors. Inhibitors directed against the human immunodeficiency virus(HIV) IN bind within the active site of IN containing viral DNA ends thus preventing integration and subsequent HIV/AIDS.

Expression and biological activity of double replica retrovirus carrier-mediated neurotrophin-3 in olfactory ensheathing cells

BACKGROUND： Previous studies have demonstrated that the combination of olfactory ensheathing cells （OECs） and neurotrophic factor-3 （NT-3） in the rat lateral ventricle can promote nerve axonal regeneration and myelin sheath repair. However, this effect remains very short-lived. OBJECTIVE： To transfect NT-3 into OECs and to observe the biological activity of OEC-expressing NT-3. DESIGN, TIME AND SETTING： This genetic engineering, in vitro experiment was performed in the Provincial Hospital Affiliated to Shandong University between January 2007 and October 2008. MATERIALS： Trizol Reagent kit was purchased from Gibco, USA; reverse transcription kit, NT-3 Emax ImmunoAssay System reagent was purchased from Promega, USA. METHODS： Neonatal Wistar rat OECs were established as primary cultures and were transfected with pN2A-NT-3 viral vector. The OECs with the highest virus titer and stable cellular growth served as the transfection group; OECs transfected with NT-3-free retrovirus carrier pN2A served as the empty vector group; un-transfected OECs served as the control group. After adherence, the logarithmically cultured PC12-TrkC cells were plated in OECs supernatant from the transfection and empty vector groups, as well as 20 μL PBS, and cultured for 4 days. MAIN OUTCOME MEASURES： NT-3 mRNA expression in OECs, fluorescence of NT-3-positive cells in the transfection group and control group; influence of OECs secreting NT-3 on the differentiation ratio of PC12-TrkC cells. RESULTS： NT-3 mRNA expression was observed 24 hours after transfection and lasted for 28 days which was greater than the control and empty vector groups （P 〈 0.01）. A large number of NT-3-positive cells were observed in the transfection group, and immunofluorescence was greater than the control and empty vector groups. PC12-TrkC cells co-cultured with OECs from the transfection group exhibited a thick and long cell process, increased cell density, and the differentiation ratio was increased （P 〈 0.01）. CONCLUSION： Recombinant double replica retrovirus NT-3 gene was stably and effectively expressed in OECs, and the expressed NT-3 possessed biological activity that promoted neuronal survival.

Recombinant Apoptin Gene Retrovirus Induces Apoptosis in Human Breast Cancer Cells 435

Objective： To construct recombinant Apoptin gene （vp3） retrovirus pLVP3 and to study its apoptosis inducing effect on human breast cancer cells 435 as well as to discuss its mechanism in vitro and in vivo. Methods： vp3 gene was cloned and recombinated into retrovirus vector pLP-LNCX-VP3 （pLVP3） at loxP site, which was transformed into package cell line PT67 and then into NIH3T3 cells for titer assay. The human breast cancer cell line 435 was infected with retrovirus pLVP3, and then MTT assay and Western blotting were adopted to detect cellular proliferation and Apoptin protein expression. Forty-eight hours after infection flow cytometry （FCM） was used for apoptosis detection and Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometry （SELDI-TOF-MS） was used for protein profile assay. Nude mice model of human breast cancer cells 435 was set up to observe the tumor inhibiting rates of pLVP3, and TUNEL assay was used to detect tumor apoptosis as well as real-time PCR for vp3 gene expression. Results： Recombinant plasmid pLVP3 was successfully constructed. Virus titer reached to 5×10^8 pfu/ml in the PT67 culture supernatant. Forty-eight hours after infection, cellular inhibition rate was 65.1% in MTT assay, higher than that in blank control （P〈0.05） and Apoptin protein expressed more in test group in Western blotting. FCM assay showed apoptotic peaks with a percentage of 15.42%. SELDI-TOF-MS findings suggested that two protein peaks, M_2544.1＋H and M_3712.4＋H, were statistically different between infection group and control group （P〈0.05）. The tumor inhibition rates in pLVP3 group were 65.52% and 68.23%, much higher than that of control group （t=4.06, P〈0.01）. TUNEL assay findings showed that positive yellow stains were seen in pLVP3 retrovirus group and 5-FU positive control group without difference （t1=1.05, t2=0.84, P〉0.05）. Conclusion： The experiment demonstrated that vp3 could induce apoptosis in tumor cells in vivo and in vitro, which laid a basis for further study on molecular mechanism of tumor cell apoptosis induced by Apoptin and provided valuable reference for tumor gene therapy.

Reducing porcine corneal graft rejection, with an emphasis on porcine endogenous retrovirus transmission safety: a review

Donor cornea shortage is a primary hurdle in the development of corneal transplantation. Of all species, porcine corneas are the ideal transplantation material for humans. However, the xenoimmune rejection induced by porcine corneal xenotransplantation compromises surgical efficacy. Although the binding of IgM/IgG in human serum to a genetically modified porcine cornea is significantly weaker than that of the wild type(WT), genetically modified porcine corneas do not display a prolonged graft survival time in vivo. Conversely, costimulatory blockade drugs, such as anti-CD40 antibodies, can reduce the xenoimmune response and prolong graft survival time in animal experiments. Moreover, porcine endothelial grafts can survive for more than 6mo with only the subconjunctival injection of a steroid-based immunosuppressants regime; therefore, they show great value for treating corneal endothelial disease. In addition, zoonotic transmission is a primary concern of xenotransplantation. Porcine endogenous retrovirus(PERV) is the most significant virus assessed by ophthalmologists. PERV integrates into the porcine genome and infects human cells in vitro. Fortunately, no evidence from in vivo studies has yet shown that PERV can be transmitted to hosts.

Retrovirus-Mediated Gene Transfer in Immortalization of Progenitor Hair Cell Lines in Newborn Rat

Objective To present an experimental method that allows isolation of greater epithelial ridge (GER) and lesser epithelial ridge(LER) cells from postnatal rat cochleae using a combinatorial approach of enzymatic digestion and mechanical separation and to investigate a retrovirus-mediated gene transfer technique for its possible utility in immortalization of the GER and LER cell lines, in an effort to establish an in vitro model system of hair cell differentiation. Methods GER and LER cells were dissected from postnatal rat cochleae and immortalized by transferring the SV40 large T antigen using a retrovirus. The established cell lines were confirmed through mor-phology observation, immunnocytochemical staining and RT-PCR analysis. The Hath1 gene was transferred into the cell lines using adenovirus-mediated techniques to explore their potential to differentiate into hair cells. Results The established cell lines were stably maintained for more than 20 passages and displayed many features similar to primary GER and LER cells. They grew in patches and assumed a polygonal morphology. Immunostaining showed labeling by SV40 large T antigen and Islet1(a specific marker for GER and LER). All passages of the cell lines expressed SV40 large T antigen on RT-PCR analysis. The cells also showed the capability to differentiate into hair cell-like cells when forced to express Hath1. Conclusion Retrovirus-mediated gene transfer can be used in establishing immortalized progenitor hair cell lines in newborn rat, which may provide an invaluable system for studying hair cell differentiation and regeneration for new treatment of sensory hearing loss caused by hair cell loss.

GENE EXPRESSION OF NOVEL RETROVIRUS ASSOCIATED WITH HUMAN ACUTE MULOID LEUKEMIA

Objective: To explore the potentiality of retroviral etiology in human acute myeloid leukemia(AML). Methods: The expression of clone 6#11 in leukemic cell samples from 19 AML cases and peripheral blood mononuclear cells (PBMNCs) from 20 controls was studied by means of Northern blot and reversal transcription polymerase chain reaction (RT-PCR). Results: Northern blot and RT-PCR analyses showed that the expression level of clone 6#11 was significantly higher in AML patients than that in control. Conclusion: Northern blot and RT-PCR analyses revealed that the expression of novel retrovirus were associated with acute myeloid leukemia.

Classification of Retroviruses Based on Genomic Data Using RVGC

Retroviruses are a large group of infectious agents with similar virion structures and replication mechanisms.AIDS,cancer,neurologic disorders,and other clinical conditions can all be fatal due to retrovirus infections.Detection of retroviruses by genome sequence is a biological problem that benefits from computational methods.The National Center for Biotechnology Information(NCBI)promotes science and health by making biomedical and genomic data available to the public.This research aims to classify the different types of rotavirus genome sequences available at the NCBI.First,nucleotide pattern occurrences are counted in the given genome sequences at the preprocessing stage.Based on some significant results,the number of features used for classification is reduced to five.The classification shall be carried out in two phases.The first phase of classification shall select only two features.Unclassified data in the first phase is transferred to the next phase,where the final decision is taken with the remaining three features.Three data sets of animals and human retroviruses are selected;the training data set is used to minimize the classifier’s number and training;the validation data set is used to validate the models.The performance of the classifier is analyzed using the test data set.Also,we use decision tree,naive Bayes,knearest neighbors,and vector support machines to compare results.The results show that the proposed approach performs better than the existing methods for the retrovirus’s imbalanced genome-sequence dataset.

Induction of anti-hepatoma immunity by recombinant retrovirus expressing B7-1 /B7-2 costimulatory molecules

Objective: To construct recombinant R7-l/B7-2 retrovirus vectors and observe the effects of B7-l/R7-2 gene expression on in ho and in for immune response against against murine hepatoma. Methods: The recombinant retrovirus vectors expressing B7-1/B7-2 were constructed by gene cloning technology to produce retrovirus-infected PE501 and PA317 cell lines and murine hepatoma Hepal-6. The expression of R7-l/B7-2 was detected by fluorescence activated cell soning analysis (FACS). B7-l/B7-2 positive Hepal-6 Cell lines were used in inducing anti-hepatoma immunity in ho and in the. Results: In contrast to the excessive growth of parental Hemal-6 tumor, the growth of B7-l/B7-2-positive Hepal-6 inoculated into syngenic mice regressed. B7-1/R7-2-positive or cytokine-treated Hepal-6 alone could only induce mild cytototicity; in contrast, B7-1/B7-2-positive Hemal-6 treated with cytokine-stimulated spleen cells and activated the cytotoxicity effectively. Immunity in mice with R7-1/B7-2-positive tumor cells or cytokine-beated Hepal-6 only provided partial protection against parental Hepa1-6 tumor, whereas pretreatment of the transfected tumor cells with IFN-r and TNF-a induced complete immunity protection in vivo. Mice receiving inoculation of cytokine-treated B7-l/R7-2-positive Hemal-6 cells presented regression of the establoshed pental tUmor and survived for more than l00 d, while those untreated mice died within 40 d. Conclu sions: B7-l/R7-2 expression is necessary but not sufficient in inducing anti-hepatoma immune response, whereas it is efficient when combined with the beatment of IFN-γ and TNF-a.

EFFECTS OF PSEUDOFYPE RETROVIRUS CONTAINING HUMAN N-RAS ANTISENSE GENE ON THE GROWTH OF HUMAN LIVER CANCER LTNM4 TRANSPLANTED IN NUDE MICE

An amphotropic pseudotype retrovirus containing human N-ras antisense gene was constructed and packaged with helper cells. It has been previously demonstrated that the virus did inhibit the growth of human hepatocarcinoma cell line PLC PRF/5 in vitro accompanied with the blockage of p21 expression. Based on these results, further study was carried on to examine the effect of these viruses on the growth of human hepatoma transplanted LTNM4 in nude mice. It has been shown that the retrovirus containing human antisense N-ras gene could inhibit the hepatoma in nude mice at a rate of 78% (P<0.05) as compared with saline control. No inhibition was observed in group treated with retrovirus which contained no N-ras sequence. These results in vivo lend further support that human N-ras antisense gene mediated by retrovirus could block the expression of the relevant oncogene and lead to the inhibition of cancer growth. It also provided the basis for further approaches of gene therapy for human cancer.

Differentiation of wild boar and domestic pig populations based on the frequency of chromosomes carrying endogenous retroviruses

Analysis of the frequencies of chromosomes carrying various classes of porcine endogenous retroviruses (PERVs) and combinations of these classes was performed in the swine species Sus scrofa L. by using maps constructed in two principal component coordinates. Four population clusters can be recognized in the maps. Cluster 1 is formed by wild boars,cluster 2 by domestic meat breeds, cluster 3 by meat-and-lard (universal) breeds, and cluster 4 by miniature pigs. The maps indicate that modern domesticated swine meat breeds are the closest to the wild type. Meat-and-lard domestic swine breeds are more distant from wild boars, and miniature pigs are diverged the most. The maps showed that microevolution processes associated with PERV carriership frequency had two basic dimensions, or vectors: the vector of fat deposition variation and the “minus” selection vector (determination of commercial traits). Thus, PERVs may cause variation in pig physiology.

Gene therapy strategies for treating brain tumors: Retroviruses are still good candidates for therapeutic vectors

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. In the past few decades, many efforts have been made to improve the prognosis of GBM, however, with limited success. Many gene therapy strategies for GBM have been developed and a few have progressed to clinical trials. Retroviral vectors have superior features for gene therapy in brain cancers, including tumor specificity, immunogenicity, and longer half-life. Early gene therapy trials in GBM patients based on transplantation of retrovirus-producing cells into the brain failed to prove efficacious. Adenoviral vectors, which can be prepared as high-titer virus solutions and undergo efficient transduction in tumor cells, failed in clinical trials, likely due to immunogenicity and instability of gene expression. Alternative therapeutics such as oncolytic viruses that specifically target and destroy cancer cells are currently under investigation. In addition to novel vectors, retroviral vectors are still attractive candidates for use in gene therapy against brain tumors. Since yields of properly-packaged viral particles from virus-producing cells have been very limited so far, gene therapy by direct injection of hightiter retroviral vectors into the patients’ brains was not possible. To overcome these disadvantages, a packaging cell line that yields high-titer retroviral solutions was established by our group, enabling the direct injection of massive retroviral vector stocks directly into the brain. Mouse glioma models were effectively cured with a combination of a suicide gene/ prodrug system and a highly-concentrated retrovirus solution. Preclinical assessments, including that of replication-competent retroviruses and tumorigenicity of the combination method, have confirmed the safety of the highly-concentrated retrovirus solution. Addi tional studies are needed to address the clinical utility of such combination gene therapies. Taken together, these data suggest that retroviral vectors are still good candidates for development in gene therapy applications.

Human endogenous retroviruses and cancer:Causality and therapeutic possibilities

A substantial part of the human genome is derived from transposable elements;remnants of ancient retroviral infections.Conservative estimates set the percentage of human endogenous retroviruses(HERVs) in the genome at 8%.For the most part,the interplay between mutations,epigenetic mechanisms and posttranscriptional regulations silence HERVs in somatic cells.We first highlight mechanisms by which activation of members of several HERV families may be associated with tumor development before discussing the arising chances for both diagnosis and therapy.It has been shown that at least in some cases,tumor cells expressing HERV open reading frames(ORFs) thus gain tumor-promoting functions.However,since these proteins are not expressed in healthy tissues,they become prime target structures.Of potential pharmacological interest are the prevention of HERV transposition,the inhibition of HERV-encoded protein expression and the interference with these proteins' activities.Evidence from recent studies unequivocally proves that HERV ORFs represent a very interesting source of novel tumor-specific antigens with even the potential to surpass entity boundaries.The development of new tumor(immune-) therapies is a very active field and true tumor-specific targets are of outstanding interest since they minimize the risk of autoimmunity and could reduce side effects.Finally,we postulate on main future research streams in order to stimulate discussion on this hot topic.

Genome-wide identification of endogenous retrovirus elements and their active transcription in mink genome

Mammalian endogenous retroviruses(ERVs)are ancient retroviruses that have been integrated into genomes.ERVs were believed to be inactive until the discovery of ERV transcription in the mouse genome.However,the transcription level and function of ERV elements in mammalian genomes are not well understood.In this study,we performed the first genome‐wide scanning of ERV loci in the American mink(Neogale vison)genome(NeoERV)followed by transcriptomic analysis to detect actively transcribed NeoERV elements.A total of 365,791 NeoERV loci were identified,and161,205(44%)of these loci were found to be actively transcribed based on transcriptomic data from three types of tissues(amygdala,trachea and lung).More than one third of the actively transcribed NeoERV loci were tissue‐specific.Furthermore,some of the active loci were associated with host gene transcription,and the level of NeoERV transcription was positively correlated with that of host genes,specifically when active loci were located in overlapped gene regions.An in‐depth analysis of the envelope protein coding env gene showed that,in general,its transcription level was higher than that of NeoERVs,which is believed to be associated with host immunity.

The diversity and evolution of retroviruses:Perspectives from viral“fossils”

Retroviruses exclusively infect vertebrates,causing a variety of diseases.The replication of retroviruses requires reverse transcription and integration into host genomes.When infecting germline cells,retroviruses become inherited vertically,forming endogenous retroviruses(ERVs).ERVs document past viral infections,providing molecular fossils for studying the evolutionary history of retroviruses.In this review,we summarize the recent advances in understanding the diversity and evolution of retroviruses from the perspectives of viral fossils,and discuss the effects of ERVs on the evolution of host biology.

山羊地方性鼻内肿瘤病毒TaqMan荧光定量RT-PCR检测方法的建立及应用

山羊地方性鼻内肿瘤(enzootic nasal tumor, ENT)是山羊的病毒性传染病,由山羊地方性鼻内肿瘤病毒(enzootic nasal tumor virus of goats, ENTV-2)所引起,感染后可以导致山羊的鼻道筛骨上皮细胞发生不可逆的癌变,已在世界范围内广泛存在,对山羊养殖业造成严重的经济损失。为建立快速、准确且能定量分析ENTV-2的检测方法,本研究根据GenBank上发布的ENTV-2 env基因(MT254061.1)保守区域设计引物和TaqMan探针,建立ENTV-2 TaqMan探针荧光定量RT-PCR检测方法,并对其特异性、灵敏性、重复性与临床检测效果进行验证。结果显示,重组质粒标准品模板浓度为6.30×10^(2)~6.30×10^(7) copies·μL^(-1)呈现良好的线性关系,相关系数R^(2)为0.996 5,扩增效率为110%,线性方程的斜率为-2.953,最低检测限度为6.30×10^(1) copies·μL^(-1);组内变异系数和组间变异系数均小于2%,重复性好;与口蹄疫病毒((foot-and-mouth disease virus, FMDV)、小反刍兽疫病毒(peste des petits ruminants virus, PPRV)、蓝舌病毒(bluetongue virus)、羊内源性逆转录病毒(endogenous retroviruses)等均无交叉反应,表明该方法具有很好的特异性。利用本研究所建立的TaqMan探针实时荧光定量RT-PCR检测方法对29份临床样品进行检测,该方法较普通PCR方法的检出率更高。综上表明,本研究成功建立了ENTV-2 TaqMan探针实时荧光定量RT-PCR检测方法,为ENTV-2的快速诊断和流行病学调查提供技术手段。

长非编码RNA lnc-ALVE1-AS1抑制ALV-J在鸡巨噬细胞中增殖

研究表明,源自内源性反转录病毒的长非编码RNA可能是巨噬细胞抗病毒免疫反应的重要组成部分。鸡内源性反转录病毒衍生的长非编码RNA lnc-ALVE1-AS1与宿主遗传抗性有关,能够激活非免疫细胞抗病毒天然免疫反应。为进一步探讨lnc-ALVE1-AS1在鸡巨噬细胞中的抗病毒作用及其机制,本研究通过荧光定量PCR发现lnc-ALVE1-AS1在ALV-J感染鸡巨噬细胞系HD11后24 h显著下调。进一步通过lnc-ALVE1-AS1过表达实验证实,lnc-ALVE1-AS1可以显著抑制ALV-J在HD11细胞中增殖。机制上,lnc-ALVE1-AS1显著上调HD11细胞dsRNA识别受体(TLR3)、Ⅰ型干扰素(IFN-α和IFN-β)和其他抗病毒天然免疫基因(IRF7、MX1、OASL和IFITM3)表达。然而,干扰TLR3或者抑制TLR3信号后lnc-ALVE1-AS1诱导Ⅰ型干扰素(IFN-α和IFN-β)的能力显著减弱。共聚焦定位分析显示在HD11细胞中lnc-ALVE1-AS1可以与TLR3蛋白直接结合。这说明激活TLR3信号可能是lnc-ALVE1-AS1抑制ALV-J在巨噬细胞中增殖的一个重要机制。本研究揭示了lnc-ALVE1-AS1在巨噬细胞中抗病毒功能及其作用机制,为抗病育种研究提供了新的遗传基础。

Human leukemia antigen-A0201-restricted epitopes of humanendogenous retrovirus W family envelope(HERV-W env)inducestrong cytotoxic T lymphocyte responses

Human endogenous retrovirus W family(HERV-W) envelope(env) has been reported to be related to several human diseases, including autoimmune disorders, and it could activate innate immunity.However, there are no reports investigating whether human leukemia antigen(HLA)-A~*0201^+restriction is involved in the immune response caused by HERV-W env in neuropsychiatric diseases. In the present study, HERV-W env-derived epitopes presented by HLA-A~*0201 are described with the potential for use in adoptive immunotherapy. Five peptides displaying HLAA~*0201-binding motifs were predicted using SYFEPITHI and BIMAS, and synthesized. A CCK-8 assay showed peptides W, Q and T promoted lymphocyte proliferation. Stimulation of peripheral blood mononuclear cells from HLA-A~*0201^+ donors with each of these peptides induced peptidespecific CD8^+ T cells. High numbers of IFN-γ-secreting T cells were also detectable after several weekly stimulations with W, Q and T. Besides lysis of HERV-W env-loaded target cells, specific apoptosis was also observed. These data demonstrate that human T cells can be sensitized toward HERV-W env peptides(W, Q and T) and, moreover, pose a high killing potential toward HERV-W env-expressing U251 cells. In conclusion, peptides W Q and T, which are HERV-W env antigenic epitopes, have both antigenicity and immunogenicity, and can cause strong T cell immune responses. Our data strengthen the view that HERV-W env should be considered as an autoantigen that can induce autoimmunity in neuropsychiatric diseases, such as multiple sclerosis and schizophrenia. These data might provide an experimental foundation for a HERV-W env peptide vaccine and new insight into the treatment of neuropsychiatric diseases.

Retrovirus-mediated delivery of an IL-4 receptor antagonist inhibits allergic responses in a murine model of asthma

This work reports the investigation of the effect of airway IL-4RA gene transfer by a recombinant retroviral vector on airway inflammation and airway responsiveness in asthmatic mice. The retrovirus-mediated delivery of IL-4RA to the airways of mice inhibited elevations of airway responsiveness and the development of allergic inflammation in asthmatic mice, and regulated the Th1/Th2 balance in OVA-sensitized and -challenged mouse models. This suggests that gene therapy is a therapeutic option for treating and controlling chronic airway inflammation and asthma symptoms.