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Enhancement of Antitumor Immunity Using a DNA-Based Replicon Vaccine Derived from Semliki Forest Virus  [PDF]
Liang Zhang, Yue Wang, Yi Xiao, Yu Wang, JinKai Dong, Kun Gao, Yan Gao, Xi Wang, Wei Zhang, YuanJi Xu, JinQi Yan, JiYun Yu
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0090551
Abstract: A DNA-based replicon vaccine derived from Semliki Forest virus, PSVK-shFcG-GM/B7.1 (Fig. 1a) was designed for tumor immunotherapy as previously constructed. The expression of the fusion tumor antigen (survivin and hCGβ-CTP37) and adjuvant molecular protein (Granulocyte-Macrophage Colony-Stimulating Factor/ GM-CSF/B7.1) genes was confirmed by Immunofluorescence assay in vitro, and immunohistochemistry assay in vivo. In this paper, the immunological effect of this vaccine was determined using immunological assays as well as animal models. The results showed that this DNA vaccine induced both humoral and cellular immune responses in C57BL/6 mice after immunization, as evaluated by the ratio of CD4+/CD8+ cells and the release of IFN-γ. Furthermore, the vaccination of C57BL/6 mice with PSVK-shFcG-GM/B7.1 significantly delayed the in vivo growth of tumors in animal models (survivin+ and hCGβ+ murine melanoma, B16) when compared to vaccination with the empty vector or the other control constructs (Fig. 1b). These data indicate that this type of replicative DNA vaccine could be developed as a promising approach for tumor immunotherapy. Meanwhile, these results provide a basis for further study in vaccine pharmacodynamics and pharmacology, and lay a solid foundation for clinical application.
Transfection of Infectious RNA and DNA/RNA Layered Vectors of Semliki Forest Virus by the Cell-Penetrating Peptide Based Reagent PepFect6  [PDF]
Kalle P?rn, Liane Viru, Taavi Lehto, Nikita Oskolkov, ülo Langel, Andres Merits
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0069659
Abstract: Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.
A prime-boost vaccination strategy using a Semliki Forest virus replicon vectored DNA vaccine followed by a recombinant adenovirus protects pigs from classical swine fever

Yuan Sun,Dafei Liu,Yufei Wang,Na Li,Hongyu Li,Bingbing Liang,Huaji Qiu,

生物工程学报 , 2009,
Abstract: We have previously evaluated a Semliki Forest virus(SFV) replicon vectored DNA vaccine(pSFV1CS2-E2) and a recombinant adenovirus(rAdV-E2) expressing the E2 glycoprotein of classical swine fever virus(CSFV) in pigs.The results showed that the immunized pigs were protected from virulent challenge, but few pigs showed short-term fever and occasional pathological changes following virulent challenge.To enhance the immunogenecity of the vaccines, we tried a prime-boost vaccination strategy using a combination of...
Phenoloxidase Activity Acts as a Mosquito Innate Immune Response against Infection with Semliki Forest Virus  [PDF]
Julio Rodriguez-Andres,Seema Rani,Margus Varjak,Margo E. Chase-Topping,Markus H. Beck,Mhairi C. Ferguson,Esther Schnettler,Rennos Fragkoudis,Gerald Barry,Andres Merits,John K. Fazakerley,Michael R. Strand ,Alain Kohl
PLOS Pathogens , 2012, DOI: 10.1371/journal.ppat.1002977
Abstract: Several components of the mosquito immune system including the RNA interference (RNAi), JAK/STAT, Toll and IMD pathways have previously been implicated in controlling arbovirus infections. In contrast, the role of the phenoloxidase (PO) cascade in mosquito antiviral immunity is unknown. Here we show that conditioned medium from the Aedes albopictus-derived U4.4 cell line contains a functional PO cascade, which is activated by the bacterium Escherichia coli and the arbovirus Semliki Forest virus (SFV) (Togaviridae; Alphavirus). Production of recombinant SFV expressing the PO cascade inhibitor Egf1.0 blocked PO activity in U4.4 cell- conditioned medium, which resulted in enhanced spread of SFV. Infection of adult female Aedes aegypti by feeding mosquitoes a bloodmeal containing Egf1.0-expressing SFV increased virus replication and mosquito mortality. Collectively, these results suggest the PO cascade of mosquitoes plays an important role in immune defence against arboviruses.
Control of the Rescue and Replication of Semliki Forest Virus Recombinants by the Insertion of miRNA Target Sequences  [PDF]
Kaspar Ratnik, Liane Viru, Andres Merits
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0075802
Abstract: Due to their broad cell- and tissue-tropism, alphavirus-based replication-competent vectors are of particular interest for anti-cancer therapy. These properties may, however, be potentially hazardous unless the virus infection is controlled. While the RNA genome of alphaviruses precludes the standard control techniques, host miRNAs can be used to down-regulate viral replication. In this study, target sites from ubiquitous miRNAs and those of miRNAs under-represented in cervical cancer cells were inserted into replication-competent DNA/RNA layered vectors of Semliki Forest virus. It was found that in order to achieve the most efficient suppression of recombinant virus rescue, the introduced target sequences must be fully complementary to those of the corresponding miRNAs. Target sites of ubiquitous miRNAs, introduced into the 3′ untranslated region of the viral vector, profoundly reduced the rescue of recombinant viruses. Insertion of the same miRNA targets into coding region of the viral vector was approximately 300-fold less effective. Viruses carrying these miRNAs were genetically unstable and rapidly lost the target sequences. This process was delayed, but not completely prevented, by miRNA inhibitors. Target sites of miRNA under-represented in cervical cancer cells had much smaller but still significant effects on recombinant virus rescue in cervical cancer-derived HeLa cells. Over-expression of miR-214, one of these miRNAs, reduced replication of the targeted virus. Though the majority of rescued viruses maintained the introduced miRNA target sequences, genomes with deletions of these sequences were also detected. Thus, the low-level repression of rescue and replication of targeted virus in HeLa cells was still sufficient to cause genetic instability.
Semliki Forest Virus: un vector viral con múltiples aplicaciones
Henao,Luis Felipe; Cortés,Fabián; Navas,María Cristina;
Colombia Médica , 2007,
Abstract: recently, alphavirus have been used as expression vectors, among these, semliki forest virus (sfv), an enveloped virus, besides replicating itself in the cytoplasm, has the property to express structural proteins separately from nonstructural proteins, allowing a greater expression control. vectors derived from sfv can have a broad range of applications. high viral titers can be obtained to efficiently express proteins in different cell lines. they can infect a wide spectrum of mammalian cells, as well as tissues. they are promising to be used on gene therapy as vehicles for specific gene delivery in vivo or in vitro, as much as in therapy against cancer as neuronal therapy, especially when a short term expression is necessary. another studied aspect is sfv vectors applications in prophylactic or therapeutic vaccine production; the generation of important immune responses against different viral and tumor diseases is still been discussed. development of new non-cytopathic vectors, temperature-regulated vectors, as well as others with persistent replication, will allow prolongation of expression. due to these new advantages and to others already known, uses for vectors derived from sfv could be extended gradually, as long as undesired effects are controlled.
Semliki Forest Virus: un vector viral con múltiples aplicaciones
Luis Felipe Henao,Fabián Cortés,María Cristina Navas
Colombia Médica , 2007,
Abstract: Se han utilizado los alfavirus como vectores de expresión, entre estos se encuentra el Semliki Forest virus (SFV), que es un virus envuelto, el cual, además de replicarse en el citoplasma, tiene la propiedad de expresar por separado las proteínas estructurales de las no estructurales, permitiendo un mayor control de la expresión. Los vectores derivados del SFV pueden tener una gama amplia de aplicaciones. Se pueden obtener altos títulos virales para la expresión eficiente de proteínas en diferentes líneas celulares. Pueden infectar un espectro amplio de células de mamíferos, así como de tejidos. Son prometedores para ser usados en la terapia génica como vehículos para el envío de genes específicos in vivo o in vitro, tanto en la terapia contra el cáncer como en la neuronal, especialmente cuando sólo sea necesaria una expresión a corto plazo. Sus aplicaciones en la producción de vacunas profilácticas o terapéuticas, es otro aspecto estudiado; se ha demostrado la generación de respuestas inmunes importantes contra diferentes enfermedades virales y tumorales. El desarrollo de nuevos vectores no citopáticos, de otros regulados por temperatura, así como también de otros con replicación persistente; permitirán la prolongación de la expresión. Debido a estas nuevas ventajas y a las ya conocidas, gradualmente se podrían ampliar los usos para los vectores derivados del SFV a medida que se controlen sus efectos no deseados.
Expression of the Hc Fragment of Clostridium botulinum Neurotoxin Serotype A in Mammalian Cells with Recombinant Semliki Forest Virus Expression System

Yunzhou Yu,Zhiwei Sun,Shuang Wang,Weiyuan Yu,

生物工程学报 , 2008,
Abstract: Expression of the Hc Fragment of Clostridium botulinum Neurotoxin Serotype A in Mammalian Cells with Recombinant Semliki Forest Virus Expression System
Virus-Receptor Mediated Transduction of Dendritic Cells by Lentiviruses Enveloped with Glycoproteins Derived from Semliki Forest Virus  [PDF]
Steven Froelich, April Tai, Katie Kennedy, Adnan Zubair, Pin Wang
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0021491
Abstract: Lentiviruses have recently attracted considerable interest for their potential as a genetic modification tool for dendritic cells (DCs). In this study, we explore the ability of lentiviruses enveloped with alphaviral envelope glycoproteins derived from Semliki Forest virus (SFV) to mediate transduction of DCs. We found that SFV glycoprotein (SFV-G)-pseudotyped lentiviruses use C-type lectins (DC-SIGN and L-SIGN) as attachment factors for transduction of DCs. Importantly, SFV-G pseudotypes appear to have enhanced transduction towards C-type lectin-expressing cells when produced under conditions limiting glycosylation to simple high-mannose, N-linked glycans. These results, in addition to the natural DC tropism of SFV-G, offer evidence to support the use of SFV-G-bearing lentiviruses to genetically modify DCs for the study of DC biology and DC-based immunotherapy.
Rab7 Associates with Early Endosomes to Mediate Sorting and Transport of Semliki Forest Virus to Late Endosomes  [PDF]
Andreas Vonderheit,Ari Helenius
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.0030233
Abstract: Semliki forest virus (SFV) is internalized by clathrin-mediated endocytosis, and transported via early endosomes to late endosomes and lysosomes. The intracellular pathway taken by individual fluorescently labeled SFV particles was followed using immunofluorescence in untransfected cells, and by video-enhanced, triple-color fluorescence microscopy in live cells transfected with GFP- and RFP-tagged Rab5, Rab7, Rab4, and Arf1. The viruses progressed from Rab5-positive early endosomes to a population of early endosomes (about 10% of total) that contained both Rab5 and Rab7. SFV were sequestered in the Rab7 domains, and they were sorted away from the early endosomes when these domains detached as separate transport carriers devoid of Rab5, Rab4, EEA1, Arf1, and transferrin. The process was independent of Arf1 and the acidic pH in early endosomes. Nocodazole treatment showed that the release of transport carriers was assisted by microtubules. Expression of constitutively inactive Rab7T22N resulted in accumulation of SFV in early endosomes. We concluded that Rab7 is recruited to early endosomes, where it forms distinct domains that mediate cargo sorting as well as the formation of late-endosome-targeted transport vesicles.
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