RESUMO
The Semliki Forest virus capsid protein (C) is an RNA binding protein which exhibits both specific and unspecific affinities to single-strand nucleic acids. The putative use of the self-amplifying RNAs (saRNAs) of alphaviruses for biotechnological purpose is one of the main studied strategies concerning RNA-based therapies or immunization. In this work, a recombinant C protein from SFV was expressed and purified from bacteria and used to associate in vitro with a saRNA derived from SFV. Results showed that the purified form of C protein can associate with the saRNA even after high temperature treatment. The C protein was associated with a modified saRNA coding for the green fluorescent protein (GFP) and delivered to murine macrophage cells which expressed the GFP, showing that the saRNA was functional after being associated with the recombinant purified C protein.
Assuntos
Proteínas do Capsídeo , Macrófagos , RNA Viral , Proteínas Recombinantes , Vírus da Floresta de Semliki , Vírus da Floresta de Semliki/genética , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Camundongos , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas Recombinantes/genética , RNA Viral/genética , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismoRESUMO
Madariaga virus (MADV) has recently been associated with severe human disease in Panama, where the closely related Venezuelan equine encephalitis virus (VEEV) also circulates. In June 2017, a fatal MADV infection was confirmed in a community of Darien Province. We conducted a cross-sectional outbreak investigation with human and mosquito collections in July 2017, where sera were tested for alphavirus antibodies and viral RNA. In addition, by applying a catalytic, force-of-infection (FOI) statistical model to two serosurveys from Darien Province in 2012 and 2017, we investigated whether endemic or epidemic alphavirus transmission occurred historically. In 2017, MADV and VEEV IgM seroprevalences were 1.6% and 4.4%, respectively; IgG antibody prevalences were MADV: 13.2%, VEEV: 16.8%, Una virus (UNAV): 16.0%, and Mayaro virus: 1.1%. Active viral circulation was not detected. Evidence of MADV and UNAV infection was found near households, raising questions about its vectors and enzootic transmission cycles. Insomnia was associated with MADV and VEEV infections, depression symptoms were associated with MADV, and dizziness with VEEV and UNAV. Force-of-infection analyses suggest endemic alphavirus transmission historically, with recent increased human exposure to MADV and VEEV in Aruza and Mercadeo, respectively. The lack of additional neurological cases suggests that severe MADV and VEEV infections occur only rarely. Our results indicate that over the past five decades, alphavirus infections have occurred at low levels in eastern Panama, but that MADV and VEEV infections have recently increased-potentially during the past decade. Endemic infections and outbreaks of MADV and VEEV appear to differ spatially in some locations of eastern Panama.
Assuntos
Encefalomielite Equina do Leste/epidemiologia , Encefalomielite Equina Venezuelana/epidemiologia , Fazendeiros/estatística & dados numéricos , Adolescente , Adulto , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Alphavirus/imunologia , Infecções por Alphavirus/epidemiologia , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/fisiopatologia , Animais , Anticorpos Antivirais/imunologia , Febre de Chikungunya/epidemiologia , Febre de Chikungunya/imunologia , Febre de Chikungunya/fisiopatologia , Vírus Chikungunya/imunologia , Criança , Pré-Escolar , Estudos Transversais , Depressão/fisiopatologia , Tontura/fisiopatologia , Vírus da Encefalite Equina do Leste/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina do Leste/imunologia , Encefalomielite Equina do Leste/fisiopatologia , Encefalomielite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/fisiopatologia , Doenças Endêmicas , Epidemias , Fadiga/fisiopatologia , Feminino , Habitação/estatística & dados numéricos , Humanos , Imunoglobulina G , Imunoglobulina M , Masculino , Pessoa de Meia-Idade , Mosquitos Vetores/virologia , Panamá/epidemiologia , Vírus da Floresta de Semliki/imunologia , Estudos Soroepidemiológicos , Distúrbios do Início e da Manutenção do Sono/fisiopatologia , Adulto JovemRESUMO
The Semliki Forest virus (SFV) viral vector has been widely used for transient protein expression. This study aimed to analyze comprehensively the capacity of SFV vector to express rabies lyssavirus glycoprotein (RVGP) in mammalian cells. The assessed parameters were transfection strategy, multiplicity of infection (MOI), harvest time and mammalian cell host. Two transfection approaches, electroporation and lipofection were evaluated to obtain the recombinant SFV, and the electroporation was found to be the most effective. Viral quantification by RT-qPCR was performed to elucidate the relation between the amount of recombinant virus utilized in the infection process and the production levels of the heterologous protein. Four different multiplicities of infection (MOIs = 1; 10; 15; 50) were evaluated using five mammalian cell lines: BHK-21, HuH-7, Vero, L929, and HEK-293T. Protein expression was assessed at two harvest times after infection (24 and 48 h). The recombinant protein generated was characterized by western blot, dot blot, and indirect immunofluorescence (IIF), while its concentration was determined by enzyme-linked immunosorbent assay (ELISA). Similar expression patterns were observed in cell lines BHK-21, HEK-293T, L929, and Vero, with higher RVGP production in the first 24 h. The BHK-21 cells showed yields of up to 4.3 µg per 106 cells when lower MOIs (1 and 10) were used. The HEK-293 T cells also showed similar production (4.3 µg per 106 cells) with MOI of 1, while the L929 and Vero cell lines showed lower expression rates of 2.82 and 1.26 µg per 106 cells, respectively. These cell lines showed lower expression levels at 48 h after infection compared to 24 h. Controversially, in the case of the HuH-7 cell line, RVGP production was higher at 48 h after infection (4.0 µg per 106 cells) and using MOIs of 15 and 50. This work may contribute to optimize the RVGP production using SFV system in mammalian cells. This study can also substantiate for example, the development of approaches that use of SFV for applications for other protein expressions and suggests values for relevant parameters and cell lines of this biotechnique.
Assuntos
Glicoproteínas/genética , Glicoproteínas/metabolismo , Vírus da Raiva/metabolismo , Vírus da Floresta de Semliki/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Eletroporação , Regulação Viral da Expressão Gênica , Células HEK293 , Humanos , Engenharia de Proteínas , Vírus da Raiva/genética , Transfecção , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
BACKGROUND: Metastatic breast cancer is a major cause of death among women worldwide; therefore efficient therapeutic strategies are extremely needed. In this work we have developed a gene therapy- and bacteria-based combined neoadjuvant approach and evaluated its antitumor effect in a clinically relevant animal model of metastatic breast cancer. METHODS: 2×10(8) particles of a Semliki Forest virus vector expressing interleukin-12 (SFV-IL-12) and/or 2×10(7) units of an aroC (-) Samonella Typhimurium strain (LVR01) were injected into 4T1 tumor nodules orthotopically implanted in mice. Tumors were surgically resected and long-term survival was determined. IL-12 and interferon-γ were quantified by Enzyme-Linked ImmunoSorbent Assay, bacteria was visualized by inmunohistochemistry and the number of lung metastasis was calculated with a clonogenic assay. RESULTS: SFV-IL-12 and LVR01 timely inoculated and followed by surgical resection of tumors succeeded in complete inhibition of lethal lung metastasis and long-term survival in 90% of treated mice. The combined therapy was markedly synergistic compared to each treatment alone, since SFV-IL-12 monotherapy showed a potent antiangiogenic effect, being able to inhibit tumor growth and extend survival, but could not prevent establishment of distant metastasis and death of tumor-excised animals. On the other hand, LVR01 alone also showed a significant, although limited, antitumor potential, despite its ability to invade breast cancer cells and induce granulocyte recruitment. The efficacy of the combined therapy depended on the order in which both factors were administered; inasmuch the therapeutic effect was only observed when SFV-IL-12 was administered previous to LVR01, whereas administration of LVR01 before SFV-IL-12 had negligible antitumor activity. Moreover, pre-treatment with LVR01 seemed to suppress SFV-IL-12 antiangiogenic effects associated to lower IL-12 expression in this group. Re-challenged mice were unable to reject a second 4T1 tumor; however 100% of them could be totally cured by applying the same neoadjuvant combined regimen. To our knowledge, these are the most encouraging results obtained to date in a post-operatory setting using the highly aggressive 4T1 animal model. CONCLUSIONS: SFV-IL-12-based gene therapy combined with Salmonella LVR01 neoadjuvant administration has a synergic antitumor effect and may be a promising therapeutic option to prevent and/or eradicate pre-operatory metastasis in locally advanced breast cancer.
Assuntos
Neoplasias da Mama/terapia , Regulação da Expressão Gênica/fisiologia , Terapia Genética , Interleucina-12/genética , Neoplasias Pulmonares/terapia , Salmonella typhimurium/genética , Vírus da Floresta de Semliki/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Terapia Combinada , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos , Imunocompetência , Imuno-Histoquímica , Interferon gama/sangue , Interleucina-12/sangue , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Terapia Neoadjuvante , Transplante de Neoplasias , Fragmentos de Peptídeos/sangue , Taxa de SobrevidaRESUMO
Recombinant replicons of Semliki Forest virus (SFV) can be used to induce high-level, transient expression of heterologous proteins in vivo. We constructed infectious but replication-deficient SFV particles carrying recombinant RNA encoding the Brucella abortus translation initiation factor 3 (IF3). The recombinant SFV particles (SFV-IF3 particles) were then evaluated for their ability to induce immune responses and to protect BALB/c mice against a challenge with B. abortus 2308 following vaccination. Animals inoculated with SFV-IF3 developed IF3-specific IgM antibodies at day 14 post-immunization. In vitro stimulation of splenocytes from vaccinated mice with either recombinant IF3 (rIF3) or crude Brucella protein extracts resulted in a T-cell proliferative response and induction of interferon gamma secretion, but not interleukin-4. In addition, mice immunized with SFV-IF3 exhibited a significant level of resistance against challenge with the virulent B. abortus strain 2308 (P<0.01). These findings indicate that an SFV-based vector carrying RNA encoding Brucella IF3 has potential for use as a vaccine to induce protection against B. abortus infections.
Assuntos
Infecções por Alphavirus/imunologia , Fatores de Iniciação em Eucariotos/imunologia , Fator de Iniciação 3 em Procariotos/imunologia , Vírus da Floresta de Semliki/imunologia , Vacinação , Infecções por Alphavirus/prevenção & controle , Animais , Brucella abortus/genética , Fatores de Iniciação em Eucariotos/genética , Engenharia Genética , Imunidade Ativa/genética , Camundongos , Camundongos Endogâmicos BALB C , Fator de Iniciação 3 em Procariotos/genética , Recombinação Genética , Vírus da Floresta de Semliki/patogenicidade , VirulênciaRESUMO
Rabies is to this date one of the most important death causing zoonotic viral diseases, with 98% of deathsreported in developing countries, where access to modern vaccines and tools for efficient diagnosticremain unaffordable. In this paper, we describe a newly engineered RNA-based rabies virus glycoprotein(RVGP) expression vector based on the Semliki Forest Virus (SFV) system. A recombinant SFV carryingan RNA coding for RVGP (SFV-RVGP) was constructed and the RVGP expression was evaluated in animalcell cultures. The mRNA coding for RVGP and the RVGP itself were assessed by qPCR, Western-blotting,confocal microscopy, flow cytometry and ELISA. Moreover, SFV-RVGP was proven to be highly efficientin expressing the functionally trimeric RVGP. SFV-RVGP is easy to produce and efficient in different celllines, making it an interesting candidate for efficient and functional viral glycoprotein expression.
Assuntos
Masculino , Feminino , Humanos , Animais , Vacina Antirrábica , Vírus da Floresta de Semliki , Vírus da RaivaRESUMO
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.
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.
Assuntos
Alphavirus , Expressão Gênica , Vírus da Floresta de Semliki , Transdução GenéticaRESUMO
We constructed infectious but replication-deficient Semliki Forest virus (SFV) particles carrying recombinant RNA encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). The recombinant SFV particles (SFV-SOD particles) were then evaluated for their ability to induce a T-cell immune response and to protect BALB/c mice against a challenge with B. abortus 2308. Intraperitoneal injection of mice with recombinant SFV-SOD particles did not lead to the induction of SOD-specific antibodies, at least until week 6 after immunization (the end of the experiment). In vitro stimulation of splenocytes from the vaccinated mice with either recombinant Cu,Zn SOD (rSOD) or crude Brucella protein resulted in a T-cell proliferative response and the induction of gamma interferon secretion but not interleukin-4. In addition, the splenocytes exhibited significant levels of cytotoxic T-lymphocyte activity against Brucella-infected cells. The SFV-SOD particles, but not the control virus particles, induced a significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308. These findings indicated that an SFV-based vector carrying the SOD gene has potential for use as a vaccine to induce resistance against B. abortus infections.
Assuntos
Vacina contra Brucelose/imunologia , Brucella abortus/imunologia , Brucelose/prevenção & controle , RNA/imunologia , Vírus da Floresta de Semliki/genética , Superóxido Dismutase/imunologia , Vacinas Sintéticas/imunologia , Vírion/genética , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Superóxido Dismutase/genética , Linfócitos T Citotóxicos/imunologia , VacinaçãoRESUMO
The use of DNA vectors based on the SFV (Semliki Forest virus) replicon have not been reported in the modality of DNA prime virus boost. In the present study, SFV DNA vectors (DNA vectors based on the SFV replicon) bearing the HIV-1 TAB9 multiepitopic polypeptide minigene were evaluated as priming DNA immunogens followed by a recombinant fowlpox expressing the TAB9 mutiepitope (FPTAB9LZ) boost. The results indicated that mice primed with pSFV(k)tab9 and boosted with FPTAB9LZ significantly decreased the HIV-1 recombinant (VVTAB13, a recombinant vaccinia virus expressing the TAB13 multiepitope) vaccinia virus replication, compared with groups given pSFV(k)tab9 vector and FPTAB9LZ virus alone. Additionally, the viral titre in ovary correlated with the number of specific gamma-interferon-secreting T-cells in spleen. These results support the possible use of SFV DNA vectors in prime-boost approaches implemented in therapeutic/prophylactic treatments for infectious diseases such as HIV-1.
Assuntos
DNA Viral/administração & dosagem , Vírus da Varíola das Aves Domésticas/genética , Terapia Genética/métodos , Infecções por HIV/prevenção & controle , Imunização Secundária/métodos , Vírus da Floresta de Semliki/genética , Vacinas de DNA/administração & dosagem , Animais , Terapia Combinada , Feminino , Vírus da Varíola das Aves Domésticas/imunologia , Infecções por HIV/genética , Infecções por HIV/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vírus da Floresta de Semliki/imunologia , Resultado do Tratamento , Vacinação/métodos , Vacinas de DNA/imunologiaRESUMO
En éste trabajo se presentan los mecanismos y las moléculas que participan de la apoptosis en células de mamífero. Se discute la función de la mitocondria, se relacionan los distintos controles del sistema con patologías humanas y se presentan algunos virus neurotrópicos donde existe una importante conexión con la apoptosis. Asimismo, se indican algunos factores participantes del proceso que tienen una veta promisoria en el tratamiento de enfermedades donde la desregulación de la muerte celular programada es la causa de la patología en cuestión
Assuntos
Humanos , Apoptose , Caspases , Genes bcl-2 , Receptores do Fator de Necrose Tumoral , Alphavirus , Infecções por Alphavirus , Biotecnologia , Infecções por Bunyaviridae , Caspases , Flavivirus , Infecções por Flavivirus , Terapia Genética , Neoplasias , Doenças Neurodegenerativas , Neurônios , Oligonucleotídeos Antissenso/uso terapêutico , Orthobunyavirus , Reoviridae , Infecções por Reoviridae , Vírus da Floresta de SemlikiRESUMO
En éste trabajo se presentan los mecanismos y las moléculas que participan de la apoptosis en células de mamífero. Se discute la función de la mitocondria, se relacionan los distintos controles del sistema con patologías humanas y se presentan algunos virus neurotrópicos donde existe una importante conexión con la apoptosis. Asimismo, se indican algunos factores participantes del proceso que tienen una veta promisoria en el tratamiento de enfermedades donde la desregulación de la muerte celular programada es la causa de la patología en cuestión (AU)
Assuntos
Humanos , Apoptose/fisiologia , Caspases/antagonistas & inibidores , Genes bcl-2/fisiologia , Receptores do Fator de Necrose Tumoral/uso terapêutico , Infecções por Flavivirus , Flavivirus/patogenicidade , Vírus da Floresta de Semliki/patogenicidade , Alphavirus/patogenicidade , Infecções por Alphavirus , Infecções por Reoviridae , Reoviridae/patogenicidade , Orthobunyavirus , Infecções por Bunyaviridae , Neurônios/virologia , Caspases/fisiologia , Terapia Genética/métodos , Neoplasias/fisiopatologia , Neoplasias/tratamento farmacológico , Oligonucleotídeos Antissenso/uso terapêutico , Doenças Neurodegenerativas/fisiopatologia , Biotecnologia/métodosRESUMO
The antiviral effect of homoeopathic drugs against two animal viruses, Chicken Embryo Virus (CEV) of fowls and Simliki Forest Virus (SFV), causing encephalitis and death in mice were investigated. In all 10 drugs in 33 potencies were tested against CEV and 8 drugs in 26 potencies showed varying degree of virus inhibition. The drugs that caused 100//inhibition of CEV were Typhoidinum 200, Hydrophobinum 1000, Tuberculinum 1000, Nux vomica 200, and Malandrinum 1000. Of the drugs tested in 11 potencies against SFV, none were found effective in either preventing disease or death of mice infected with this virus
Assuntos
Animais , Camundongos , Antivirais , Vírus , Vírus da Floresta de Semliki , Índia , /farmacologia , Malandrinum/farmacologia , Tuberculinum koch/farmacologia , Lyssinum/farmacologia , Pesquisa Homeopática BásicaRESUMO
Complexes of formalinized Venezuelan equine encephalomyelitis (VEE) virus vaccine and specific IgG formed at antigen-antibody equivalence enhanced the immune responses of rhesus monkeys (Macaca mulatta). The predomonant class of antibody elicited by complexes was IgG. In contrast, lower titers of antibody and a more biphasic (IgG-IgM) response were observed after exposure of monkeys to the vaccine alone. In comparison to the response of monkeys primed with antigen, a more rapid secondary response was obtained in monkeys primed with the complexes of antigen and antibody formed at equivalence. A sustained level of protection of 88% was afforded mice 24 hr after immunization with antigen-antibody complexes; development of protection after administration of antigen required eight days to reach this level. Passive protection (80%-100%) was conferred by IgG controls for seven to eight days after immunization. This level of protection was not significantly affected by X-irradiation 24 hr prior to administration of IgG; however, protection in mice similarly irradiated prior to immunization with antigen-antibody complexes was significantly decreased. Early protection afforded by the complexes was not nonspecific (interferon) but was mediated by specific immunologic mechanisms and may be caused by an early formation of IgG.