RESUMEN
The outbreaks of Zika virus (ZIKV) infection in Brazil, 2015-2016, were associated with severe congenital malformations. Our translational study aimed to test the efficacy of the antiviral agent sofosbuvir (SOF) against vertical transmission of ZIKV and the associated congenital syndrome (CZS), using a rhesus monkey model. Eight pregnant macaques were successfully infected during the organogenesis phase with a Brazilian ZIKV strain; five of them received SOF from two to fifteen days post-infection. Both groups of dams showed ZIKV-associated clinical signals, detectable ZIKV RNA in several specimens, specific anti-ZIKV IgM and IgG antibodies, and maternal neutralizing antibodies. However, malformations occurred only among non-treated dam offspring. Compared to non-treated animals, all SOF-treated dams had a shorter ZIKV viremia and four of five neonates had undetectable ZIKV RNA in blood and tissue samples. These results support further clinical evaluations aiming for the prevention of CZS.
Asunto(s)
Antivirales/uso terapéutico , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Sofosbuvir/uso terapéutico , Infección por el Virus Zika/prevención & control , Infección por el Virus Zika/transmisión , Virus Zika/efectos de los fármacos , Animales , Anticuerpos Antivirales/sangre , Antivirales/administración & dosificación , Brasil , Femenino , Macaca mulatta , Embarazo , Complicaciones Infecciosas del Embarazo/prevención & control , Complicaciones Infecciosas del Embarazo/virología , Sofosbuvir/administración & dosificación , Investigación Biomédica Traslacional , Viremia/tratamiento farmacológico , Viremia/prevención & control , Virus Zika/inmunología , Infección por el Virus Zika/congénito , Infección por el Virus Zika/tratamiento farmacológicoRESUMEN
BACKGROUND: HIV-infected individuals have deficient responses to Yellow Fever vaccine (YFV) and may be at higher risk for adverse events (AE). Chronic immune activation-characterized by low CD4/CD8 ratio or high indoleamine 2,3-dioxygenase-1 (IDO) activity-may influence vaccine response in this population. METHODS: We prospectively assessed AE, viremia by the YFV virus and YF-specific neutralizing antibodies (NAb) in HIV-infected (CD4>350) and -uninfected adults through 1 year after vaccination. The effect of HIV status on initial antibody response to YFV was measured during the first 3 months following vaccination, while the effect on persistence of antibody response was measured one year following vaccination. We explored CD4/CD8 ratio, IDO activity (plasma kynurenine/tryptophan [KT] ratio) and viremia by Human Pegivirus as potential predictors of NAb response to YFV among HIV-infected participants with linear mixed models. RESULTS: 12 HIV-infected and 45-uninfected participants were included in the final analysis. HIV was not significantly associated with AE, YFV viremia or NAb titers through the first 3 months following vaccination. However, HIV-infected participants had 0.32 times the NAb titers observed for HIV-uninfected participants at 1 year following YFV (95% CI 0.13 to 0.83, p = 0.021), independent of sex, age and prior vaccination. In HIV-infected participants, each 10% increase in CD4/CD8 ratio predicted a mean 21% higher post-baseline YFV Nab titer (p = 0.024). Similarly, each 10% increase in KT ratio predicted a mean 21% lower post-baseline YFV Nab titer (p = 0.009). Viremia by Human Pegivirus was not significantly associated with NAb titers. CONCLUSIONS: HIV infection appears to decrease the durability of NAb responses to YFV, an effect that may be predicted by lower CD4/CD8 ratio or higher KT ratio.
Asunto(s)
Relación CD4-CD8 , Infecciones por VIH/inmunología , Inmunogenicidad Vacunal , Quinurenina/sangre , Triptófano/sangre , Vacuna contra la Fiebre Amarilla/inmunología , Fiebre Amarilla/prevención & control , Adulto , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Femenino , Infecciones por VIH/virología , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Viremia , Fiebre Amarilla/inmunología , Fiebre Amarilla/virología , Vacuna contra la Fiebre Amarilla/efectos adversos , Virus de la Fiebre Amarilla/inmunologíaRESUMEN
BACKGROUND: Yellow fever vaccine (YFV) induces weaker immune responses in HIV-infected individuals. However, little is known about YFV responses among antiretroviral-treated patients and potential immunological predictors of YFV response in this population. METHODS: We enrolled 34 antiretroviral therapy (ART)-treated HIV-infected and 58 HIV-uninfected adults who received a single YFV dose to evaluate antibody levels and predictors of immunity, focusing on CD4(+) T-cell count, CD4(+)/CD8(+) ratio, and Human Pegivirus (GBV-C) viremia. Participants with other immunosuppressive conditions were excluded. RESULTS: Median time since YFV was nonsignificantly shorter in HIV-infected participants than in HIV-uninfected participants (42 and 69 months, respectively, P = 0.16). Mean neutralizing antibody (NAb) titers was lower in HIV-infected participants than HIV-uninfected participants (3.3 vs. 3.6 log10mIU/mL, P = 0.044), a difference that remained significant after adjustment for age, sex, and time since vaccination (P = 0.024). In HIV-infected participants, lower NAb titers were associated with longer time since YFV (rho: -0.38, P = 0.027) and lower CD4(+)/CD8(+) ratio (rho: 0.42, P = 0.014), but not CD4(+) T-cell count (P = 0.52). None of these factors were associated with NAb titers in HIV-uninfected participant. GBV-C viremia was not associated with difference in NAb titers overall or among HIV-infected participants. CONCLUSIONS: ART-treated HIV-infected individuals seem to have impaired and/or less durable responses to YFV than HIV-uninfected individuals, which were associated with lower CD4(+)/CD8(+) ratio, but not with CD4(+) T-cell count. These results supports the notion that low CD4(+)/CD8(+) ratio, a marker linked to persistent immune activation, is a better indicator of functional immune disturbance than CD4(+) T-cell count in patients with successful ART.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Anticuerpos Antivirales/sangre , Relación CD4-CD8 , Infecciones por VIH/inmunología , Vacuna contra la Fiebre Amarilla/inmunología , Adulto , Anticuerpos Neutralizantes/sangre , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Estudios de Casos y Controles , Femenino , Infecciones por VIH/tratamiento farmacológico , Humanos , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Carga ViralRESUMEN
Yellow fever is an acute infectious disease caused by prototype virus of the genus Flavivirus. It is endemic in Africa and South America where it represents a serious public health problem causing epidemics of hemorrhagic fever with mortality rates ranging from 20% to 50%. There is no available antiviral therapy and vaccination is the primary method of disease control. Although the attenuated vaccines for yellow fever show safety and efficacy it became necessary to develop a new yellow fever vaccine due to the occurrence of rare serious adverse events, which include visceral and neurotropic diseases. The new inactivated vaccine should be safer and effective as the existing attenuated one. In the present study, the immunogenicity of an inactivated 17DD vaccine in C57BL/6 mice was evaluated. The yellow fever virus was produced by cultivation of Vero cells in bioreactors, inactivated with ß-propiolactone, and adsorbed to aluminum hydroxide (alum). Mice were inoculated with inactivated 17DD vaccine containing alum adjuvant and followed by intracerebral challenge with 17DD virus. The results showed that animals receiving 3 doses of the inactivated vaccine (2 µg/dose) with alum adjuvant had neutralizing antibody titers above the cut-off of PRNT50 (Plaque Reduction Neutralization Test). In addition, animals immunized with inactivated vaccine showed survival rate of 100% after the challenge as well as animals immunized with commercial attenuated 17DD vaccine.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vacuna contra la Fiebre Amarilla/inmunología , Fiebre Amarilla/prevención & control , Virus de la Fiebre Amarilla/crecimiento & desarrollo , Animales , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Reactores Biológicos/virología , Chlorocebus aethiops , Desinfectantes/farmacología , Inmunidad Humoral , Esquemas de Inmunización , Ratones Endogámicos C57BL , Pruebas de Neutralización , Propiolactona/farmacología , Análisis de Supervivencia , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunología , Células Vero , Cultivo de Virus , Vacuna contra la Fiebre Amarilla/administración & dosificación , Virus de la Fiebre Amarilla/inmunología , Virus de la Fiebre Amarilla/aislamiento & purificación , Virus de la Fiebre Amarilla/patogenicidadRESUMEN
Yellow fever (YF) is an endemic disease in some tropical areas of South America and Africa that presents lethality rate between 20 and 50%. There is no specific treatment and to control this disease a highly effective live-attenuated egg based vaccine is widely used for travelers and residents of areas where YF is endemic. However, recent reports of rare, sometimes fatal, adverse events post-vaccination have raised concerns. In order to increase safety records, alternative strategies should be considered, such as developing a new inactivated vaccine using a cell culture based technology, capable of meeting the demands in cases of epidemic. With this goal, the production of YF virus in Vero cells grown on microcarriers and its subsequent purification by chromatographic techniques was studied. In this work we investigate the capture step of the purification process of the YF virus. At first, virus stability was studied over a wide pH range, showing best results for the alkaline region. Considering this result and the pI of the envelope protein previously determined in silico, a strong anion exchanger was considered most suitable. Due to the easy scalability, simplicity to handle, absence of diffusional limitations and suitability for virus handling of membrane adsorbers, a Q membrane was evaluated. The amount of antigen adsorbed onto the membrane was investigated within the pH range for virus stability, and the best pH for virus adsorption was considered to be 8.5. Finally, studies on gradient and step elution allowed to determine the most adequate salt concentration for washing (0.15M) and virus elution (0.30 M). Under these operating conditions, it was shown that this capture step is quite efficient, showing high product recovery (93.2±30.3%) and efficient DNA clearance (0.9±0.3 ng/dose).
Asunto(s)
Cultivo de Virus/métodos , Virus de la Fiebre Amarilla/aislamiento & purificación , Adsorción , Animales , Chlorocebus aethiops , Cromatografía por Intercambio Iónico , Concentración de Iones de Hidrógeno , Membranas/química , Células VeroRESUMEN
The dengue envelope glycoprotein (E) is the major component of virion surface and its ectodomain is composed of domains I, II and III. This protein is the main target for the development of a dengue vaccine with induction of neutralizing antibodies. In the present work, we tested two different vaccination strategies, with combined immunizations in a prime/booster regimen or simultaneous inoculation with a DNA vaccine (pE1D2) and a chimeric yellow fever/dengue 2 virus (YF17D-D2). The pE1D2 DNA vaccine encodes the ectodomain of the envelope DENV2 protein fused to t-PA signal peptide, while the YF17D-D2 was constructed by replacing the prM and E genes from the 17D yellow fever vaccine virus by those from DENV2. Balb/c mice were inoculated with these two vaccines by different prime/booster or simultaneous immunization protocols and most of them induced a synergistic effect on the elicited immune response, mainly in neutralizing antibody production. Furthermore, combined immunization remarkably increased protection against a lethal dose of DENV2, when compared to each vaccine administered alone. Results also revealed that immunization with the DNA vaccine, regardless of the combination with the chimeric virus, induced a robust cell immune response, with production of IFN-γ by CD8+ T lymphocytes.
Asunto(s)
Vacunas contra el Dengue/uso terapéutico , Dengue/prevención & control , Vacunas de ADN/uso terapéutico , Proteínas del Envoltorio Viral/inmunología , Virus de la Fiebre Amarilla/genética , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Virus del Dengue/genética , Interferón gamma/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Estructura Terciaria de Proteína , Vacunación/métodosRESUMEN
OBJECTIVE: To verify if the Bio-Manguinhos 17DD yellow fever vaccine (17DD-YFV) used in lower doses is as immunogenic and safe as the current formulation. RESULTS: Doses from 27,476 IU to 587 IU induced similar seroconversion rates and neutralizing antibodies geometric mean titers (GMTs). Immunity of those who seroconverted to YF was maintained for 10 mo. Reactogenicity was low for all groups. METHODS: Young and healthy adult males (n = 900) were recruited and randomized into 6 groups, to receive de-escalating doses of 17DD-YFV, from 27,476 IU to 31 IU. Blood samples were collected before vaccination (for neutralization tests to yellow fever, serology for dengue and clinical chemistry), 3 to 7 d after vaccination (for viremia and clinical chemistry) and 30 d after vaccination (for new yellow fever serology and clinical chemistry). Adverse events diaries were filled out by volunteers during 10 d after vaccination. Volunteers were retested for yellow fever and dengue antibodies 10 mo later. Seropositivity for dengue was found in 87.6% of volunteers before vaccination, but this had no significant influence on conclusions. CONCLUSION: In young healthy adults Bio-Manguinhos/Fiocruz yellow fever vaccine can be used in much lower doses than usual. INTERNATIONAL REGISTER: ISRCTN 38082350.
Asunto(s)
Relación Dosis-Respuesta Inmunológica , Vacunación/métodos , Vacuna contra la Fiebre Amarilla/administración & dosificación , Vacuna contra la Fiebre Amarilla/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Método Doble Ciego , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Voluntarios Sanos , Humanos , Masculino , Factores de Tiempo , Vacunación/efectos adversos , Vacuna contra la Fiebre Amarilla/efectos adversos , Adulto JovenRESUMEN
The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection.
Asunto(s)
Virus del Dengue/inmunología , Virus del Dengue/metabolismo , Vacunas de ADN/inmunología , Proteínas no Estructurales Virales/inmunología , Animales , Western Blotting , Línea Celular , Cricetinae , Dengue/inmunología , Dengue/prevención & control , Virus del Dengue/genética , Virus del Dengue/patogenicidad , Técnica del Anticuerpo Fluorescente , Masculino , Ratones , Ratones Endogámicos BALB C , Plásmidos/genética , Vacunas de ADN/metabolismo , Vacunas de ADN/uso terapéutico , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Two DNA vaccines were constructed encoding the ectodomain (domains I, II and III) of the DENV2 envelope protein (pE1D2) or only its domain III (pE2D2), fused to the human tissue plasminogen activator signal peptide (t-PA). The expression and secretion of recombinant proteins was confirmed in vitro in BHK cells transfected with the two plasmids, detected by immunofluorescence or immunoprecipitation of metabolically labeled gene products, using polyclonal and monoclonal antibodies against DENV2. Besides, results reveal that the ectodomain of the E protein can be efficiently expressed in vivo, in a mammalian system, without the prM protein that is hypothesized to act as a chaperonin during dengue infection. Balb/c mice were immunized with the DNA vaccines and challenged with a lethal dose of DENV2. All pE1D2-vaccinated mice survived challenge, while 45% of animals immunized with the pE2D2 died after infection. Furthermore, only 10% of pE1D2-immunized mice presented some clinical signs of infection after challenge, whereas most of animals inoculated with the pE2D2 showed effects of the disease with high morbidity degrees. Levels of neutralizing antibodies were significantly higher in pE1D2-vaccinated mice than in pE2D2-immunized animals, also suggesting that the pE1D2 vaccine was more protective than the pE2D2.
Asunto(s)
Virus del Dengue/inmunología , Vacunas de ADN/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Animales , Línea Celular , Cricetinae , Dengue/inmunología , Virus del Dengue/patogenicidad , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Ratones , Ratones Endogámicos BALB C , Plásmidos , Proteínas del Envoltorio Viral/genéticaRESUMEN
Viruses are a structurally diverse group of infectious agents that differ widely in their sensitivities to high hydrostatic pressure (HHP). Studies on picornaviruses have demonstrated that these viruses are extremely resistant to HHP treatments, with poliovirus appearing to be the most resistant. Here, the three attenuated poliovirus serotypes were compared with regard to pressure and thermal resistance. We found that HHP does not inactivate any of the three serotypes studied (1-3). Rather, HHP treatment was found to stabilize poliovirus by increasing viral thermal resistance at 37 degrees C. Identification of new methods that stabilize poliovirus against heat inactivation would aid in the design of a more heat-stable vaccine, circumventing the problems associated with refrigeration during storage and transport of the vaccine prior to use.
Asunto(s)
Calor , Presión Hidrostática , Poliovirus/fisiología , Animales , Chlorocebus aethiops , Humanos , Vacunas contra Poliovirus/farmacología , Preservación Biológica , Células Vero , Replicación ViralRESUMEN
In this work, the propagation of the 17DD yellow fever virus in Vero cells grown on Cytodex-1 microcarriers was evaluated. After verifying that upon infection the virus adsorption step could be performed under continuous agitation, experiments were carried out in spinners and sparged lab-scale stirred-tank bioreactor to evaluate the use of a commercial serum-free medium (VP-SFM) and to investigate the effects of multiplicity of infection (MOI) and time of infection (TOI) on virus production. Virus titers as high as 8.4 x 10(8)pfu/mL were obtained upon infection with MOI of 0.02 and TOI of 3 days, using the serum-free medium in the sparged bioreactor.
Asunto(s)
Técnicas de Cultivo de Célula , Cultivo de Virus/métodos , Virus de la Fiebre Amarilla/crecimiento & desarrollo , Animales , Reactores Biológicos , Chlorocebus aethiops , Medio de Cultivo Libre de Suero , Células VeroRESUMEN
A comparison of dengue virus (DENV) antibody levels in paired serum samples collected from predominantly DENV-naive residents in an agricultural settlement in Brazilian Amazonia (baseline seroprevalence, 18.3%) showed a seroconversion rate of 3.67 episodes/100 person-years at risk during 12 months of follow-up. Multivariate analysis identified male sex, poverty, and migration from extra-Amazonian states as significant predictors of baseline DENV seropositivity, whereas male sex, a history of clinical diagnosis of dengue fever, and travel to an urban area predicted subsequent seroconversion. The laboratory surveillance of acute febrile illnesses implemented at the study site and in a nearby town between 2004 and 2006 confirmed 11 DENV infections among 102 episodes studied with DENV IgM detection, reverse transcriptase-polymerase chain reaction, and virus isolation; DENV-3 was isolated. Because DENV exposure is associated with migration or travel, personal protection measures when visiting high-risk urban areas may reduce the incidence of DENV infection in this rural population.
Asunto(s)
Dengue/etiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antivirales/sangre , Brasil , Niño , Preescolar , Estudios Transversales , Dengue/diagnóstico , Dengue/prevención & control , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , Análisis Multivariante , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , Estudios Seroepidemiológicos , Caracteres SexualesRESUMEN
For the development of safe live attenuated flavivirus vaccines one of the main properties to be established is viral replication. We have used real-time reverse transcriptase-polymerase chain reaction and virus titration by plaque assay to determine the replication of yellow fever 17DD virus (YFV 17DD) and recombinant yellow fever 17D viruses expressing envelope proteins of dengue virus serotypes 2 and 4 (17D-DENV-2 and 17D-DENV-4). Serum samples from rhesus monkeys inoculated with YFV 17DD and 17D-DENV chimeras by intracerebral or subcutaneous route were used to determine and compare the viremia induced by these viruses. Viral load quantification in samples from monkeys inoculated by either route with YFV 17DD virus suggested a restricted capability of the virus to replicate reaching not more than 2.0 log10 PFU mL-1 or 3.29 log10 copies mL-1. Recombinant 17D-dengue viruses were shown by plaquing and real-time PCR to be as attenuated as YF 17DD virus with the highest mean peak titer of 1.97 log10 PFU mL-1 or 3.53 log10 copies mL-1. These data serve as a comparative basis for the characterization of other 17D-based live attenuated candidate vaccines against other diseases.
Uma das principais propriedades a serem estabelecidas para o desenvolvimento de vacinas seguras e atenuadas de flavivirus,é a taxa de replicação viral. Neste trabalho, aplicamos a metodologia de amplificação pela reação em cadeia da polimerase em tempo real e titulação viral por plaqueamento para determinação da replicação do vírus 17DD (FA 17DD) e recombinantes, expressando proteínas do envelope de dengue sorotipos 2 e 4 (17D-DENV-2 e 17D-DENV-4). As amostras de soros de macacos inoculados por via intracerebral ou subcutânea com FA 17DD ou 17D-DENV foram usadas para determinar e comparar a viremia induzida por estes vírus. A quantificação da carga viral em amostras de macacos inoculados por ambas as vias com FA 17DD sugere restrita capacidade de replicação com taxa não superior a 2,0 log10 PFU mL-1 ou 3,29 log10 cópias/mL-1. Os vírus recombinantes 17D-DENV mostraram-se tão atenuados quanto o vírus 17DD, tanto porRT-PCR em tempo real quanto por plaqueamento, com título médio máximo de 1,97 log10 PFU mL-1 ou 3,53 log10 cópias/mL-1. Estes dados servem como base comparativapara caracterização de outros vírus vivos atenuados, derivados do vírus 17D, candidatos a vacinas contra outras doenças.
Asunto(s)
Animales , Anticuerpos Antivirales , Virus del Dengue/fisiología , ARN Viral/inmunología , Replicación Viral , Viremia/inmunología , Virus de la Fiebre Amarilla/fisiología , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Macaca mulatta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ARN Viral/sangre , Recombinación Genética/inmunología , Carga Viral , Vacunas Atenuadas/inmunología , Vacuna contra la Fiebre Amarilla/inmunología , Virus de la Fiebre Amarilla/inmunologíaRESUMEN
For the development of safe live attenuated flavivirus vaccines one of the main properties to be established is viral replication. We have used real-time reverse transcriptase-polymerase chain reaction and virus titration by plaque assay to determine the replication of yellow fever 17DD virus (YFV 17DD) and recombinant yellow fever 17D viruses expressing envelope proteins of dengue virus serotypes 2 and 4 (17D-DENV-2 and 17D-DENV-4). Serum samples from rhesus monkeys inoculated with YFV 17DD and 17D-DENV chimeras by intracerebral or subcutaneous route were used to determine and compare the viremia induced by these viruses. Viral load quantification in samples from monkeys inoculated by either route with YFV 17DD virus suggested a restricted capability of the virus to replicate reaching not more than 2.0 log10 PFU mL(-1) or 3.29 log10 copies mL(-1). Recombinant 17D-dengue viruses were shown by plaquing and real-time PCR to be as attenuated as YF 17DD virus with the highest mean peak titer of 1.97 log10 PFU mL(-1) or 3.53 log10 copies mL(-1). These data serve as a comparative basis for the characterization of other 17D-based live attenuated candidate vaccines against other diseases.
Asunto(s)
Anticuerpos Antivirales , Virus del Dengue/fisiología , ARN Viral/inmunología , Viremia/inmunología , Replicación Viral , Virus de la Fiebre Amarilla/fisiología , Animales , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Macaca mulatta , ARN Viral/sangre , Recombinación Genética/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vacunas Atenuadas/inmunología , Carga Viral , Vacuna contra la Fiebre Amarilla/inmunología , Virus de la Fiebre Amarilla/inmunologíaRESUMEN
The successful Yellow Fever (YF) vaccine consists of the live attenuated 17D-204 or 17DD viruses. Despite its excellent record of efficacy and safety, serious adverse events have been recorded and influenced extensive vaccination in endemic areas. Therefore, alternative strategies should be considered, which may include inactivated whole virus. High hydrostatic pressure has been described as a method for viral inactivation and vaccine development. The present study evaluated whether high hydrostatic pressure would inactivate the YF 17DD virus. YF 17DD virus was grown in Vero cells in roller bottle cultures and subjected to 310MPa for 3h at 4 degrees C. This treatment abolished YF infectivity and eliminated the ability of the virus to cause disease in mice. Pressure-inactivated virus elicited low level of neutralizing antibody titers although exhibited complete protection against an otherwise lethal challenge with 17DD virus in the murine model. The data warrant further development of pressure-inactivated vaccine against YF.
Asunto(s)
Inactivación de Virus , Vacuna contra la Fiebre Amarilla/efectos adversos , Vacuna contra la Fiebre Amarilla/inmunología , Virus de la Fiebre Amarilla/fisiología , Animales , Anticuerpos Antivirales/sangre , Chlorocebus aethiops , Presión Hidrostática , Ratones , Viabilidad Microbiana , Pruebas de Neutralización , Análisis de Supervivencia , Células Vero , Ensayo de Placa Viral , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/inmunologíaRESUMEN
To evaluate the genetic stability of the CAM-70 measles vaccine strain we have performed 10 serial passages of the seed lot virus FMS-7 in chicken embryo fibroblasts primary cultures (CEF) under production conditions. The nucleotide sequences of the seed lot virus, the virus from a vaccine vial (third passage) and from the 10th passage were determined and compared with each other and with sequences from other sources. The full genome analysis of the CAM-70 vaccine still considers it as the most divergent among all vaccine strains. The nucleotide sequence analyses of viral genomes from the three CAM-70 passage levels have demonstrated that they are identical. This study shows that the measles CAM-70 vaccine virus is highly adapted to its cultivation conditions and that its genetic stability contributes, in part, to the safety profile of the vaccine.
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Genoma Viral , Vacuna Antisarampión/genética , Virus del Sarampión/genética , Animales , Secuencia de Bases , Línea Celular , Embrión de Pollo , Fibroblastos , Humanos , Sarampión/virología , Virus del Sarampión/fisiología , Datos de Secuencia Molecular , Mutación , Alineación de Secuencia , Pase SeriadoRESUMEN
BACKGROUND: The yellow fever virus, a member of the genus Flavivirus, is an arthropod-borne pathogen causing severe disease in humans. The attenuated yellow fever 17D virus strain has been used for human vaccination for 70 years and has several characteristics that are desirable for the development of new, live attenuated vaccines. We described here a methodology to construct a viable, and immunogenic recombinant yellow fever 17D virus expressing a green fluorescent protein variant (EGFP). This approach took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor. RESULTS: YF 17D EGFP recombinant virus was grew in Vero cells and reached a peak titer of approximately 6.45 +/- 0.4 log10 PFU/mL at 96 hours post-infection. Immunoprecipitation and confocal laser scanning microscopy demonstrated the expression of the EGFP, which was retained in the endoplasmic reticulum and not secreted from infected cells. The association with the ER compartment did not interfere with YF assembly, since the recombinant virus was fully competent to replicate and exit the cell. This virus was genetically stable up to the tenth serial passage in Vero cells. The recombinant virus was capable to elicit a neutralizing antibody response to YF and antibodies to EGFP as evidenced by an ELISA test. The applicability of this cloning strategy to clone gene foreign sequences in other flavivirus genomes was demonstrated by the construction of a chimeric recombinant YF 17D/DEN4 virus. CONCLUSION: This system is likely to be useful for a broader live attenuated YF 17D virus-based vaccine development for human diseases. Moreover, insertion of foreign genes into the flavivirus genome may also allow in vivo studies on flavivirus cell and tissue tropism as well as cellular processes related to flavivirus infection.
Asunto(s)
Flavivirus/genética , Vectores Genéticos , Proteínas del Envoltorio Viral/genética , Proteínas no Estructurales Virales/genética , Animales , Quimera/genética , Quimera/inmunología , Chlorocebus aethiops , Flavivirus/inmunología , Ingeniería Genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal , Vacunas Sintéticas/genética , Vacunas Sintéticas/aislamiento & purificación , Células Vero , Proteínas del Envoltorio Viral/metabolismo , Vacunas Virales/genética , Vacunas Virales/aislamiento & purificaciónRESUMEN
The yellow fever (YF) 17D vaccine is a live attenuated virus, and its genetic manipulation constitutes a new platform for vaccine development. In this article, we review one of the possible approaches to enable this development, which is the insertion of foreign protein epitopes into different locations of the genome. We describe the three-dimensional (3D) modeling of the YF 17D virus E protein structure based on tick-borne encephalitis (TBE) and the identification of a potential insertion site located at the YF 17D fg loop. Further 3D analysis revealed that it is possible to accommodate inserts of different sizes and amino acid composition in the flavivirus E protein fg loop. We demonstrate that seven YF 17D viruses bearing foreign epitopes that vary in sequence and length show differential growth characteristics in cell culture. The testing of recombinant viruses for mouse neurovirulence suggests that insertions at the 17D E protein fg loop do not compromise the attenuated phenotype of YF 17D virus, further confirming the potential use of this site for the development of new live attenuated 17D virus-based vaccines.
Asunto(s)
Modelos Moleculares , Vacunas Atenuadas/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacuna contra la Fiebre Amarilla/inmunología , Virus de la Fiebre Amarilla/química , Virus de la Fiebre Amarilla/inmunología , Animales , Clonación Molecular , ADN Complementario , Virus de la Encefalitis Transmitidos por Garrapatas/genética , Epítopos/inmunología , Ratones , Conformación Proteica , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vacunas Atenuadas/química , Proteínas del Envoltorio Viral/química , Vacuna contra la Fiebre Amarilla/químicaRESUMEN
Over the last 17 years, the yellow fever (YF) 17DD vaccine secondary seed lot 102/84 was used to produce many million doses of vaccine but it was recently used up. In the absence of other lots at the same passage level a large vaccine batch produced from 102/84 was turned into a new working seed. This new seed was characterized with regard to attenuation in the recommended internationally accepted monkey neurovirulence test (MNVT) using the 102/84 virus as reference. All rhesus monkeys (Macaca mulatta) developed limited viremia and comparable neutralizing antibody titers. Clinical evaluation and histological examination of the central nervous system (CNS) according to WHO criteria for acceptability gave consistent data that demonstrated an attenuated phenotype for the YF 17DD 993FB013Z (13Z) vaccine batch. It is concluded that the additional chicken embryo passage did not lead to any genetic change and the new working seed virus retained its attenuation for monkeys comparable to the 102/84 reference virus.
Asunto(s)
Vacuna contra la Fiebre Amarilla/genética , Vacuna contra la Fiebre Amarilla/toxicidad , Animales , Anticuerpos Antivirales/sangre , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/patología , Embrión de Pollo , Chlorocebus aethiops , Fiebre/virología , Macaca mulatta , Fenotipo , Análisis de Secuencia de ADN , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/toxicidad , Células Vero , Viremia/virología , Vacuna contra la Fiebre Amarilla/inmunología , Virus de la Fiebre Amarilla/genética , Virus de la Fiebre Amarilla/inmunologíaRESUMEN
Dengue is one of the most important arboviral diseases in humans, and although efforts over the last decades have dealt with the development of a vaccine, this vaccine is not available yet. In order to evaluate the potential of a DNA vaccine based on the non-structural 1 (NS1) protein against dengue virus (DENV), we constructed the pcTPANS1 plasmid which contains the secretory signal sequence derived from human tissue plasminogen activator (t-PA) fused to the full length of the DENV-2 NS1 gene. Results indicate that pcTPANS1 promotes correct expression of NS1 in eukaryotic cells and drives secretion of the recombinant protein to the surrounding medium in a dimeric form. Balb/c mice, intramuscularly inoculated with this plasmid, presented high levels of antibodies, recognizing mainly surface-exposed conformational epitopes present in the NS1 protein expressed by insect cells. Long-term antibody response was observed in animals 56 weeks after the first plasmid inoculation, and a rapid, efficient secondary response was observed after a DNA boost. Vaccinated animals were challenged against DENV-2 in two murine models, based on intracerebral (i.c.) and intraperitoneal (i.p.) virus inoculations, and in both cases, pcTPANS1-immunized mice were protected. Overall, these results provide further support for the use of such a plasmid in a possible approach for the development of a vaccine against DENV.