RESUMEN
We performed Ebola virus disease diagnosis and viral load estimation for Ebola cases in Sierra Leone during the late stage of the 2014-2015 outbreak (January-March 2015) and analyzed antibody and cytokine levels and the viral genome sequences. Ebola virus disease was confirmed in 86 of 1001 (9.7%) patients, with an overall case fatality rate of 46.8%. Fatal cases exhibited significantly higher levels of viral loads, cytokines, and chemokines at late stages of infection versus early stage compared with survivors. The viruses converged in a new clade within sublineage 3.2.4, which had a significantly lower case fatality rate.
Asunto(s)
Ebolavirus/genética , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/inmunología , Carga Viral , Anticuerpos Antivirales/sangre , Citocinas/sangre , Brotes de Enfermedades , Genoma Viral , Humanos , Sierra Leona/epidemiología , SobrevivientesRESUMEN
The kinetics of dengue virus (DENV)-specific IgA antibody in urine and the potential correlation with disease severity remain elusive. In this study, 262 serial urine samples from 78 laboratory-confirmed patients were assayed by a commercial immunoglobulin A (IgA) kit against DENV. All cases were classified into dengue fever (DF) and severe dengue (SD) according to the 2009 WHO/TDR guideline. The total positive rate of IgA in urine was 59%. DENV-specific IgA was detected in urine from day 2 to day 13 after the onset of illness in DF patients; While for SD patients, anti-DENV IgA could be detected till day 14. The positive rate of IgA in patients with secondary infection was higher than that in patients with primary infection. Importantly, during 4-7 days after the onset of illness, the IgA positive rate of SD patients was significantly higher than that of DF patients. Especially, the intensity of IgA signal in SD patients was obviously stronger than that in DF patient at the recovery stage. Overall, our results suggested that the existence of DENV-specific IgA antibodies in urine might be a warning sign for the severity of disease and its measurement might provide valuable guidance for proper patient management.
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Anticuerpos Antivirales/orina , Virus del Dengue/inmunología , Dengue/inmunología , Dengue/patología , Inmunoglobulina A/orina , Adulto , Anciano , Anciano de 80 o más Años , China , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Developing vaccine formulations with excellent thermostability and immunogenicity remains a great challenge. By in situ encapsulating a live-attenuated strain of human enterovirus 71 (EV71) in alumina, we obtained a robust vaccine formulation named EV71@NanoAlum, which features significantly enhanced thermostability and immunogenicity. This attempt follows a material-based tactic for vaccine improvement.
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Óxido de Aluminio/química , Enterovirus Humano A/química , Inmunogenicidad Vacunal/inmunología , Temperatura , Vacunas/química , Vacunas/inmunología , Óxido de Aluminio/inmunología , Enterovirus Humano A/inmunología , Tamaño de la Partícula , Estabilidad ProteicaRESUMEN
The dengue virus (DENV) envelope protein domain III (ED3) has been suggested to contain receptor recognition sites and the critical neutralizing epitopes. Up to date, relatively little work has been done on fine mapping of neutralizing epitopes on ED3 for DENV4. In this study, a novel mouse type-specific neutralizing antibody 1G6 against DENV4 was obtained with both prophylactic and therapeutic effects. The epitope was mapped to residues 387-390 of DENV4 envelope protein. Furthermore, site-directed mutagenesis assay identified two critical residues (T388 and H390). The epitope is variable among different DENV serotypes but is highly conserved among four DENV4 genotypes. Affinity measurement showed that naturally occurring variations in ED3 outside the epitope region did not alter the binding of mAb 1G6. These findings expand our understanding of the interactions between neutralizing antibodies and the DENV4 and may be valuable for rational design of DENV vaccines and antiviral drugs.
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Virus del Dengue/inmunología , Epítopos/inmunología , Pruebas de Neutralización , Animales , Línea Celular , Cricetinae , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Live-attenuated influenza vaccines (LAIVs) are now available for the prevention of influenza, with LAIV strains generally derived from serial passage in cultures or by reverse genetics (RG). The receptor-binding domain (RBD) in haemagglutinin (HA) of influenza virus is responsible for viral binding to the avian-type 2,3-α-linked or human-type 2,6-α-linked sialic acid receptor; however, the virulence determinants in the RBD of H5N1 virus remain largely unknown. In the present study, serial passage of H5N1 virus A/Vietnam/1194/2004 in Madin-Darby canine kidney cells resulted in the generation of adapted variants with large-plaque morphology, and genomic sequencing of selected variants revealed two specific amino acid substitutions (K193E and G225E) in the RBD. RG was used to generate H5N1 viruses containing either single or double substitutions in HA. The RG virus containing K193E and G225E mutations (rVN-K193E/G225E) demonstrated large-plaque morphology, enhanced replication and genetic stability after serial passage, without changing the receptor-binding preference. Importantly, in vivo virulence assessment demonstrated that rVN-K193E/G225E was significantly attenuated in mice. Microneutralization and haemagglutination inhibition assays demonstrated that immunization with rVN-K193E/G225E efficiently induced a robust antibody response against WT H5N1 virus in mice. Taken together, our experiments demonstrated that K193E and G225E mutations synergistically attenuated H5N1 virus without enhancing the receptor-binding avidity, and that the RG virus rVN-K193E/G225E represents a potential H5N1 LAIV strategy that deserves further development. These findings identify the RBD as a novel attenuation target for live vaccine development and highlight the complexity of RBD interactions.
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Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H5N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Gripe Humana/virología , Mutación Missense , Animales , Anticuerpos Antivirales , Perros , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/administración & dosificación , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Humanos , Inmunización , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/química , Gripe Humana/inmunología , Ratones , Ratones Endogámicos BALB C , Estructura Terciaria de Proteína , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/química , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas ViralesRESUMEN
Human enterovirus 71 (EV71) infection has emerged as a major threat to children; however, no effective antiviral treatment or vaccine is currently available. Antibody-based treatment shows promises to control this growing public health problem of EV71 infection, and a few potent monoclonal antibodies (mAbs) targeting viral capsid protein have been well described. Here, we generated an EV71-specific mouse mAb 2G8 that conferred full protection against lethal EV71 challenge in a suckling mouse model. 2G8 belonged to IgM isotype and neutralized EV71 at the attachment stage. Biochemical assays mapped the binding epitope of 2G8 to the SP70 peptide, which spanning amino acid residues 208-222 on the VP1 protein. Alanine scanning mutagenesis defined the essential roles of multiple residues, including Y208, T210, G212, K215, K218, L220, E221, and Y222, for 2G8 binding. Then, a panel of single mutation was individually introduced into the EV71 infectious clone by reverse genetics, and three mutant viruses, K215A, K218A, and L220A, were successfully recovered and characterized. Biochemical and neutralization assays revealed that K218A mutant partially escaped 2G8 neutralization, while L220A completely abolished 2G8 binding and neutralization. In particular, neutralization assays with human sera demonstrated that K218A and L220A substitutions are also critical for antibody neutralization in natural infection population. These findings not only generate a protective mAb candidate with therapeutic potential but also provide insights into antibody-mediated EV71 neutralization mechanism.
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Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Enterovirus Humano A/inmunología , Infecciones por Enterovirus/terapia , Sustitución de Aminoácidos , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Enterovirus Humano A/genética , Evasión Inmune , Inmunización Pasiva , Inmunoglobulina M/inmunología , Inmunoglobulina M/aislamiento & purificación , Inmunoglobulina M/uso terapéutico , Ratones , Pruebas de Neutralización , Unión Proteica , Genética Inversa , Análisis de Supervivencia , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/inmunologíaRESUMEN
Heat-lability is a key roadblock that strangles the widespread applications of many biological products. In nature, archaeal and extremophilic organisms utilize amorphous silica as a protective biomineral and exhibit considerable thermal tolerance. Here we present a bioinspired approach to generate thermostable virus by introducing an artificial hydrated silica exterior on individual virion. Similar to thermophiles, silicified viruses can survive longer at high temperature than their wild-type relatives. Virus inactivation assays showed that silica hydration exterior of the modified virus effectively prolonged infectivity of viruses by â¼ 10-fold at room temperature, achieving a similar result as that obtained by storing native ones at 4 °C. Mechanistic studies indicate that amorphous silica nanoclusters stabilize the inner virion structure by forming a layer that restricts molecular mobility, acting as physiochemical nanoanchors. Notably, we further evaluate the potential application of this biomimetic strategy in stabilizing clinically approved vaccine, and the silicified polio vaccine that can retain 90% potency after the storage at room temperature for 35 days was generated by this biosilicification approach and validated with in vivo experiments. This approach not only biomimetically connects inorganic material and living virus but also provides an innovative resolution to improve the thermal stability of biological agents using nanomaterials.
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Biomimética/métodos , Calor , Dióxido de Silicio/química , Vacunas Virales/química , Agua/química , Animales , Línea Celular , Cricetinae , Enterovirus Humano A/química , Enterovirus Humano A/inmunología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformación Molecular , Nanoestructuras/química , Virión/químicaRESUMEN
Hand-foot-and-mouth disease (HFMD) has been recognized as an important global public health issue, which is predominantly caused by enterovirus 71 (EV-A71) and coxsackievirus A16 (CVA16). There is no available vaccine against HFMD. An ideal HFMD vaccine should be bivalent against both EV-A71 and CVA16. Here, a novel strategy to produce bivalent HFMD vaccine based on chimeric EV-A71 virus-like particles (ChiEV-A71 VLPs) was proposed and illustrated. The neutralizing epitope SP70 within the capsid protein VP1 of EV-A71 was replaced with that of CVA16 in ChiEV-A71 VLPs. Structural modeling revealed that the replaced CVA16-SP70 epitope is well exposed on the surface of ChiEV-A71 VLPs. These VLPs produced in Saccharomyces cerevisiae exhibited similarity in both protein composition and morphology as naive EV-A71 VLPs. Immunization with ChiEV-A71 VLPs in mice elicited robust Th1/Th2 dependent immune responses against EV-A71 and CVA16. Furthermore, passive immunization with anti-ChiEV-A71 VLPs sera conferred full protection against lethal challenge of both EV-A71 and CVA16 infection in neonatal mice. These results suggested that this chimeric vaccine, ChiEV-A71 might have the potential to be further developed as a bivalent HFMD vaccine in the near future. Such chimeric enterovirus VLPs provide an alternative platform for bivalent HFMD vaccine development.
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Anticuerpos Neutralizantes/inmunología , Enfermedad de Boca, Mano y Pie/inmunología , Enfermedad de Boca, Mano y Pie/prevención & control , Vacunas de Partículas Similares a Virus/inmunología , Animales , Anticuerpos Neutralizantes/administración & dosificación , Enterovirus/inmunología , Enterovirus/patogenicidad , Enterovirus Humano A/inmunología , Enterovirus Humano A/patogenicidad , Epítopos/inmunología , Enfermedad de Boca, Mano y Pie/virología , Humanos , Ratones , Vacunación , Vacunas de Partículas Similares a Virus/uso terapéuticoRESUMEN
BACKGROUND: The emerged human infection with avian influenza A (H7N9) virus in China since 2013 has aroused global concerns. There is great demand for simple and rapid diagnostic method for early detection of H7N9 to provide timely treatment and disease control. The aim of the current study was to develop a rapid, accurate and feasible reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of H7N9 virus. RESULTS: The detection limits of the H7- and N9-specific RT-LAMP assay were both approximately 0.2 PFU per reaction. No cross-reactivity was observed with other subtype of influenza viruses or common respiratory viral pathogens. The assay worked well with clinical specimens from patients and chickens, and exhibited high specificity and sensitivity. CONCLUSIONS: The H7/N9 specific RT-LAMP assay was sensitive and accurate, which could be a useful alternative in clinical diagnostics of influenza A (H7N9) virus, especially in the hospitals and laboratories without sophisticated diagnostic systems.
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Subtipo H7N9 del Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/diagnóstico , Gripe Humana/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Animales , Pollos , China , Humanos , Subtipo H7N9 del Virus de la Influenza A/genética , Gripe Aviar/virología , Gripe Humana/virología , Sensibilidad y Especificidad , Virología/métodosRESUMEN
Human adenovirus type 55 (HAdV-B55) represents a re-emerging human pathogen, and this adenovirus has been reported to cause outbreaks of acute respiratory diseases among military trainees and in school populations around the world. HAdV-B55 has been revealed to have evolved from homologous recombination between human adenovirus type 14 (HAdV-B14) and type 11 (HAdV-B11), but it presents different clinical manifestations from parental virus HAdV-B11. In the present paper, we report the distinct biological features of HAdV-B55 in comparison with the parental viruses HAdV-B11 and HAdV-B14 in cell cultures. The results showed that HAdV-B55 replicated well in various cells, similar to HAdV-B11 and HAdV-B14, but that its processing had a slower and milder cytopathic effect in the early stages of infection. Viral fitness analysis showed that HAdV-B55 exhibited higher levels of replication in respiratory cells than did either of its parents. Cytotoxicity and apoptosis analyses in A549 cells indicated that HAdV-B55 was less cytotoxic than HAdV-B11 and HAdV-B14 were and induced milder apoptosis. Finally, thermal sensitivity analysis revealed that HAdV-B55 exhibited lower thermostability than did either HAdV-B11 or HAdV-B14, which may limit the transmission of HAdV-B55 in humans. Together, the findings described here expand current knowledge about this re-emerging recombinant HAdV, shedding light on the pathogenesis of HAdV-B55.
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Adenovirus Humanos/fisiología , Adenovirus Humanos/crecimiento & desarrollo , Adenovirus Humanos/patogenicidad , Apoptosis , Línea Celular Tumoral , Efecto Citopatogénico Viral , Calor , Humanos , CinéticaRESUMEN
BACKGROUND: Human Enterovirus 71 (EV71) has emerged as the leading cause of viral encephalitis in children, especially in the Asia-Pacific regions. EV71 vaccine development is of high priority at present, and neutralization antibodies have been documented to play critical roles during in vitro and in vivo protection against EV71 infection. RESULTS: In this study, a novel strategy to produce EV71 vaccine candidate based on recombinant multiple tandem linear neutralizing epitopes (mTLNE) was proposed. The three well identified EV71 linear neutralizing epitopes in capsid proteins, VP1-SP55, VP1-SP70 and VP2-SP28, were sequentially linked by a Gly-Ser linker ((G4S)3), and expressed in E.coli in fusion with the Trx and His tag at either terminal. The recombinant protein mTLNE was soluble and could be purified by standard affinity chromatography. Following three dosage of immunization in adult mice, EV71-specific IgG and neutralization antibodies were readily induced by recombinant mTLNE. IgG subtyping demonstrated that lgG1 antibodies dominated the mTLNE-induced humoral immune response. Especially, cytokine profiling in spleen cells from the mTLNE-immunized mice revealed high production of IL-4 and IL-6. Finally, in vivo challenge experiments showed that passive transfer with anti-mTLNE sera conferred full protection against lethal EV71 challenge in neonatal mice. CONCLUSION: Our results demonstrated that this rational designed recombinant mTLNE might have the potential to be further developed as an EV71 vaccine in the future.
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Proteínas de la Cápside/inmunología , Enterovirus Humano A/inmunología , Infecciones por Enterovirus/prevención & control , Epítopos de Linfocito B/inmunología , Proteínas Recombinantes de Fusión/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Proteínas de la Cápside/genética , Cromatografía de Afinidad , Citocinas/análisis , Modelos Animales de Enfermedad , Infecciones por Enterovirus/inmunología , Escherichia coli/genética , Femenino , Expresión Génica , Inmunización Pasiva , Inmunoglobulina G/sangre , Leucocitos Mononucleares/inmunología , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/genética , Análisis de Supervivencia , Vacunación/métodos , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
Human enterovirus 71 (HEV71) has emerged as the leading cause of viral encephalitis in children in most Asian countries. The roles of host miRNAs in the neurological pathogenesis of HEV71 infection remain unknown. In the present study, comprehensive miRNA expression profiling in HEV71-infected human neuroblastoma SH-SY5Y cells was performed using the Affymetrix Gene Chip microarray assay and was validated using real-time RT-PCR. Among the 69 differentially expressed miRNAs, miR-1246 was specifically induced by HEV71 infection in human neuroblastoma cells, but inhibition of miR-1246 failed to affect HEV71 replication. Parallel mRNA and microRNA profiling based on the 35 K Human Genome Array identified 182 differentially regulated genes. Target prediction of miR-1246 and network modeling revealed 14 potential target genes involved in cell death and cell signaling. Finally, a combined analysis of the results from mRNA profiling and miR-1246 target predication led to the identification of disc-large homolog 3 (DLG3), which is associated with neurological disorders, for further validation. Sequence alignment and luciferase reporter assay showed that miR-1246 directly bound with the 3'-UTR of DLG3 gene. Down-regulation of miR-1246 induced significant changes in DLG3 expression levels in HEV71-infected SHSY5Y cells. Together, these results suggested that miR-1246 might play a role in neurological pathogenesis of HEV71 by regulating DLG3 gene in infected cells. These findings provide new information on the miRNA and mRNA profiles of HEV71-infected neuroblastoma cells. The biological significance of miR-1246 and DLG3 during the course of HEV71 infection deserves further investigation.
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Enterovirus Humano A/genética , Redes Reguladoras de Genes , Interacciones Huésped-Patógeno/genética , MicroARNs/genética , Neuronas/metabolismo , Proteínas Nucleares/genética , Factores de Transcripción/genética , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Chlorocebus aethiops , Enterovirus Humano A/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Luciferasas/genética , Luciferasas/metabolismo , MicroARNs/metabolismo , Datos de Secuencia Molecular , Neuronas/patología , Neuronas/virología , Proteínas Nucleares/metabolismo , Mapeo de Interacción de Proteínas , Alineación de Secuencia , Transducción de Señal , Factores de Transcripción/metabolismo , Células VeroRESUMEN
The live attenuated Japanese encephalitis (JE) vaccine SA14-14-2 was licensed decades ago and now approved for clinical use in most JE endemic countries. Large-scale clinical trials demonstrate ideal safety and efficacy profile of this Chinese vaccine. The SA14-14-2 vaccine was derived from a virulent strain SA14 after hundreds of serial passaging in cells and animals, concern about virulence reversion remains exist. In the present study, to study the in vitro and in vivo genetic and attenuation stability of the vaccine virus, SA14-14-2 was serially passaged in Vero cells and mouse brain followed by sequence comparison and attenuation phenotype analysis. The results showed that no significant mutation was acquired after serial passaging in Vero cells except a single Ser66Leu mutation within capsid protein, which had no effect on viral virulence in mice. Importantly, serial passaging of SA14-14-2 in suckling mouse brain resulted in emergence of adaptive mutations and increased virulence in mice. Population and plaque-purified clone consensus sequence analysis showed four adaptive mutations in envelope (E) protein, F107L, K138E, T226R and I270T, sequentially occurred and become predominant during serial passaging in suckling mouse brain. Especially, these adaptive mutations were close related with the enhanced neurovirulence and neuroinvasiveness in mice. Our results provide experimental evidence of highly genetic and attenuation stability of SA14-14-2 following passaging in Vero cells, and reveal the potential virulence reversion during passaging in mouse brain in association with critical adaptive mutations in E protein. These findings are important for quality control and evaluation of live JE vaccines and will help understand the attenuation mechanism of flavivirus vaccine.
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Virus de la Encefalitis Japonesa (Especie)/genética , Vacunas contra la Encefalitis Japonesa/genética , Virulencia/genética , Animales , Encéfalo/virología , Chlorocebus aethiops , Virus de la Encefalitis Japonesa (Especie)/patogenicidad , Genoma Viral , Ratones , Ratones Endogámicos BALB C , Mutación , Pase Seriado , Vacunas Atenuadas/genética , Células Vero , Ensayo de Placa ViralAsunto(s)
Antiinflamatorios/uso terapéutico , Subtipo H5N1 del Virus de la Influenza A , Metilprednisolona/uso terapéutico , Neumonía Viral/prevención & control , Animales , Antiinflamatorios/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Pulmón/virología , Metilprednisolona/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Tasa de Supervivencia , Insuficiencia del Tratamiento , Carga Viral , Pérdida de PesoAsunto(s)
Brotes de Enfermedades , Enterovirus Humano A/genética , Enfermedad de Boca, Mano y Pie/epidemiología , ARN Viral/análisis , Preescolar , China/epidemiología , Ciudades/epidemiología , Enterovirus Humano A/clasificación , Enterovirus Humano A/aislamiento & purificación , Enfermedad de Boca, Mano y Pie/virología , Humanos , Filogeografía , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Dengue virus (DENV) still poses a global public health threat, and no vaccine or antiviral therapy is currently available. Antibody plays distinct roles in controlling DENV infections. Neutralizing antibody is protective against DENV infection, whereas sub-neutralizing concentration of antibody can increase DENV infection, termed antibody-dependent enhancement (ADE). Plaque-based assay represents the most widely accepted method measuring neutralizing or enhancing antibodies. RESULTS: In this study, a novel reporter virus-based system was developed for measuring neutralization and ADE activity. A stable Renilla luciferase reporter DENV (Luc-DENV) that can produce robust luciferase signals in BHK-21 and K562 cells were used to establish the assay and validated against traditional plaque-based assay. Luciferase value analysis using various known DENV-specific monoclonal antibodies showed good repeatability and a well linear correlation with conventional plaque-based assays. The newly developed assay was finally validated with clinical samples from infected animals and individuals. CONCLUSIONS: This reporter virus-based assay for neutralizing and enhancing antibody evaluation is rapid, lower cost, and high throughput, and will be helpful for laboratory detection and epidemiological investigation for DENV antibodies.
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Anticuerpos Bloqueadores/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunología , Virología/métodos , Animales , Línea Celular , Genes Reporteros , Humanos , Inmunoensayo/métodos , Luciferasas de Renilla/análisis , Reproducibilidad de los ResultadosRESUMEN
The worldwide expansion of four serotypes of dengue virus (DENV) poses great risk to global public health. Several vaccine candidates are under development. However, none is yet available for humans. In the present study, a novel strategy to produce tetravalent DENV vaccine based on envelope protein domain III (EDIII) was proposed. Tandem EDIIIs of two serotypes (type 1-2 and type 3-4) of DENV connected by a Gly-Ser linker ((Gly4Ser)3) were expressed in E. coli, respectively. Then, the two bivalent recombinant EDIIIs were equally mixed to form the tetravalent vaccine candidate MixBiEDIII, and used to immunize BALB/c mice. The results showed that specific IgG and neutralizing antibodies against all four serotypes of DENV were successfully induced in the MixBiEDIII employing Freund adjuvant immunized mice. Furthermore, in the suckling mouse model, sera from mice immunized with MixBiEDIII provided significant protection against four serotypes of DENV challenge. Our data demonstrated that MixBiEDIII, as a novel form of subunit vaccine candidates, might have the potential to be further developed as a tetravalent dengue vaccine in the near future.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Vacunas de Subunidad/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Células Cultivadas , Cricetinae , Culicidae/inmunología , Escherichia coli/genética , Femenino , Inmunización/métodos , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas del Envoltorio Viral/genéticaRESUMEN
Human coxsackievirus A16 (CA16) infection results in hand, foot, and mouth disease (HFMD) along with other severe neurological diseases in children and poses an important public health threat in Asian countries. During an HFMD epidemic in 2009 in Guangdong, China, two CA16 strains (GD09/119 and GD09/24) were isolated and characterized. Although both strains were similar in plaque morphology and growth properties in vitro, the two isolates exhibited distinct pathogenicity in neonatal mice upon intraperitoneal or intracranial injection. Complete genome sequences of both CA16 strains were determined, and the possible virulence determinants were analyzed and predicted. Phylogenetic analysis revealed that these CA16 isolates from Guangdong belonged to the B1b genotype and were closely related to other recent CA16 strains isolated in mainland China. Similarity and bootscanning analyses of these CA16 strains detected homologous recombination with the EV71 prototype strain BrCr in the non-structural gene regions and the 3'-untranslated regions. Together, the phenotypic and genomic characterizations of the two clinical CA16 isolates circulating in China were compared in detail, and the potential amino acid residues responsible for CA16 virulence in mice were predicted. These findings will help explain the evolutionary relationship of the CA16 strains circulating in China, warranting future studies investigating enterovirus virulence.
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Enterovirus Humano A/genética , Enterovirus Humano A/patogenicidad , Genoma Viral , Enfermedad de Boca, Mano y Pie/virología , Secuencia de Aminoácidos , Animales , Enterovirus Humano A/clasificación , Enterovirus Humano A/fisiología , Femenino , Genómica , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fenotipo , Filogenia , Alineación de Secuencia , VirulenciaRESUMEN
Hand, foot, and mouth disease (HFMD) has caused significant morbidity and mortality in the Asia-Pacific regions, particularly in infants and young children. Coxsackievirus A16 (CA16) represents one of the major causative agents for HFMD, and the development of a safe and effective vaccine preventing CA16 infections has become a public health priority. In this study, we have developed a yeast system for the production of virus-like particles (VLPs) for CA16 by co-expressing P1 and 3CD of CA16 in Saccharomyces cerevisiae. These VLPs exhibit similarity in both protein composition and morphology as empty particles from CA16-infected cells. Immunization with CA16 VLPs in mice potently induced CA16-specific IgG and neutralization antibodies in a dose-dependent manner. IgG subclass isotyping revealed that IgG1 and lgG2b were dominantly induced by VLPs. Meanwhile, cytokine profiling demonstrated that immunization with VLPs significantly induced the secretion of IFN-γ, indicating potent cellular immune response. Furthermore, in vivo challenge experiments showed that passive immunization with anti-VLPs sera conferred full protection against lethal CA16 challenge in neonate mice. Taken together, our data demonstrated that VLPs produced in yeast might have the potential to be further developed as a vaccine candidate against HFMD.