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1.
Proc Natl Acad Sci U S A ; 120(3): e2218899120, 2023 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-36638211

RESUMEN

Cleavage of the flavivirus premembrane (prM) structural protein during maturation can be inefficient. The contribution of partially mature flavivirus virions that retain uncleaved prM to pathogenesis during primary infection is unknown. To investigate this question, we characterized the functional properties of newly-generated dengue virus (DENV) prM-reactive monoclonal antibodies (mAbs) in vitro and using a mouse model of DENV disease. Anti-prM mAbs neutralized DENV infection in a virion maturation state-dependent manner. Alanine scanning mutagenesis and cryoelectron microscopy of anti-prM mAbs in complex with immature DENV defined two modes of attachment to a single antigenic site. In vivo, passive transfer of intact anti-prM mAbs resulted in an antibody-dependent enhancement of disease. However, protection against DENV-induced lethality was observed when the transferred mAbs were genetically modified to inhibit their ability to interact with Fcγ receptors. These data establish that in addition to mature forms of the virus, partially mature infectious prM+ virions can also contribute to pathogenesis during primary DENV infections.


Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antivirales , Virus del Dengue , Dengue , Microscopía por Crioelectrón , Proteínas del Envoltorio Viral/metabolismo , Virión/metabolismo , Animales , Ratones
2.
PLoS Pathog ; 9(2): e1003157, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23459315

RESUMEN

Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) are life-threatening complications following infection with one of the four serotypes of dengue virus (DENV). At present, no vaccine or antiviral therapies are available against dengue. Here, we characterized a panel of eight human or mouse-human chimeric monoclonal antibodies (MAbs) and their modified variants lacking effector function and dissected the mechanism by which some protect against antibody-enhanced lethal DENV infection. We found that neutralizing modified MAbs that recognize the fusion loop or the A strand epitopes on domains II and III of the envelope protein, respectively, act therapeutically by competing with and/or displacing enhancing antibodies. By analyzing these relationships, we developed a novel in vitro suppression-of-enhancement assay that predicts the ability of modified MAbs to act therapeutically against antibody-enhanced disease in vivo. These studies provide new insight into the biology of DENV pathogenesis and the requirements for antibodies to treat lethal DENV disease.


Asunto(s)
Anticuerpos Bloqueadores/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunología , Dengue/prevención & control , Receptores de IgG/inmunología , Animales , Dengue/inmunología , Dengue/virología , Mapeo Epitopo , Epítopos/inmunología , Humanos , Células K562 , Ratones , Pruebas de Neutralización , Conformación Proteica , Receptores de IgG/metabolismo , Tasa de Supervivencia , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología
3.
J Virol ; 87(13): 7747-53, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23658441

RESUMEN

Dengue viruses are the most common arthropod-transmitted viral infection, with an estimated 390 million human infections annually and ∼3.6 billion people at risk. Currently, there are no approved vaccines or therapeutics available to control the global dengue virus disease burden. In this study, we demonstrate the binding, neutralizing activity, and therapeutic capacity of a novel bispecific dual-affinity retargeting molecule (DART) that limits infection of all four serotypes of dengue virus.


Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Virus del Dengue/inmunología , Dengue/tratamiento farmacológico , Terapia Molecular Dirigida/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Reacciones Cruzadas/inmunología , Dengue/inmunología , Epítopos/genética , Humanos , Región Variable de Inmunoglobulina/genética , Estimación de Kaplan-Meier , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Pruebas de Neutralización , Especificidad de la Especie , Estadísticas no Paramétricas , Vacunas Virales/inmunología
4.
J Virol ; 87(16): 8826-42, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23785205

RESUMEN

Although prior studies have characterized the neutralizing activities of monoclonal antibodies (MAbs) against dengue virus (DENV) serotypes 1, 2, and 3 (DENV-1, DENV-2, and DENV-3), few reports have assessed the activity of MAbs against DENV-4. Here, we evaluated the inhibitory activity of 81 new mouse anti-DENV-4 MAbs. We observed strain- and genotype-dependent differences in neutralization of DENV-4 by MAbs mapping to epitopes on domain II (DII) and DIII of the envelope (E) protein. Several anti-DENV-4 MAbs inefficiently inhibited at least one strain and/or genotype, suggesting that the exposure or sequence of neutralizing epitopes varies within isolates of this serotype. Remarkably, flavivirus cross-reactive MAbs, which bound to the highly conserved fusion loop in DII and inhibited infection of DENV-1, DENV-2, and DENV-3, more weakly neutralized five different DENV-4 strains encompassing the genetic diversity of the serotype after preincubation at 37°C. However, increasing the time of preincubation at 37°C or raising the temperature to 40°C enhanced the potency of DII fusion loop-specific MAbs and some DIII-specific MAbs against DENV-4 strains. Prophylaxis studies in two new DENV-4 mouse models showed that neutralization titers of MAbs after preincubation at 37°C correlated with activity in vivo. Our studies establish the complexity of MAb recognition against DENV-4 and suggest that differences in epitope exposure relative to other DENV serotypes affect antibody neutralization and protective activity.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunología , Epítopos/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Reacciones Cruzadas , Dengue/prevención & control , Modelos Animales de Enfermedad , Mapeo Epitopo , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Temperatura , Factores de Tiempo
5.
J Virol ; 86(14): 7637-49, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22573866

RESUMEN

Dengue viruses (DENV) are transmitted to humans by the bite of Aedes aegypti or Aedes albopictus mosquitoes, with millions of infections annually in over 100 countries. The diseases they produce, which occur exclusively in humans, are dengue fever (DF) and dengue hemorrhagic fever (DHF). We previously developed a humanized mouse model of DF in which mice transplanted with human hematopoietic stem cells produced signs of DENV disease after injection with low-passage, wild-type isolates. Using these mice, but now allowing infected A. aegypti to transmit dengue virus during feeding, we observed signs of more severe disease (higher and more sustained viremia, erythema, and thrombocytopenia). Infected mice mounted innate (gamma interferon [IFN-γ] and soluble interleukin 2 receptor alpha [sIL-2Rα]) and adaptive (anti-DENV antibodies) immune responses that failed to clear viremia until day 56, while a mosquito bite alone induced strong immunomodulators (tumor necrosis factor alpha [TNF-α], IL-4, and IL-10) and thrombocytopenia. This is the first animal model that allows an evaluation of human immunity to DENV infection after mosquito inoculation.


Asunto(s)
Virus del Dengue/inmunología , Virus del Dengue/patogenicidad , Dengue/inmunología , Modelos Animales de Enfermedad , Aedes/virología , Animales , Dengue/transmisión , Dengue/virología , Femenino , Trasplante de Células Madre Hematopoyéticas , Humanos , Mordeduras y Picaduras de Insectos/virología , Insectos Vectores/virología , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Interleucina-10/inmunología , Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Interleucina-4/biosíntesis , Interleucina-4/inmunología , Masculino , Ratones , Ratones Endogámicos NOD , Trombocitopenia/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis
6.
PLoS Pathog ; 6(4): e1000823, 2010 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-20369024

RESUMEN

Antibody protection against flaviviruses is associated with the development of neutralizing antibodies against the viral envelope (E) protein. Prior studies with West Nile virus (WNV) identified therapeutic mouse and human monoclonal antibodies (MAbs) that recognized epitopes on domain III (DIII) of the E protein. To identify an analogous panel of neutralizing antibodies against DENV type-1 (DENV-1), we immunized mice with a genotype 2 strain of DENV-1 virus and generated 79 new MAbs, 16 of which strongly inhibited infection by the homologous virus and localized to DIII. Surprisingly, only two MAbs, DENV1-E105 and DENV1-E106, retained strong binding and neutralizing activity against all five DENV-1 genotypes. In an immunocompromised mouse model of infection, DENV1-E105 and DENV1-E106 exhibited therapeutic activity even when administered as a single dose four days after inoculation with a heterologous genotype 4 strain of DENV-1. Using epitope mapping and X-ray crystallographic analyses, we localized the neutralizing determinants for the strongly inhibitory MAbs to distinct regions on DIII. Interestingly, sequence variation in DIII alone failed to explain disparities in neutralizing potential of MAbs among different genotypes. Overall, our experiments define a complex structural epitope on DIII of DENV-1 that can be recognized by protective antibodies with therapeutic potential.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Virus del Dengue/genética , Virus del Dengue/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Epítopos de Linfocito B/inmunología , Genotipo , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Estructura Terciaria de Proteína , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética
7.
J Exp Med ; 219(4)2022 04 04.
Artículo en Inglés | MEDLINE | ID: mdl-35297953

RESUMEN

Venezuelan equine encephalitis virus (VEEV) remains a risk for epidemic emergence or use as an aerosolized bioweapon. To develop possible countermeasures, we isolated VEEV-specific neutralizing monoclonal antibodies (mAbs) from mice and a human immunized with attenuated VEEV strains. Functional assays and epitope mapping established that potently inhibitory anti-VEEV mAbs bind distinct antigenic sites in the A or B domains of the E2 glycoprotein and block multiple steps in the viral replication cycle including attachment, fusion, and egress. A 3.2-Å cryo-electron microscopy reconstruction of VEEV virus-like particles bound by a human Fab suggests that antibody engagement of the B domain may result in cross-linking of neighboring spikes to prevent conformational requirements for viral fusion. Prophylaxis or postexposure therapy with these mAbs protected mice against lethal aerosol challenge with VEEV. Our study defines functional and structural mechanisms of mAb protection and suggests that multiple antigenic determinants on VEEV can be targeted for vaccine or antibody-based therapeutic development.


Asunto(s)
Virus de la Encefalitis Equina Venezolana , Encefalomielitis Equina Venezolana , Vacunas Virales , Aerosoles , Animales , Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Microscopía por Crioelectrón , Encefalomielitis Equina Venezolana/prevención & control , Caballos , Ratones
8.
J Virol ; 84(20): 10630-43, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20702644

RESUMEN

Dengue viruses (DENV) comprise a family of related positive-strand RNA viruses that infect up to 100 million people annually. Currently, there is no approved vaccine or therapy to prevent infection or diminish disease severity. Protection against DENV is associated with the development of neutralizing antibodies that recognize the viral envelope (E) protein. Here, with the goal of identifying monoclonal antibodies (MAbs) that can function as postexposure therapy, we generated a panel of 82 new MAbs against DENV-3, including 24 highly neutralizing MAbs. Using yeast surface display, we localized the epitopes of the most strongly neutralizing MAbs to the lateral ridge of domain III (DIII) of the DENV type 3 (DENV-3) E protein. While several MAbs functioned prophylactically to prevent DENV-3-induced lethality in a stringent intracranial-challenge model of mice, only three MAbs exhibited therapeutic activity against a homologous strain when administered 2 days after infection. Remarkably, no MAb in our panel protected prophylactically against challenge by a strain from a heterologous DENV-3 genotype. Consistent with this, no single MAb neutralized efficiently the nine different DENV-3 strains used in this study, likely because of the sequence variation in DIII within and between genotypes. Our studies suggest that strain diversity may limit the efficacy of MAb therapy or tetravalent vaccines against DENV, as neutralization potency generally correlated with a narrowed genotype specificity.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Variación Antigénica/genética , Antígenos Virales/química , Antígenos Virales/genética , Línea Celular , Reacciones Cruzadas , Dengue/inmunología , Dengue/prevención & control , Dengue/virología , Virus del Dengue/clasificación , Virus del Dengue/genética , Virus del Dengue/patogenicidad , Mapeo Epitopo , Epítopos/química , Epítopos/genética , Genotipo , Humanos , Inmunización Pasiva , Técnicas In Vitro , Ratones , Ratones Noqueados , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Estructura Terciaria de Proteína , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología
9.
J Virol ; 84(18): 9227-39, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20592088

RESUMEN

Dengue virus (DENV) is the most prevalent insect-transmitted viral disease in humans globally, and currently no specific therapy or vaccine is available. Protection against DENV and other related flaviviruses is associated with the development of antibodies against the viral envelope (E) protein. Although prior studies have characterized the neutralizing activity of monoclonal antibodies (MAbs) against DENV type 2 (DENV-2), none have compared simultaneously the inhibitory activity against a genetically diverse range of strains in vitro, the protective capacity in animals, and the localization of epitopes. Here, with the goal of identifying MAbs that can serve as postexposure therapy, we investigated in detail the functional activity of a large panel of new anti-DENV-2 mouse MAbs. Binding sites were mapped by yeast surface display and neutralization escape, cell culture inhibition assays were performed with homologous and heterologous strains, and prophylactic and therapeutic activity was evaluated with two mouse models. Protective MAbs localized to epitopes on the lateral ridge of domain I (DI), the dimer interface, lateral ridge, and fusion loop of DII, and the lateral ridge, C-C' loop, and A strand of DIII. Several MAbs inefficiently inhibited at least one DENV-2 strain of a distinct genotype, suggesting that recognition of neutralizing epitopes varies with strain diversity. Moreover, antibody potency generally correlated with a narrowed genotype and serotype specificity. Five MAbs functioned efficiently as postexposure therapy when administered as a single dose, even 3 days after intracranial infection of BALB/c mice. Overall, these studies define the structural and functional complexity of antibodies against DENV-2 with protective potential.


Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Sitios de Unión , Línea Celular , Cricetinae , Dengue/prevención & control , Dengue/terapia , Virus del Dengue/genética , Mapeo Epitopo , Humanos , Inmunoterapia/métodos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Pruebas de Neutralización , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Relación Estructura-Actividad , Proteínas del Envoltorio Viral/genética
10.
J Virol ; 81(23): 12816-26, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17881453

RESUMEN

Neutralization of flaviviruses in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Previous studies demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral ridge of domain III (DIII) of the West Nile virus (WNV) E protein strongly protect against infection in animals. Based on X-ray crystallography and sequence analysis, an analogous type-specific neutralizing epitope for individual serotypes of the related flavivirus dengue virus (DENV) was hypothesized. Using yeast surface display of DIII variants, we defined contact residues of a panel of type-specific, subcomplex-specific, and cross-reactive MAbs that recognize DIII of DENV type 2 (DENV-2) and have different neutralizing potentials. Type-specific MAbs with neutralizing activity against DENV-2 localized to a sequence-unique epitope on the lateral ridge of DIII, centered at the FG loop near residues E383 and P384, analogous in position to that observed with WNV-specific strongly neutralizing MAbs. Subcomplex-specific MAbs that bound some but not all DENV serotypes and neutralized DENV-2 infection recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310. In contrast, several MAbs that had poor neutralizing activity against DENV-2 and cross-reacted with all DENV serotypes and other flaviviruses recognized an epitope with residues in the AB loop of DIII, a conserved region that is predicted to have limited accessibility on the mature virion. Overall, our experiments define adjacent and structurally distinct epitopes on DIII of DENV-2 which elicit type-specific, subcomplex-specific, and cross-reactive antibodies with different neutralizing potentials.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunología , Epítopos/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Reacciones Cruzadas , Mapeo Epitopo , Pruebas de Neutralización
11.
mBio ; 4(6): e00873-13, 2013 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-24255124

RESUMEN

UNLABELLED: Following natural dengue virus (DENV) infection, humans produce some antibodies that recognize only the serotype of infection (type specific) and others that cross-react with all four serotypes (cross-reactive). Recent studies with human antibodies indicate that type-specific antibodies at high concentrations are often strongly neutralizing in vitro and protective in animal models. In general, cross-reactive antibodies are poorly neutralizing and can enhance the ability of DENV to infect Fc receptor-bearing cells under some conditions. Type-specific antibodies at low concentrations also may enhance infection. There is an urgent need to determine whether there are conserved antigenic sites that can be recognized by cross-reactive potently neutralizing antibodies. Here, we describe the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) directed to the DENV domain II fusion loop (FL) envelope protein region from subjects following vaccination or natural infection. Most of the FL-specific antibodies exhibited a conventional phenotype, characterized by low-potency neutralizing function and antibody-dependent enhancing activity. One clone, however, recognized the bc loop of domain II adjacent to the FL and exhibited a unique phenotype of ultrahigh potency, neutralizing all four serotypes better than any other previously described MAb recognizing this region. This antibody not only neutralized DENV effectively but also competed for binding against the more prevalent poor-quality antibodies whose binding was focused on the FL. The 1C19 human antibody could be a promising component of a preventative or therapeutic intervention. Furthermore, the unique epitope revealed by 1C19 suggests a focus for rational vaccine design based on novel immunogens presenting cross-reactive neutralizing determinants. IMPORTANCE: With no effective vaccine available, the incidence of dengue virus (DENV) infections worldwide continues to rise, with more than 390 million infections estimated to occur each year. Due to the unique roles that antibodies are postulated to play in the pathogenesis of DENV infection and disease, there is consensus that a successful DENV vaccine must protect against all four serotypes. If conserved epitopes recognized by naturally occurring potently cross-neutralizing human antibodies could be identified, monovalent subunit vaccine preparations might be developed. We characterized 30 DENV cross-neutralizing human monoclonal antibodies (MAbs) and identified one (1C19) that recognized a novel conserved site, known as the bc loop. This antibody has several desirable features, as it neutralizes DENV effectively and competes for binding against the more common low-potency fusion loop (FL) antibodies, which are believed to contribute to antibody-mediated disease. To our knowledge, this is the first description of a potent serotype cross-neutralizing human antibody to DENV.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas , Virus del Dengue/inmunología , Epítopos de Linfocito B/inmunología , Proteínas del Envoltorio Viral/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Humanos
12.
PLoS Negl Trop Dis ; 5(6): e1188, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21713020

RESUMEN

Humans who experience a primary dengue virus (DENV) infection develop antibodies that preferentially neutralize the homologous serotype responsible for infection. Affected individuals also generate cross-reactive antibodies against heterologous DENV serotypes, which are non-neutralizing. Dengue cross-reactive, non-neutralizing antibodies can enhance infection of Fc receptor bearing cells and, potentially, exacerbate disease. The actual binding sites of human antibody on the DENV particle are not well defined. We characterized the specificity and neutralization potency of polyclonal serum antibodies and memory B-cell derived monoclonal antibodies (hMAbs) from 2 individuals exposed to primary DENV infections. Most DENV-specific hMAbs were serotype cross-reactive and weakly neutralizing. Moreover, many hMAbs bound to the viral pre-membrane protein and other sites on the virus that were not preserved when the viral envelope protein was produced as a soluble, recombinant antigen (rE protein). Nonetheless, by modifying the screening procedure to detect rare antibodies that bound to rE, we were able to isolate and map human antibodies that strongly neutralized the homologous serotype of DENV. Our MAbs results indicate that, in these two individuals exposed to primary DENV infections, a small fraction of the total antibody response was responsible for virus neutralization.


Asunto(s)
Anticuerpos Antivirales/sangre , Virus del Dengue/inmunología , Dengue/inmunología , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Reacciones Cruzadas , Humanos , Factores de Tiempo
13.
Cell Host Microbe ; 8(3): 271-83, 2010 Sep 16.
Artículo en Inglés | MEDLINE | ID: mdl-20833378

RESUMEN

Antibodies protect against homologous Dengue virus (DENV) infection but can precipitate severe dengue by promoting heterotypic virus entry via Fcγ receptors (FcγR). We immortalized memory B cells from individuals after primary or secondary infection and analyzed anti-DENV monoclonal antibodies (mAbs) thus generated. MAbs to envelope (E) protein domain III (DIII) were either serotype specific or cross-reactive and potently neutralized DENV infection. DI/DII- or viral membrane protein prM-reactive mAbs neutralized poorly and showed broad cross-reactivity with the four DENV serotypes. All mAbs enhanced infection at subneutralizing concentrations. Three mAbs targeting distinct epitopes on the four DENV serotypes and engineered to prevent FcγR binding did not enhance infection and neutralized DENV in vitro and in vivo as postexposure therapy in a mouse model of lethal DENV infection. Our findings reveal an unexpected degree of cross-reactivity in human antibodies against DENV and illustrate the potential for an antibody-based therapy to control severe dengue.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Acrecentamiento Dependiente de Anticuerpo , Virus del Dengue/inmunología , Dengue/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Linfocitos B/inmunología , Western Blotting , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Immunoblotting , Ratones , Pruebas de Neutralización , Receptores de IgG/inmunología , Serotipificación , Proteínas no Estructurales Virales
14.
J Gen Virol ; 90(Pt 4): 799-809, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19264660

RESUMEN

The flavivirus envelope glycoprotein (E) is responsible for viral attachment and entry by membrane fusion. Its ectodomain is the primary target of the humoral immune response. In particular, the C-terminal Ig-like domain III of E, which is exposed at the surface of the viral particle, forms an attractive antigen for raising protective monoclonal antibodies (mAb). 9F12, a mouse mAb raised against a dengue virus (DENV) serotype 2 recombinant domain III, cross-reacts with corresponding domains from the other three DENV serotypes and also with West Nile virus. mAb 9F12 binds with nanomolar affinity to a conserved epitope that maps to the viral surface comprising residues 305, 307, 310 and 330 of the E protein. mAb 9F12 neutralizes all four DENV serotypes in plaque reduction assays. We expressed a single-chain Fv from 9F12 that retains the binding activity of the parent mAb. Adsorption and fusion inhibition assays indicate that mAb 9F12 prevents early steps of viral entry. Its virus inhibition activity and broad cross-reactivity makes mAb 9F12 a suitable candidate for optimization and humanization into a therapeutic antibody to treat severe infections by dengue.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Virus del Dengue/clasificación , Virus del Dengue/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Cricetinae , Reacciones Cruzadas , Dengue/inmunología , Virus del Dengue/genética , Mapeo Epitopo , Fusión de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pruebas de Neutralización , Serotipificación , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética
15.
Virology ; 394(2): 175-82, 2009 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-19833371

RESUMEN

Severe dengue virus (DENV) infection is epidemiologically linked to pre-existing anti-DENV antibodies acquired by maternal transfer or primary infection. A possible explanation is that DENV immune complexes evade neutralization by engaging Fcgamma receptors (FcgammaR) on monocytes, natural targets for DENV in humans. Using epitope-matched humanized monoclonal antibodies (mAbs) and stable FcgammaR-transfected CV-1 cells, we found that DENV neutralization by IgG1, IgG3, and IgG4 mAbs was enhanced in high-affinity FcgammaRIA transfectants and diminished in low-affinity FcgammaRIIA transfectants, whereas neutralization by IgG2 mAbs (low-affinity ligands for both FcgammaRs) was diminished equally. In FcgammaR-negative Vero cells, IgG3 mAbs exhibited the strongest neutralizing activity and IgG2, the weakest. Our results demonstrate that DENV neutralization is modulated by the Fc region in an IgG subclass manner, likely through effects on virion and FcgammaR binding. Thus, the IgG antibody subclass profile generated by DENV infection or vaccination may independently influence the magnitude of the neutralizing response.


Asunto(s)
Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , Virus del Dengue/inmunología , Inmunoglobulina G/metabolismo , Receptores de IgG/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/clasificación , Anticuerpos Antivirales/clasificación , Línea Celular , Chlorocebus aethiops , Dengue/inmunología , Dengue/virología , Humanos , Inmunoglobulina G/clasificación , Técnicas In Vitro , Ratones , Pruebas de Neutralización , Pan troglodytes , Receptores de IgG/clasificación , Proteínas Recombinantes de Fusión/inmunología , Células Vero
16.
Nat Struct Mol Biol ; 15(3): 312-7, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18264114

RESUMEN

The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37 degrees C, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the organization of the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Virus del Dengue/química , Virus del Dengue/inmunología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/ultraestructura , Anticuerpos Antivirales/química , Anticuerpos Antivirales/ultraestructura , Sitios de Unión , Microscopía por Crioelectrón , Cristalografía por Rayos X , Virus del Dengue/ultraestructura , Glicoproteínas de Membrana/ultraestructura , Pruebas de Neutralización , Temperatura
17.
J Bacteriol ; 188(11): 3870-7, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16707679

RESUMEN

Haemophilus influenzae type b is a common cause of invasive bacterial disease, especially among children in underdeveloped countries. The type b polysaccharide capsule is a polymer of ribose and ribitol-5-phosphate and is a critical determinant of virulence. Expression of the type b capsule is dependent upon the cap b locus, which consists of three functionally distinct regions, designated regions 1 to 3. Region 3 contains the hcsA and hcsB genes, which share significant homology with genes that have been implicated in encapsulation in other pathogenic bacteria but have unclear functions. In this study, we inactivated hcsA alone, hcsB alone, and both hcsA and hcsB together and examined the effects of these mutations on polysaccharide transport and bacterial virulence properties. Inactivation of hcsA alone resulted in accumulation of polysaccharide in the periplasm and a partial decrease in surface-associated polysaccharide, whereas inactivation of hcsB alone or of both hcsA and hcsB together resulted in accumulation of polysaccharide in the periplasm and complete loss of surface-associated polysaccharide. All mutations eliminated serum resistance and abrogated bacteremia and mortality in neonatal rats. These results indicate that the hcsA and hcsB gene products have complementary functions involved in the transport of polysaccharide across the outer membrane and are essential for virulence.


Asunto(s)
Proteínas Bacterianas/genética , Haemophilus influenzae tipo b/genética , Proteínas de Transporte de Membrana/genética , Polisacáridos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Proteínas del Sistema Complemento/fisiología , Haemophilus influenzae tipo b/patogenicidad , Haemophilus influenzae tipo b/ultraestructura , Humanos , Proteínas de Transporte de Membrana/metabolismo , Microscopía Inmunoelectrónica , Mutagénesis , Fosfolípidos/metabolismo , Virulencia
18.
J Virol ; 80(24): 12149-59, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17035317

RESUMEN

Previous studies have demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral surface of domain III (DIII) of the West Nile virus (WNV) envelope (E) strongly protect against infection in animals. Herein, we observed significantly less efficient neutralization by 89 MAbs that recognized domain I (DI) or II (DII) of WNV E protein. Moreover, in cells expressing Fc gamma receptors, many of the DI- and DII-specific MAbs enhanced infection over a broad range of concentrations. Using yeast surface display of E protein variants, we identified 25 E protein residues to be critical for recognition by DI- or DII-specific neutralizing MAbs. These residues cluster into six novel and one previously characterized epitope located on the lateral ridge of DI, the linker region between DI and DIII, the hinge interface between DI and DII, and the lateral ridge, central interface, dimer interface, and fusion loop of DII. Approximately 45% of DI-DII-specific MAbs showed reduced binding with mutations in the highly conserved fusion loop in DII: 85% of these (34 of 40) cross-reacted with the distantly related dengue virus (DENV). In contrast, MAbs that bound the other neutralizing epitopes in DI and DII showed no apparent cross-reactivity with DENV E protein. Surprisingly, several of the neutralizing epitopes were located in solvent-inaccessible positions in the context of the available pseudoatomic model of WNV. Nonetheless, DI and DII MAbs protect against WNV infection in mice, albeit with lower efficiency than DIII-specific neutralizing MAbs.


Asunto(s)
Especificidad de Anticuerpos/inmunología , Epítopos/genética , Proteínas del Envoltorio Viral/inmunología , Virus del Nilo Occidental/genética , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/genética , Línea Celular , Mapeo Epitopo , Ratones , Mutación/genética , Pruebas de Neutralización , Estructura Terciaria de Proteína/genética , Receptores de IgG/inmunología , Proteínas del Envoltorio Viral/genética , Virus del Nilo Occidental/inmunología , Levaduras
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