RESUMEN
The envelope (E) protein of dengue virus (DENV) is composed of three domains (EDI, EDII, EDIII) and is the main target of neutralizing antibodies. Many monoclonal antibodies that bind EDIII strongly neutralize DENV. However in vitro studies indicate that anti-EDIII antibodies contribute little to the neutralizing potency of human DENV-immune serum. In this study, we assess the role of anti-EDIII antibodies in mouse and human DENV-immune serum in neutralizing or enhancing DENV infection in mice. We demonstrate that EDIII-depleted human DENV-immune serum was protective against homologous DENV infection in vivo. Although EDIII-depleted DENV-immune mouse serum demonstrated decreased neutralization potency in vitro, reduced protection in some organs, and enhanced disease in vivo, administration of increased volumes of EDIII-depleted serum abrogated these effects. These data indicate that anti-EDIII antibodies contribute to protection and minimize enhancement when present, but can be replaced by neutralizing antibodies targeting other epitopes on the dengue virion.
Asunto(s)
Anticuerpos Antivirales/inmunología , Dengue/inmunología , Dengue/prevención & control , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Línea Celular , Dengue/virología , Virus del Dengue/química , Virus del Dengue/genética , Virus del Dengue/inmunología , Femenino , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Estructura Terciaria de Proteína , Especificidad de la Especie , Proteínas del Envoltorio Viral/genéticaRESUMEN
Dengue is a mosquito-borne flavivirus that is spreading at an unprecedented rate and has developed into a major health and economic burden in over 50 countries. Even though infected individuals develop potent and long-lasting serotype-specific neutralizing antibodies (Abs), the epitopes engaged by human neutralizing Abs have not been identified. Here, we demonstrate that the dengue virus (DENV)-specific serum Ab response in humans consists of a large fraction of cross-reactive, poorly neutralizing Abs and a small fraction of serotype-specific, potently inhibitory Abs. Although many mouse-generated, strongly neutralizing monoclonal antibodies (mAbs) recognize epitopes that are present on recombinant DENV envelope (E) proteins, unexpectedly, the majority of neutralizing Abs in human immune sera bound to intact virions but not to the ectodomain of purified soluble E proteins. These conclusions with polyclonal Abs were confirmed with newly generated human mAbs derived from DENV-immune individuals. Two of three strongly neutralizing human mAbs bound to E protein epitopes that were preserved on the virion but not on recombinant E (rE) protein. We propose that humans produce Abs that neutralize DENV infection by binding a complex, quaternary structure epitope that is expressed only when E proteins are assembled on a virus particle. Mapping studies indicate that this epitope has a footprint that spans adjacent E protein dimers and includes residues at the hinge between domains I and II of E protein. These results have significant implications for the DENV Ab and vaccine field.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunología , Dengue/inmunología , Epítopos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , Especificidad de Anticuerpos/inmunología , Chlorocebus aethiops , Dengue/virología , Virus del Dengue/genética , Virus del Dengue/metabolismo , Ensayo de Inmunoadsorción Enzimática , Epítopos/metabolismo , Humanos , Sueros Inmunes/inmunología , Macaca mulatta , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Pruebas de Neutralización , Unión Proteica/inmunología , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Células Vero , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Virión/inmunologíaRESUMEN
Humans develop polyclonal, serotype-specific neutralizing antibody responses after dengue virus (DENV) infection. Many mouse antibodies that neutralize DENV bind to the lateral ridge or A strand epitopes on domain III of the viral envelope (EDIII) protein. It has been assumed that these epitopes are also the main target of human neutralizing antibodies. Using recombinant dengue serotype 2 viruses with altered EDIII epitopes, we demonstrate that EDIII epitopes are not the main target of human neutralizing antibody.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Virus del Dengue/inmunología , Dengue/inmunología , Epítopos/inmunología , Sueros Inmunes/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/inmunología , Dengue/virología , Virus del Dengue/química , Virus del Dengue/genética , Epítopos/genética , Humanos , Ratones , Estructura Terciaria de Proteína , Proteínas del Envoltorio Viral/genéticaRESUMEN
The number of cases and severity of disease associated with dengue infection in Sri Lanka has been increasing since 1989, when the first epidemic of dengue hemorrhagic fever was recorded. We identified a new dengue virus 1 strain circulating in Sri Lanka that coincided with the 2009 dengue epidemic.
Asunto(s)
Virus del Dengue/genética , Dengue/epidemiología , Genotipo , Dengue Grave/epidemiología , Dengue/virología , Virus del Dengue/clasificación , Virus del Dengue/aislamiento & purificación , Genes Virales , Humanos , Filogenia , ARN Viral/química , Dengue Grave/virología , Sri Lanka/epidemiologíaRESUMEN
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 TiempoRESUMEN
Dengue viruses (DENV) are the causative agents of dengue fever (DF) and dengue hemorrhagic fever (DHF). Here we review the current state of knowledge about the human antibody response to dengue and identify important knowledge gaps. A large body of work has demonstrated that antibodies can neutralize or enhance DENV infection. Investigators have mainly used mouse monoclonal antibodies (MAbs) to study interactions between DENV and antibodies. These studies indicate that antibody neutralization of DENVs is a "multi-hit" phenomenon that requires the binding of multiple antibodies to neutralize a virion. The most potently neutralizing mouse MAbs bind to surface exposed epitopes on domain III of the dengue envelope (E) protein. One challenge facing the dengue field now is to extend these studies with mouse MAbs to better understand the human antibody response. The human antibody response is complex as it involves a polyclonal response to primary and secondary infections with 4 different DENV serotypes. Here we review studies conducted with immune sera and MAbs isolated from people exposed to dengue infections. Most dengue-specific antibodies in human immune sera are weakly neutralizing and bind to multiple DENV serotypes. The human antibodies that potently and type specifically neutralize DENV represent a small fraction of the total DENV-specific antibody response. Moreover, these neutralizing antibodies appear to bind to novel epitopes including complex, quaternary epitopes that are only preserved on the intact virion. These studies establish that human and mouse antibodies recognize distinct epitopes on the dengue virion. The leading theory proposed to explain the increased risk of severe disease in secondary cases is antibody dependent enhancement (ADE), which postulates that weakly neutralizing antibodies from the first infection bind to the second serotype and enhance infection of FcγR bearing myeloid cells such as monocytes and macrophages. Here we review results from human, animal and cell culture studies relevant to the ADE hypothesis. By understanding how human antibodies neutralize or enhance DENV, it will be possible to better evaluate existing vaccines and develop the next generation of novel vaccines.
Asunto(s)
Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunología , Dengue/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Formación de Anticuerpos/inmunología , Antígenos Virales/inmunología , Linfocitos B/inmunología , Epítopos/inmunología , Humanos , Ratones , Dengue Grave/inmunologíaRESUMEN
Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge "type specific" epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Virus del Dengue/genética , Virus del Dengue/inmunología , Dengue/inmunología , Dengue/virología , Aedes/citología , Animales , Anticuerpos Monoclonales/metabolismo , Chlorocebus aethiops , Virus del Dengue/clasificación , Mapeo Epitopo , Epítopos/inmunología , Genotipo , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Pruebas de Neutralización , Unión Proteica/inmunología , Células U937 , Células Vero , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Recent emergence of dengue hemorrhagic fever in the Indian subcontinent has been well documented in Sri Lanka. We compare recent (2003-2006) and past (1980-1997) dengue surveillance data for Sri Lanka. The 4 dengue virus (DENV) serotypes have been cocirculating in Sri Lanka for >30 years. Over this period, a new genotype of DENV-1 has replaced an old genotype. Moreover, new clades of DENV-3 genotype III viruses have replaced older clades. Emergence of new clades of DENV-3 in 1989 and 2000 coincided with abrupt increases in the number of reported dengue cases, implicating this serotype in severe epidemics. In 1980-1997, most reported dengue cases were in children. Recent epidemics have been characterized by many cases in children and adults. Changes in local transmission dynamics and genetic changes in DENV-3 are likely increasing emergence of severe dengue epidemics in Sri Lanka.