RESUMEN
Understanding adaptive immunity to SARS-CoV-2 is important for vaccine development, interpreting coronavirus disease 2019 (COVID-19) pathogenesis, and calibration of pandemic control measures. Using HLA class I and II predicted peptide "megapools," circulating SARS-CoV-2-specific CD8+ and CD4+ T cells were identified in â¼70% and 100% of COVID-19 convalescent patients, respectively. CD4+ T cell responses to spike, the main target of most vaccine efforts, were robust and correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titers. The M, spike, and N proteins each accounted for 11%-27% of the total CD4+ response, with additional responses commonly targeting nsp3, nsp4, ORF3a, and ORF8, among others. For CD8+ T cells, spike and M were recognized, with at least eight SARS-CoV-2 ORFs targeted. Importantly, we detected SARS-CoV-2-reactive CD4+ T cells in â¼40%-60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating "common cold" coronaviruses and SARS-CoV-2.
Asunto(s)
Betacoronavirus/fisiología , Infecciones por Coronavirus/inmunología , Epítopos de Linfocito T , Neumonía Viral/inmunología , Betacoronavirus/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , COVID-19 , Vacunas contra la COVID-19 , Convalecencia , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/virología , Reacciones Cruzadas , Humanos , Leucocitos Mononucleares/inmunología , Pandemias , Neumonía Viral/sangre , Neumonía Viral/metabolismo , Neumonía Viral/virología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismo , Proteínas Virales/metabolismo , Vacunas Virales/inmunologíaRESUMEN
The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.
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Betacoronavirus/genética , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Neumonía Viral/patología , Neumonía Viral/virología , Sistema Respiratorio/virología , Genética Inversa/métodos , Anciano , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Betacoronavirus/inmunología , Betacoronavirus/patogenicidad , COVID-19 , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/terapia , Fibrosis Quística/patología , ADN Recombinante , Femenino , Furina/metabolismo , Humanos , Inmunización Pasiva , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Masculino , Persona de Mediana Edad , Mucosa Nasal/metabolismo , Mucosa Nasal/patología , Mucosa Nasal/virología , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/inmunología , Sistema Respiratorio/patología , SARS-CoV-2 , Serina Endopeptidasas/metabolismo , Células Vero , Virulencia , Replicación Viral , Sueroterapia para COVID-19RESUMEN
We currently have an incomplete understanding of why only a fraction of human antibodies that bind to flaviviruses block infection of cells. Here we define the footprint of a strongly neutralizing human monoclonal antibody (mAb G9E) with Zika virus (ZIKV) by both X-ray crystallography and cryo-electron microscopy. Flavivirus envelope (E) glycoproteins are present as homodimers on the virion surface, and G9E bound to a quaternary structure epitope spanning both E protomers forming a homodimer. As G9E mainly neutralized ZIKV by blocking a step after viral attachment to cells, we tested if the neutralization mechanism of G9E was dependent on the mAb cross-linking E molecules and blocking low-pH triggered conformational changes required for viral membrane fusion. We introduced targeted mutations to the G9E paratope to create recombinant antibodies that bound to the ZIKV envelope without cross-linking E protomers. The G9E paratope mutants that bound to a restricted epitope on one protomer poorly neutralized ZIKV compared to the wild-type mAb, demonstrating that the neutralization mechanism depended on the ability of G9E to cross-link E proteins. In cell-free low pH triggered viral fusion assay, both wild-type G9E, and epitope restricted paratope mutant G9E bound to ZIKV but only the wild-type G9E blocked fusion. We propose that, beyond antibody binding strength, the ability of human antibodies to cross-link E-proteins is a critical determinant of flavivirus neutralization potency.
Asunto(s)
Infección por el Virus Zika , Virus Zika , Humanos , Virus Zika/genética , Epítopos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Subunidades de Proteína , Microscopía por Crioelectrón , Proteínas del Envoltorio Viral/genética , Anticuerpos MonoclonalesRESUMEN
Dengue viruses (DENV serotypes 1-4) and Zika virus (ZIKV) are related flaviviruses that continue to be a public health concern, infecting hundreds of millions of people annually. The traditional live-attenuated virus vaccine approach has been challenging for the four DENV serotypes because of the need to achieve balanced replication of four independent vaccine components. Subunit vaccines represent an alternative approach that may circumvent problems inherent with live-attenuated DENV vaccines. In mature virus particles, the envelope (E) protein forms a homodimer that covers the surface of the virus and is the major target of neutralizing antibodies. Many neutralizing antibodies bind to quaternary epitopes that span across both E proteins in the homodimer. For soluble E (sE) protein to be a viable subunit vaccine, the antigens should be easy to produce and retain quaternary epitopes recognized by neutralizing antibodies. However, WT sE proteins are primarily monomeric at conditions relevant for vaccination and exhibit low expression yields. Previously, we identified amino acid mutations that stabilize the sE homodimer from DENV2 and dramatically raise expression yields. Here, we tested whether these same mutations raise the stability of sE from other DENV serotypes and ZIKV. We show that the mutations raise thermostability for sE from all the viruses, increase production yields from 4-fold to 250-fold, stabilize the homodimer, and promote binding to dimer-specific neutralizing antibodies. Our findings suggest that these sE variants could be valuable resources in the efforts to develop effective subunit vaccines for DENV serotypes 1 to 4 and ZIKV.
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Virus del Dengue , Vacunas de Subunidad , Proteínas del Envoltorio Viral , Vacunas Virales , Virus Zika , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Reacciones Cruzadas , Dengue/prevención & control , Virus del Dengue/genética , Epítopos , Humanos , Mutación , Vacunas Atenuadas , Vacunas de Subunidad/genética , Proteínas del Envoltorio Viral/genética , Vacunas Virales/genética , Virus Zika/genética , Infección por el Virus Zika/prevención & controlRESUMEN
Zika virus (ZIKV), a mosquito-transmitted flavivirus, caused a large epidemic in Latin America between 2015 and 2017. Effective ZIKV vaccines and treatments are urgently needed to prevent future epidemics and severe disease sequelae. People infected with ZIKV develop strongly neutralizing antibodies linked to viral clearance and durable protective immunity. To understand the mechanisms of protective immunity and to support the development of ZIKV vaccines, we characterize here a strongly neutralizing antibody, B11F, isolated from a patient who recovered from ZIKV. Our results indicate that B11F targets a complex epitope on the virus that spans domains I and III of the envelope glycoprotein. While previous studies point to quaternary epitopes centered on domain II of the ZIKV E glycoprotein as targets of strongly neutralizing and protective human antibodies, we uncover a new site spanning domains I and III as a target of strongly neutralizing human antibodies.IMPORTANCE People infected with Zika virus develop durable neutralizing antibodies that prevent repeat infections. In the current study, we characterize a ZIKV-neutralizing human monoclonal antibody isolated from a patient after recovery. Our studies establish a novel site on the viral envelope that is targeted by human neutralizing antibodies. Our results are relevant to understanding how antibodies block infection and to guiding the design and evaluation of candidate vaccines.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antivirales , Epítopos , Proteínas del Envoltorio Viral , Infección por el Virus Zika , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Chlorocebus aethiops , Epítopos/inmunología , Humanos , Unión Proteica , Dominios Proteicos , Células Vero , Envoltura Viral/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Virus Zika/inmunología , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virologíaRESUMEN
The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses of humans. The interactions between DENVs and the human host that lead to asymptomatic, mild, or severe disease are poorly understood, in part, because laboratory models are poor surrogates for human DENV disease. Virologists are interested in how the properties of DENVs replicating in people compare with virions propagated on laboratory cell lines, which are widely used for research and vaccine development. Using clinical samples from a DENV type 1 epidemic in Sri Lanka and new ultrasensitive assays, we compared the properties of DENVs in human plasma and after one passage on laboratory cell lines. DENVs in plasma were 50- to 700-fold more infectious than cell culture-grown viruses. DENVs produced by laboratory cell lines were structurally immature and hypersensitive to neutralization by human antibodies compared with DENVs circulating in people. Human plasma and cell culture-derived virions had identical genome sequences, indicating that these phenotypic differences were due to the mature state of plasma virions. Several dengue vaccines are under development. Recent studies indicate that vaccine-induced antibodies that neutralized DENVs in cell culture assays were not sufficient for protecting people from DENV infections. Our results about structural differences between DENVs produced in humans versus cell lines may be key to understanding vaccine failure and developing better models for vaccine evaluation.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunología , Dengue/inmunología , Animales , Células Cultivadas , Chlorocebus aethiops , Reacciones Cruzadas , Dengue/epidemiología , Dengue/virología , Virus del Dengue/genética , Virus del Dengue/patogenicidad , Genoma Viral/genética , Humanos , Reacción en Cadena de la Polimerasa , Sri Lanka/epidemiología , Células VeroRESUMEN
BACKGROUND: Neurodevelopmental outcomes of asymptomatic children exposed to Zika virus (ZIKV) in utero are not well characterized. METHODS: We prospectively followed 129 newborns without evidence of congenital Zika syndrome (CZS) up to 24 months of age. Participants were classified as ZIKV exposed or ZIKV unexposed. The Mullen Scales of Early Learning (MSEL) was administered in the participants' homes at 6, 12, 15, 18, 21, and 24 months of age by trained psychologists. Sociodemographic data, medical history, and infant anthropometry at birth were collected at each home visit. Our primary outcome was the Mullen Early Learning Composite Score (ECL) at 24 months of age between our 2 exposure groups. Secondary outcomes were differences in MSEL subscales over time and at 24 months. RESULTS: Of 129 infants in whom exposure status could be ascertained, 32 (24.8%) met criteria for in utero ZIKV exposure and 97 (75.2%) did not. There were no differences in maternal age, maternal educational attainment, birthweight, or gestational age at birth between the 2 exposure groups. The adjusted means and standard errors (SEs) for the ELC score between the ZIKV-exposed children compared to ZIKV-unexposed children were 91.4 (SE, 3.1) vs 96.8 (SE, 2.4) at 12 months and 93.3 (SE, 2.9) vs 95.9 (SE, 2.3) at 24 months. In a longitudinal mixed model, infants born to mothers with an incident ZIKV infection (P = .01) and low-birthweight infants (<2500 g) (P = .006) had lower composite ECL scores. CONCLUSIONS: In this prospective cohort of children without CZS, children with in utero ZIKV exposure had lower neurocognitive scores at 24 months.
Asunto(s)
Complicaciones Infecciosas del Embarazo , Infección por el Virus Zika , Virus Zika , Niño , Femenino , Humanos , Lactante , Recién Nacido , Nicaragua/epidemiología , Embarazo , Complicaciones Infecciosas del Embarazo/epidemiología , Estudios Prospectivos , Infección por el Virus Zika/epidemiologíaRESUMEN
Zika virus (ZIKV) is a member of the Flaviviridae family, which includes other clinically notable viruses such as the 4 dengue virus serotypes (DENV-1-4). Distinguishing DENVs from ZIKV using the established serologic assays widely used for monitoring DENV transmission is difficult because of antibody cross-reactivity between these closely related flaviviruses. We describe a modified and improved recombinant envelope domain III-based serologic assay for detecting ZIKV type-specific antibodies in regions with endemic DENV transmission. When the assay was used to measure ZIKV seroprevalence in 2017 among children 9-14 years of age living in a region of the Philippines with endemic DENV transmission, we observed a ZIKV seroprevalence of 18%. Investigators should consider using the ZIKV envelope domain III-based assay, which is simple and readily adaptable for use in standard clinical and public health laboratories, to assess ZIKV seroprevalence in areas with endemic DENV transmission.
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Virus del Dengue , Dengue , Infección por el Virus Zika , Virus Zika , Anticuerpos Antivirales , Niño , Reacciones Cruzadas , Dengue/diagnóstico , Dengue/epidemiología , Virus del Dengue/genética , Humanos , Filipinas/epidemiología , Estudios Seroepidemiológicos , Virus Zika/genética , Infección por el Virus Zika/diagnóstico , Infección por el Virus Zika/epidemiologíaRESUMEN
BACKGROUND: The natural antibody responses to B-cell epitopes from dengue structural proteins were assessed using immune sera from people having well-defined past dengue infections with one of the four serotypes. METHOD: Based on an immune-computational analysis previously conducted, nineteen epitopes from the envelope (E) and eight epitopes from pre-membrane (prM), which were more than 50% conserved across all the four DENV serotypes, were selected. Peptides to represent these B-cell epitopes were obtained from commercially available arrays, and were subjected to enzyme linked immunosorbent assay with sera obtained from dengue seropositive healthy volunteers (DENV1 n = 12: DENV2 n = 12: DENV3 n = 12 and DENV4 n = 12), and 10 dengue seronegative healthy volunteers from Sri Lanka. The cut-off value for the positive antibody response was set by taking the mean response of a peptide to the negative sera plus three standard deviations. The peptides (N = 7) showing the broad immune responses were used to generate antibodies in three mice (Balb/c) batches. The mice antisera were then subjected to microneutralization assays against all the four DENV serotypes. An EC50 viral neutralization ≥ 40 times the serum dilution was considered as neutralizing. RESULTS: Five of the E-peptide and two prM peptides were recognised by most individuls exposed to infections with each of the four serotypes, showing a serotype cross-reactive broad antibody response. The mice immune sera against the peptides representing the five E protein epitopes neutralized all the four DENV serotypes. Two of these five epitopes are from the Domain II, whereas one of them includes the whole bc-loop region. CONCLUSION: The antibody responses of highly conserved epitopes across the serotypes, were broadly responsive with sera of all four DENV serotypes collected from individuals infected with only one DENV serotype. Weakly conserved epitopes showed rather specific antibody responses dominated by one or few serotypes.
Asunto(s)
Biología Computacional/métodos , Virus del Dengue/fisiología , Dengue/inmunología , Epítopos de Linfocito B/genética , Proteínas del Envoltorio Viral/genética , Proteínas Virales/genética , Animales , Anticuerpos Neutralizantes/metabolismo , Secuencia Conservada/genética , Reacciones Cruzadas , Mapeo Epitopo , Epítopos de Linfocito B/inmunología , Voluntarios Sanos , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales/inmunologíaRESUMEN
Dengue virus (DENV) is responsible for the most prevalent and significant arthropod-borne viral infection of humans. The leading DENV vaccines are based on tetravalent live-attenuated virus platforms. In practice, it has been challenging to induce balanced and effective responses to each of the four DENV serotypes because of differences in the replication efficiency and immunogenicity of individual vaccine components. Unlike live vaccines, tetravalent DENV envelope (E) protein subunit vaccines are likely to stimulate balanced immune responses, because immunogenicity is replication independent. However, E protein subunit vaccines have historically performed poorly, in part because the antigens utilized were mainly monomers that did not display quaternary-structure epitopes found on E dimers and higher-order structures that form the viral envelope. In this study, we compared the immunogenicity of DENV2 E homodimers and DENV2 E monomers. The stabilized DENV2 homodimers, but not monomers, were efficiently recognized by virus-specific and flavivirus cross-reactive potently neutralizing antibodies that have been mapped to quaternary-structure epitopes displayed on the viral surface. In mice, the dimers stimulated 3-fold-higher levels of virus-specific neutralizing IgG that recognized epitopes different from those recognized by lower-level neutralizing antibodies induced by monomers. The dimer induced a stronger E domain I (EDI)- and EDII-targeted response, while the monomer antigens stimulated an EDIII epitope response and induced fusion loop epitope antibodies that are known to facilitate antibody-dependent enhancement (ADE). This study shows that DENV E subunit antigens that have been designed to mimic the structural organization of the viral surface are better vaccine antigens than E protein monomers.IMPORTANCE Dengue virus vaccine development is particularly challenging because vaccines have to provide protection against four different dengue virus stereotypes. The leading dengue virus vaccine candidates in clinical testing are all based on live-virus vaccine platforms and struggle to induce balanced immunity. Envelope subunit antigens have the potential to overcome these limitations but have historically performed poorly as vaccine antigens, because the versions tested previously were presented as monomers and not in their natural dimer configuration. This study shows that the authentic presentation of DENV2 E-based subunits has a strong impact on antibody responses, underscoring the importance of mimicking the complex protein structures that are found on DENV particle surfaces when designing subunit vaccines.
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Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antivirales/biosíntesis , Vacunas contra el Dengue/administración & dosificación , Dengue/prevención & control , Epítopos/inmunología , Vacunación/métodos , Proteínas del Envoltorio Viral/inmunología , Animales , Acrecentamiento Dependiente de Anticuerpo , Chlorocebus aethiops , Reacciones Cruzadas , Dengue/inmunología , Dengue/patología , Dengue/virología , Vacunas contra el Dengue/genética , Vacunas contra el Dengue/inmunología , Virus del Dengue/efectos de los fármacos , Virus del Dengue/genética , Virus del Dengue/inmunología , Modelos Animales de Enfermedad , Epítopos/química , Epítopos/genética , Femenino , Células HEK293 , Humanos , Inmunogenicidad Vacunal , Ratones , Ratones Endogámicos BALB C , Isoformas de Proteínas/administración & dosificación , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Multimerización de Proteína/efectos de los fármacos , Vacunas de Subunidad , Células Vero , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/genéticaRESUMEN
The short-chain flavin reductases BorF and AbeF reduce FAD to FADH2, which is then used by flavin-dependent halogenases (BorH and AbeH respectively) to regioselectively chlorinate tryptophan in the biosynthesis of indolotryptoline natural products. Recombinant AbeF and BorF were overexpressed and purified as homodimers from E. coli, and copurified with substoichiometric amounts of FAD, which could be easily removed. AbeF and BorF can reduce FAD, FMN, and riboflavin in vitro and are selective for NADH over NADPH. Initial velocity studies in the presence and absence of inhibitors showed that BorF proceeds by a sequential ordered kinetic mechanism in which FAD binds first, while AbeF follows a random-ordered sequence of substrate binding. Fluorescence quenching experiments verified that NADH does not bind BorF in the absence of FAD, and that both AbeF and BorF bind FAD with higher affinity than FADH2. pH-rate profiles of BorF and AbeF were bell-shaped with maximum kcat at pH 7.5, and site-directed mutagenesis of BorF implicated His160 and Arg38 as contributing to the catalytic activity and the pH dependence.
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Proteínas de Escherichia coli/química , Escherichia coli/enzimología , FMN Reductasa/química , Mononucleótido de Flavina/química , Flavina-Adenina Dinucleótido/química , Riboflavina/química , CinéticaRESUMEN
In a Nicaraguan population-based cohort, SARS-CoV-2 seroprevalence reached 28% in the first 6 months of the country's epidemic and reached 35% 6 months later. Immune waning was uncommon. Individuals with a seropositive household member were over three times as likely to be seropositive themselves, suggesting the importance of household transmission.
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COVID-19/epidemiología , SARS-CoV-2/aislamiento & purificación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nicaragua/epidemiología , Prevalencia , Estudios Seroepidemiológicos , Población Urbana/estadística & datos numéricos , Adulto JovenRESUMEN
In 2017, a dengue epidemic of unexpected magnitude occurred in Sri Lanka. A total of 186,101 suspected cases and 440 dengue-related deaths occurred. We conducted a comprehensive analysis of this epidemic by comparing national surveillance data for 2017 with data from the preceding 5 years. In all Sri Lanka districts, dengue incidence in 2017 increased significantly over incidence during the previous 5 years. Older schoolchildren and young adults were more clinically symptomatic than those at extremes of age. Limited virologic surveillance showed the dominant circulating variant was dengue virus type 2 cosmopolitan genotype in the most affected district. One quarter of total annual cases were reported 5 weeks after the southwest monsoon started. Changes in vector abundance were not predictive of the increased incidence. Direct government expenditures on dengue control activities in 2017 were US $12.7 million. The lessons learned from this outbreak are useful for other tropical nations facing increasing dengue incidence.
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Virus del Dengue , Dengue , Epidemias , Dengue Grave , Niño , Dengue/epidemiología , Virus del Dengue/genética , Humanos , Dengue Grave/diagnóstico , Dengue Grave/epidemiología , Sri Lanka/epidemiología , Adulto JovenRESUMEN
Dengue virus (DENV) infection causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome. It is estimated that a third of the world's population is at risk for infection, with an estimated 390 million infections annually. Dengue virus serotype 2 (DENV2) causes severe epidemics, and the leading tetravalent dengue vaccine has lower efficacy against DENV2 compared to the other 3 serotypes. In natural DENV2 infections, strongly neutralizing type-specific antibodies provide protection against subsequent DENV2 infection. While the epitopes of some human DENV2 type-specific antibodies have been mapped, it is not known if these are representative of the polyclonal antibody response. Using structure-guided immunogen design and reverse genetics, we generated a panel of recombinant viruses containing amino acid alterations and epitope transplants between different serotypes. Using this panel of recombinant viruses in binding, competition, and neutralization assays, we have finely mapped the epitopes of three human DENV2 type-specific monoclonal antibodies, finding shared and distinct epitope regions. Additionally, we used these recombinant viruses and polyclonal sera to dissect the epitope-specific responses following primary DENV2 natural infection and monovalent vaccination. Our results demonstrate that antibodies raised following DENV2 infection or vaccination circulate as separate populations that neutralize by occupying domain III and domain I quaternary epitopes. The fraction of neutralizing antibodies directed to different epitopes differs between individuals. The identification of these epitopes could potentially be harnessed to evaluate epitope-specific antibody responses as correlates of protective immunity, potentially improving vaccine design.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Virus del Dengue/inmunología , Epítopos/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Formación de Anticuerpos , Especificidad de Anticuerpos , Células Cultivadas , Chlorocebus aethiops , Reacciones Cruzadas , Dengue/inmunología , Dengue/virología , Vacunas contra el Dengue/inmunología , Epítopos/química , Epítopos/inmunología , Humanos , Estructura Cuaternaria de Proteína , Serogrupo , Vacunación , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Zika virus (ZIKV) constitutes an increasing public health problem. Previous studies have shown that CD8+ T cells play an important role in ZIKV-specific protective immunity. We have previously defined antigenic targets of the ZIKV-specific CD8+ T cell response in humans. In this study, we characterized the quality and phenotypes of these responses by a combined use of flow cytometry and transcriptomic methods, using PBMCs from donors deriving from different geographical locations collected in the convalescent phase of infection. We show that ZIKV-specific CD8+ T cells are characterized by a polyfunctional IFN-γ signature with upregulation of TNF-α, TNF receptors, and related activation markers, such as CD69, as well as a cytotoxic signature characterized by strong upregulation of GZMB and CRTAM. The signature is stable and not influenced by previous dengue virus exposure, geographical location, or time of sample collection postinfection. To our knowledge, this work elucidates the first in-depth characterization of human CD8+ T cells responding to ZIKV infection.
Asunto(s)
Linfocitos T CD8-positivos/fisiología , Infección por el Virus Zika/inmunología , Virus Zika/fisiología , Antígenos Virales/inmunología , Células Cultivadas , Citotoxicidad Inmunológica , Epítopos de Linfocito T/inmunología , Perfilación de la Expresión Génica , Granzimas/genética , Humanos , Inmunoglobulinas/genética , Inmunofenotipificación , Interferón gamma/genética , Receptores del Factor de Necrosis Tumoral/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: Dengue virus is an emerging mosquito-borne flavivirus responsible for considerable morbidity and mortality worldwide. The Division of Intramural Research, National Institute of Allergy and Infectious Diseases of the US National Institutes of Health (NIH) has developed live attenuated vaccines to each of the 4 serotypes of dengue virus (DENV1-4). While overall levels of DENV neutralizing antibodies (nAbs) in humans have been correlated with protection, these correlations vary depending on DENV serotype, prevaccination immunostatus, age, and study site. By combining both the level and molecular specificity of nAbs to each serotype, it may be possible to develop more robust correlates that predict long-term outcome. METHODS: Using depletions and recombinant chimeric epitope transplant DENVs, we evaluate the molecular specificity and mapped specific epitopes and antigenic regions targeted by vaccine-induced nAbs in volunteers who received the NIH monovalent vaccines against each DENV serotype. RESULTS: After monovalent vaccination, subjects developed high levels of nAbs that mainly targeted epitopes that are unique (type-specific) to each DENV serotype. The DENV1, 2, and 4 monovalent vaccines induced type-specific nAbs directed to quaternary structure envelope epitopes known to be targets of strongly neutralizing antibodies induced by wild-type DENV infections. CONCLUSIONS: Our results reported here on the molecular specificity of NIH vaccine-induced antibodies enable new strategies, beyond the absolute levels of nAbs, for determining correlates and mechanisms of protective immunity.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Dengue/inmunología , Epítopos/inmunología , Secuencia de Aminoácidos , Dengue/virología , Mapeo Epitopo/métodos , Humanos , National Institutes of Health (U.S.) , Serogrupo , Estados Unidos , Vacunación/métodos , Vacunas Atenuadas/inmunología , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
The spread of dengue (DENV) and Zika virus (ZIKV) is a major public health concern. The primary target of antibodies that neutralize DENV and ZIKV is the envelope (E) glycoprotein, and there is interest in using soluble recombinant E (sRecE) proteins as subunit vaccines. However, the most potent neutralizing antibodies against DENV and ZIKV recognize epitopes on the virion surface that span two or more E proteins. Therefore, to create effective DENV and ZIKV vaccines, presentation of these quaternary epitopes may be necessary. The sRecE proteins from DENV and ZIKV crystallize as native-like dimers, but studies in solution suggest that these dimers are marginally stable. To better understand the challenges associated with creating stable sRecE dimers, we characterized the thermostability of sRecE proteins from ZIKV and three DENV serotypes, DENV2-4. All four proteins irreversibly unfolded at moderate temperatures (46-53 °C). At 23 °C and low micromolar concentrations, DENV2 and ZIKV were primarily dimeric, and DENV3-4 were primarily monomeric, whereas at 37 °C, all four proteins were predominantly monomeric. We further show that the dissociation constant for DENV2 dimerization is very temperature-sensitive, ranging from <1 µm at 25 °C to 50 µm at 41 °C, due to a large exothermic enthalpy of binding of -79 kcal/mol. We also found that quaternary epitope antibody binding to DENV2-4 and ZIKV sRecE is reduced at 37 °C. Our observation of reduced sRecE dimerization at physiological temperature highlights the need for stabilizing the dimer as part of its development as a subunit vaccine.
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Virus del Dengue/química , Multimerización de Proteína , Proteínas del Envoltorio Viral/química , Virus Zika/química , Temperatura Corporal , Dengue/virología , Humanos , Estabilidad Proteica , Proteínas Recombinantes/química , Vacunas de Subunidad/química , Vacunas Virales/química , Infección por el Virus Zika/virologíaRESUMEN
Epidemics of dengue, Zika, and other arboviral diseases are increasing in frequency and severity. Current efforts to rapidly identify and manage these epidemics are limited by the short diagnostic window in acute infection, the extensive serologic cross-reactivity among flaviviruses, and the lack of point-of-care diagnostic tools to detect these viral species in primary care settings. The Partnership for Dengue Control organized a workshop to review the current landscape of Flavivirus diagnostic tools, identified current gaps, and developed strategies to accelerate the adoption of promising novel technologies into national programs. The rate-limiting step to bringing new diagnostic tools to the market is access to reference materials and well-characterized clinical samples to facilitate performance evaluation. We suggest the creation of an international laboratory-response consortium for flaviviruses with a decentralized biobank of well-characterized samples to facilitate assay validation. Access to proficiency panels are needed to ensure quality control, in additional to in-country capacity building.
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Anticuerpos Antivirales/sangre , Dengue/diagnóstico , Infección por el Virus Zika/diagnóstico , Anticuerpos Antivirales/inmunología , Seguridad de Productos para el Consumidor , Dengue/historia , Dengue/virología , Virus del Dengue/genética , Virus del Dengue/inmunología , Virus del Dengue/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/historia , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/tendencias , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Vigilancia de la Población , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/historia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/tendencias , Sensibilidad y Especificidad , Virus Zika/genética , Virus Zika/inmunología , Virus Zika/aislamiento & purificación , Infección por el Virus Zika/historia , Infección por el Virus Zika/virologíaRESUMEN
Background: Dengue virus serotypes 1-4 (DENV-1-4) are the most common vector-borne viral pathogens of humans and the etiological agents of dengue fever and dengue hemorrhagic syndrome. A live-attenuated tetravalent dengue vaccine (TDV) developed by Takeda Vaccines has recently progressed to phase 3 safety and efficacy evaluation. Methods: We analyzed the qualitative features of the neutralizing antibody (nAb) response induced in naive and DENV-immune individuals after TDV administration. Using DENV-specific human monoclonal antibodies (mAbs) and recombinant DENV displaying different serotype-specific Ab epitopes, we mapped the specificity of TDV-induced nAbs against DENV-1-3. Results: Nearly all subjects had high levels of DENV-2-specific nAbs directed to epitopes centered on domain III of the envelope protein. In some individuals, the vaccine induced nAbs that tracked with a DENV-1-specific neutralizing epitope centered on domain I of the envelope protein. The vaccine induced binding Abs directed to a DENV-3 type-specific neutralizing epitope, but findings of mapping of DENV-3 type-specific nAbs were inconclusive. Conclusion: Here we provide qualitative measures of the magnitude and epitope specificity of the nAb responses to TDV. This information will be useful for understanding the performance of TDV in clinical trials and for identifying correlates of protective immunity.
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Anticuerpos Antivirales/sangre , Formación de Anticuerpos/inmunología , Dengue Grave/sangre , Dengue Grave/inmunología , Vacunas Atenuadas/inmunología , Adolescente , Adulto , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Línea Celular Tumoral , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Método Doble Ciego , Femenino , Humanos , Inmunidad/inmunología , Masculino , Persona de Mediana Edad , Células U937 , Vacunación/métodos , Adulto JovenRESUMEN
Background: The 4 dengue virus serotypes (DENV1-4) and Zika virus (ZIKV) are related mosquito-borne flaviviruses of major importance globally. While monoclonal antibodies and plasma from DENV-immune donors can neutralize or enhance ZIKV in vitro and in small-animal models, and vice versa, the extent, duration, and significance of cross-reactivity in humans remains unknown, particularly in flavivirus-endemic regions. Methods: We studied neutralizing antibodies to ZIKV and DENV1-4 in longitudinal serologic specimens collected through 3 years after infection from people in Latin America and Asia with laboratory-confirmed DENV infections. We also evaluated neutralizing antibodies to ZIKV and DENV1-4 in patients with Zika through 6 months after infection. Results: In patients with Zika, the highest neutralizing antibody titers were to ZIKV, with low-level cross-reactivity to DENV1-4 that was greater in DENV-immune individuals. We found that, in primary and secondary DENV infections, neutralizing antibody titers to ZIKV were markedly lower than to the infecting DENV and heterologous DENV serotypes. Cross-neutralization was greatest in early convalescence, then ZIKV neutralization decreased, remaining at low levels over time. Conclusions: Patterns of antibody cross-neutralization suggest that ZIKV lies outside the DENV serocomplex. Neutralizing antibody titers can distinguish ZIKV from DENV infections when all viruses are analyzed simultaneously. These findings have implications for understanding natural immunity and vaccines.