RESUMEN

Limited knowledge exists on the quality of polyclonal antibody responses generated following Marburg virus (MARV) infection and its evolution in survivors. In this study, we evaluate MARV proteome-wide antibody repertoire longitudinally in convalescent phase approximately every six months for five years following MARV infection in ten human survivors. Differential kinetics were observed for IgM vs IgG vs IgA epitope diversity, antibody binding, antibody affinity maturation and Fc-receptor interaction to MARV proteins. Durability of MARV-neutralizing antibodies is low in survivors. MARV infection induces a diverse epitope repertoire with predominance against GP, VP40, VP30 and VP24 that persisted up to 5 years post-exposure. However, the IgM and IgA repertoire declines over time. Within MARV-GP, IgG recognize antigenic sites predominantly in the amino-terminus, wing domain and GP2-heptad repeat. Interestingly, MARV infection generates robust durable FcɣRI, FcɣRIIA and FcɣRIIIA IgG-Fc receptor interactions. Immunization with immunodominant MARV epitopes reveals conserved wing region between GP1 and GP2, induces neutralizing antibodies against MARV. These findings demonstrate that MARV infection generates a diverse, long-lasting, non-neutralizing, IgG antibody repertoire that perturbs disease by FcɣR activity. This information, along with discovery of neutralizing immunogen in wing domain, could aid in development of effective therapeutics and vaccines against Marburg virus.

Asunto(s)

Anticuerpos Neutralizantes , Anticuerpos Antivirales , Enfermedad del Virus de Marburg , Marburgvirus , Proteoma , Marburgvirus/inmunología , Humanos , Enfermedad del Virus de Marburg/inmunología , Enfermedad del Virus de Marburg/virología , Anticuerpos Antivirales/inmunología , Anticuerpos Neutralizantes/inmunología , Proteoma/inmunología , Femenino , Vacunas Virales/inmunología , Inmunoglobulina G/inmunología , Masculino , Epítopos/inmunología , Adulto , Inmunoglobulina M/inmunología , Persona de Mediana Edad , Estudios Longitudinales , Inmunoglobulina A/inmunología , Desarrollo de Vacunas , Proteínas del Envoltorio Viral/inmunología

RESUMEN

BACKGROUND: Ebola virus (EBOV) vaccines containing glycoprotein (GP) provide protection against severe Ebola virus disease (EVD). EBO vaccinations elicit antibodies that are detectable in Ebola serodiagnostic tests, as EBOV GP is a major target antigen. This vaccine-induced seropositivity presents issues with early detection of natural EBOV infections, following vaccination and during surveillance, leading to 'uninfected' vaccine trial participants being falsely diagnosed as 'EBOV infected' potentially resulting in long-term social and economic distress. Since mass vaccinations are being employed to curtail the recurrent EBOV epidemics in multiple African countries, it is, therefore, essential to differentiate vaccine-induced from natural infection-induced antibodies by a differential serodiagnosis assay for accurate detection of Ebola virus infections. METHODS: To develop a serodiagnostic test that can differentiate between individuals with EBOV infection-induced antibodies and individuals with EBOV vaccine-induced antibodies, we analysed peptides of EBOV viral protein 40 (VP40), viral protein 35 (VP35) and nucleocapsid protein (NP) using an ELISA with a panel of 181 human sera collected from healthy controls, EBO vaccinees, and EBOV-infected survivors. Receiver Operating Characteristic (ROC) curve analysis was used to calculate sensitivity and specificity of the assay. A simple peptide-based serodiagnostic assay was used to evaluate detection of breakthrough EBOV infections in vaccinated non-human primates (NHP) in EBOV challenge studies. FINDINGS: We identified conserved peptide sequences in EBOV VP40, VP35 and NP, produced soon after EBOV infection that are not part of the current EBO vaccine target antigens. The new ELISA-based differential serodetection assay termed 'EBOV-Detect' demonstrated >94% specificity and 96% sensitivity for diagnosis of EBOV infection. Importantly, the uninfected vaccine-trial participants scored negative in 'EBOV-Detect' assay. The results from the NHPs EBOV challenge study established that post-EBO vaccination serum scored negative in 'EBOV-Detect' and all NHPs with Ebola breakthrough infections, following EBOV challenge, were serodiagnosed positively with EBOV-Detect. INTERPRETATION: The new 'EBOV-Detect' is a simple and sensitive serodiagnostic assay that can specifically differentiate between natural Ebola virus infected and those with vaccine-induced immunity. This could potentially be implemented as a robust diagnostic tool for epidemiology and surveillance of EBOV infections during and after outbreaks, especially in countries with mass Ebola vaccinations. FUNDING: The antibody characterization work described in this manuscript was supported by FDA Office of Counterterrorism and Emerging Threats (OCET) - Medical Countermeasures initiative (MCMi) grant- OCET 2019-1018 and Defense Threat Reduction Agency (HDTRA1930447) funds to S.K.

Asunto(s)

Vacunas contra el Virus del Ébola , Ebolavirus , Fiebre Hemorrágica Ebola , Anticuerpos Antivirales , Ebolavirus/inmunología , Glicoproteínas , Fiebre Hemorrágica Ebola/diagnóstico , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/prevención & control , Humanos , Proteínas de la Nucleocápside , Pruebas Serológicas , Proteínas Virales

RESUMEN

BACKGROUND: Limited knowledge exists in post-partum women regarding durability of SARS-CoV-2 vaccine-induced antibody responses and their neutralising ability against SARS-CoV-2 variants of concern (VOC). METHODS: We elucidated longitudinal mRNA vaccination-induced antibody profiles of 13 post-partum and 13 non-post-partum women (control). FINDINGS: The antibody neutralisation titres against SARS-CoV-2 WA-1 strain were comparable between post-partum and non-post-partum women and these levels were sustained up to four months post-second vaccination in both groups. However, neutralisation titers declined against several VOCs, including Beta and Delta. Higher antibody binding was observed against SARS-CoV-2 receptor-binding domain (RBD) mutants with key VOC amino acids when tested with post-second vaccination plasma from post-partum women compared with controls. Importantly, post-vaccination plasma antibody affinity against VOCs RBDs was significantly higher in post-partum women compared with controls. INTERPRETATION: This study demonstrates that there is a differential vaccination-induced immune responses in post-partum women compared with non-post-partum women, which could help inform future vaccination strategies for these groups. FUNDING: The antibody characterisation work described in this manuscript was supported by FDA's Medical Countermeasures Initiative (MCMi) grant #OCET 2021-1565 to S.K and intramural FDA-CBER COVID-19 supplemental funds.

Asunto(s)

Vacunas contra la COVID-19 , COVID-19 , Anticuerpos Antivirales , Afinidad de Anticuerpos , COVID-19/prevención & control , Femenino , Humanos , Inmunoglobulina G , Periodo Posparto , SARS-CoV-2/genética , Vacunas Sintéticas , Vacunas de ARNm

RESUMEN

Respiratory syncytial virus (RSV) vaccines primarily focused on surface fusion (F) protein are under development. Therefore, to identify RSV-F protective epitopes, we evaluated 14 antigenic sites recognized following primary human RSV infection. BALB/c mice were vaccinated with F peptides, F proteins, or RSV-A2, followed by rA2-Line19F challenge. F peptides generated binding antibodies with minimal in vitro neutralization titers. However, several F peptides (including Site II) reduced lung viral loads and lung pathology scores in animals, suggesting partial protection from RSV disease. Interestingly, animals vaccinated with peptides (aa 101-121 and 110-136) spanning the F-p27 sequence, which is only present in unprocessed F0 protein, showed control of viral loads with significantly reduced pathology compared with mock-vaccinated controls. Furthermore, we observed F-p27 expression on the surface of RSV-infected cells as well as lungs from RSV-infected mice. The anti-p27 antibodies demonstrated antibody-dependent cellular cytotoxicity (ADCC) of RSV-infected A549 cells. These findings suggest that p27-mediated immune response may play a role in control of RSV disease in vivo, and F-p27 should be considered for inclusion in an effective RSV vaccine.

Asunto(s)

Infecciones por Virus Sincitial Respiratorio , Vacunas contra Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Ratones , Ratones Endogámicos BALB C , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/química , Vacunas contra Virus Sincitial Respiratorio/genética , Virus Sincitial Respiratorio Humano/genética , Proteínas Virales de Fusión/genética

RESUMEN

BACKGROUND: Limited knowledge exists regarding antibody affinity maturation following mRNA vaccination in naïve vs. COVID-19 recovered individuals and potential sex differences. METHODS: We elucidated post-vaccination antibody profiles of 69 naïve and 17 COVID-19 convalescent adults using pseudovirus neutralization assay (PsVNA) covering SARS-CoV-2 WA-1, variants of concern (VOCs) and variants of interest (VOIs). Surface Plasmon Resonance (SPR) was used to measure antibody affinity against prefusion spike and receptor binding domain (RBD) and RBD mutants. FINDINGS: Higher neutralizing antibodies were observed in convalescent vs. naïve adults against, WA-1, VOCs, and VOIs. Antibody binding to RBD and RBD mutants showed lower binding of post-vaccination sera from naïve compared with convalescent individuals. Moreover, we observed early antibody affinity maturation in convalescent individuals after one vaccine dose and higher antibody affinity after two doses compared with the naïve group. Among the naïve participants, antibody affinity against the SARS-CoV-2 prefusion spike was significantly higher for males than females even though there were no difference in neutralization titers between sexes. INTERPRETATION: This study demonstrates the impact of prior infection on vaccine-induced antibody affinity maturation and difference in antibody affinity between males and females. Further studies are needed to determine whether antibody affinity may contribute to correlates of protection against SARS-CoV-2 and its variants. FUNDING: The antibody characterization work described in this manuscript was supported by FDA's Medical Countermeasures Initiative (MCMi) grant #OCET 2021-1565 to S.K and intramural FDA-CBER COVID-19 supplemental funds. The SPARTA program was supported by the National Institute of Allergy and Infectious Diseases (NIAID), U.S. National Institutes of Health (NIH), Department of Health and Human Services contract 75N93019C00052, and the University of Georgia (US) grant UGA-001. T.M.R is also supported by the Georgia Research Alliance (US) grant GRA-001. The CTRU was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR002378.

Asunto(s)

Vacuna nCoV-2019 mRNA-1273/inmunología , Anticuerpos Neutralizantes/sangre , Afinidad de Anticuerpos/inmunología , Vacuna BNT162/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Antivirales/sangre , COVID-19/inmunología , Línea Celular , Femenino , Humanos , Masculino , Pruebas de Neutralización , Dominios Proteicos/inmunología , Resonancia por Plasmón de Superficie , Vacunación , Vacunas de ARNm/inmunología

RESUMEN

There is limited understanding of the viral antibody fingerprint following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children. Herein, SARS-CoV-2 proteome-wide immunoprofiling of children with mild/moderate or severe coronavirus disease 2019 (COVID-19) versus multisystem inflammatory syndrome in children versus hospitalized control patients revealed differential cytokine responses, IgM/IgG/IgA epitope diversity, antibody binding and avidity. Apart from spike and nucleocapsid, IgG/IgA recognized epitopes in nonstructural protein (NSP) 2, NSP3, NSP12-NSP14 and open reading frame (ORF) 3a-ORF9. Peptides representing epitopes in NSP12, ORF3a and ORF8 demonstrated SARS-CoV-2 serodiagnosis. Antibody-binding kinetics with 24 SARS-CoV-2 proteins revealed antibody parameters that distinguish children with mild/moderate versus severe COVID-19 or multisystem inflammatory syndrome in children. Antibody avidity to prefusion spike correlated with decreased illness severity and served as a clinical disease indicator. The fusion peptide and heptad repeat 2 region induced SARS-CoV-2-neutralizing antibodies in rabbits. Thus, we identified SARS-CoV-2 antibody signatures in children associated with disease severity and delineate promising serodiagnostic and virus neutralization targets. These findings might guide the design of serodiagnostic assays, prognostic algorithms, therapeutics and vaccines in this important but understudied population.

Asunto(s)

Prueba Serológica para COVID-19/métodos , COVID-19/complicaciones , COVID-19/inmunología , SARS-CoV-2/inmunología , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Adolescente , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , COVID-19/diagnóstico , Niño , Preescolar , Progresión de la Enfermedad , Epítopos/metabolismo , Femenino , Hospitalización , Humanos , Inmunidad Humoral , Inmunoglobulina A/metabolismo , Inmunoglobulina G/metabolismo , Inmunoglobulina M/metabolismo , Masculino , Pronóstico , Proteoma , Índice de Severidad de la Enfermedad , Síndrome de Respuesta Inflamatoria Sistémica/diagnóstico

RESUMEN

Mucosal immunity plays a key role in prevention of SARS-CoV-2 virus spread to the lungs. In this study, we evaluated systemic and mucosal immune signatures in asymptomatic SARS-CoV-2­infected versus symptomatic COVID-19 adults compared with RSV-infected adults. Matched serum and nasal wash pairs were subjected to cytokine/chemokine analyses and comprehensive antibody profiling including epitope repertoire analyses, antibody kinetics to SARS-CoV-2 prefusion spike and spike RBD mutants, and neutralization of SARS-CoV-2 variants of concern. The data suggest independent evolution of antibody responses in the mucosal sites as reflected in differential IgM/IgG/IgA epitope repertoire compared with serum. Antibody affinity against SARS-CoV-2 prefusion spike for both serum and nasal washes was significantly higher in asymptomatic adults compared with symptomatic COVID-19 patients. Last, the cytokine/chemokine responses in the nasal washes were more robust than in serum. These data underscore the importance of evaluating mucosal immune responses for better therapeutics and vaccines against SARS-CoV-2.

RESUMEN

Hyperimmune immunoglobulin (hCoV-2IG) generated from SARS-CoV-2 convalescent plasma (CP) are under evaluation in clinical trials. Here we explored the antibody epitope repertoire, and virus neutralizing capacity of six hCoV-2IG batches as well as nine CP against SARS-CoV-2 and emerging variants of concern (VOCs). Epitope-mapping by gene-fragment phage display library spanning the SARS-CoV-2 spike demonstrated broad recognition of multiple antigenic sites spanning the entire spike that was higher for hCoV-2IG than CP, with predominant binding to the fusion peptide. In the pseudovirus neutralization assay and in the wild-type SARS-CoV-2 PRNT assay, hCoV-2IG lots showed higher titers against the WA-1 strain compared with CP. Neutralization of VOCs were reduced to different extent by hCoV-2IG lots but were higher than CP. Significant reduction of hCoV-2IG binding was observed to RBD-E484K followed by RBD-N501Y (but not RBD-K417N). This study suggests that post-exposure treatment with hCoV-2IG could be preferable to CP.

RESUMEN

Limited knowledge exists on immune markers associated with disease severity or recovery in patients with coronavirus disease 2019 (COVID-19). Here, we elucidated longitudinal evolution of SARS-CoV-2 antibody repertoire in patients with acute COVID-19. Differential kinetics was observed for immunoglobulin M (IgM)/IgG/IgA epitope diversity, antibody binding, and affinity maturation in "severe" versus "mild" COVID-19 patients. IgG profile demonstrated immunodominant antigenic sequences encompassing fusion peptide and receptor binding domain (RBD) in patients with mild COVID-19 who recovered early compared with "fatal" COVID-19 patients. In patients with severe COVID-19, high-titer IgA were observed, primarily against RBD, especially in patients who succumbed to SARS-CoV-2 infection. The patients with mild COVID-19 showed marked increase in antibody affinity maturation to prefusion SARS-CoV-2 spike that associated with faster recovery from COVID-19. This study revealed antibody markers associated with disease severity and resolution of clinical disease that could inform development and evaluation of effective immune-based countermeasures against COVID-19.

Asunto(s)

Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Biomarcadores/sangre , COVID-19/inmunología , COVID-19/patología , SARS-CoV-2/fisiología , Índice de Severidad de la Enfermedad , Afinidad de Anticuerpos/inmunología , Formación de Anticuerpos/inmunología , COVID-19/sangre , COVID-19/virología , Citocinas/sangre , Células HEK293 , Hospitalización , Humanos , Cambio de Clase de Inmunoglobulina , Cinética , Pruebas de Neutralización , Unión Proteica , Dominios Proteicos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Carga Viral

RESUMEN

Hospitalized COVID-19 patients often present with a large spectrum of clinical symptoms. There is a critical need to better understand the immune responses to SARS-CoV-2 that lead to either resolution or exacerbation of the clinical disease. Here, we examine longitudinal plasma samples from hospitalized COVID-19 patients with differential clinical outcome. We perform immune-repertoire analysis including cytokine, hACE2-receptor inhibition, neutralization titers, antibody epitope repertoire, antibody kinetics, antibody isotype and antibody affinity maturation against the SARS-CoV-2 prefusion spike protein. Fatal cases demonstrate high plasma levels of IL-6, IL-8, TNFα, and MCP-1, and sustained high percentage of IgA-binding antibodies to prefusion spike compared with non-ICU survivors. Disease resolution in non-ICU and ICU patients associates with antibody binding to the receptor binding motif and fusion peptide, and antibody affinity maturation to SARS-CoV-2 prefusion spike protein. Here, we provide insight into the immune parameters associated with clinical disease severity and disease-resolution outcome in hospitalized patients that could inform development of vaccine/therapeutics against COVID-19.

Asunto(s)

Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Afinidad de Anticuerpos/inmunología , COVID-19/inmunología , Inmunoglobulina A/inmunología , SARS-CoV-2/inmunología , Adulto , Anciano , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , COVID-19/sangre , COVID-19/virología , Estudios de Cohortes , Citocinas/sangre , Citocinas/inmunología , Citocinas/metabolismo , Epítopos/inmunología , Femenino , Hospitalización/estadística & datos numéricos , Humanos , Inmunoglobulina A/sangre , Masculino , Persona de Mediana Edad , Pruebas de Neutralización , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/inmunología , Resonancia por Plasmón de Superficie

RESUMEN

Multiple vaccine candidates against SARS-CoV-2 based on viral spike protein are under development. However, there is limited information on the quality of antibody responses generated with these vaccine modalities. To better understand antibody responses induced by spike protein-based vaccines, we performed a qualitative study by immunizing rabbits with various SARS-CoV-2 spike protein antigens: S ectodomain (S1+S2; amino acids 16 to 1213), which lacks the cytoplasmic and transmembrane domains (CT-TM), the S1 domain (amino acids 16 to 685), the receptor binding domain (RBD) (amino acids 319 to 541), and the S2 domain (amino acids 686 to 1213, lacking the RBD, as control). Resulting antibody quality and function were analyzed by enzyme-linked immunosorbent assay (ELISA), RBD competition assay, surface plasmon resonance (SPR) against different spike proteins in native conformation, and neutralization assays. All three antigens (S1+S2 ectodomain, S1 domain, and RBD), but not S2, generated strong neutralizing antibodies against SARS-CoV-2. Vaccination-induced antibody repertoire was analyzed by SARS-CoV-2 spike genome fragment phage display libraries (SARS-CoV-2 GFPDL), which identified immunodominant epitopes in the S1, S1-RBD, and S2 domains. Furthermore, these analyses demonstrated that the RBD immunogen elicited a higher antibody titer with five-fold higher affinity antibodies to native spike antigens compared with other spike antigens, and antibody affinity correlated strongly with neutralization titers. These findings may help guide rational vaccine design and facilitate development and evaluation of effective therapeutics and vaccines against COVID-19 disease.

Asunto(s)

Anticuerpos Antivirales/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Formación de Anticuerpos/inmunología , Antígenos Virales/inmunología , Epítopos/inmunología , Femenino , Inmunización , Pruebas de Neutralización , Conejos

RESUMEN

Limited knowledge exists on the quality of polyclonal antibody response generated following Ebola virus (EBOV) infection compared with vaccination. Polyclonal antibody repertoire in plasma following EBOV infection in survivors was compared with ChAd3-MVA prime-boost human vaccination. Higher antibody binding and affinity to GP was observed in survivors compared with vaccinated plasma that correlated with EBOV neutralization. Surprisingly, a predominant IgM response was generated after prime-boost vaccination, whereas survivors demonstrated IgG-dominant antibody response. EBOV infection induced more diverse antibody epitope repertoire compared with vaccination. A strong binding to antigenic sites in the fusion peptide and another in the highly conserved GP2-HR2 domain was preferentially recognized by EBOV survivors than vaccinated individuals that correlated strongly with EBOV neutralization titers. These findings will help development and evaluation of effective Ebola countermeasures including therapeutics and vaccines.

RESUMEN

Evolution of antibody repertoire against the Ebola virus (EBOV) proteome was characterized in an acutely infected patient receiving supportive care alone to elucidate virus-host interactions over time. Differential kinetics are observed for IgM-IgG-IgA epitope diversity, antibody binding, and affinity maturation to EBOV proteins. During acute illness, antibodies predominate to VP40 and glycoprotein (GP). At day 13 of clinical illness, a marked increase in antibody titers to most EBOV proteins and affinity maturation to GP is associated with rapid decline in viral replication and illness severity. At one year, despite undetectable virus, a diverse IgM repertoire against VP40 and GP epitopes is observed suggesting occult viral persistence. Rabbit immunization experiments identify key immunodominant sites of GP, while challenge studies in mice found these epitopes induce EBOV-neutralizing antibodies and protect against lethal EBOV challenge. This study reveals markers of viral persistence and provides promising approaches for development and evaluation of vaccines and therapeutics.

Asunto(s)

Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/inmunología , Animales , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Epítopos/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Fiebre Hemorrágica Ebola/terapia , Humanos , Inmunoglobulina A/aislamiento & purificación , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina M/aislamiento & purificación , Ratones , Proteoma/inmunología , Conejos , Sobrevivientes , Proteínas del Envoltorio Viral/inmunología , Proteínas de la Matriz Viral/inmunología , Proteínas Virales/inmunología , Vacunas Virales

RESUMEN

Zika virus (ZIKV) outbreak in Americas led to extensive efforts to develop vaccines and ZIKV-specific diagnostics. In the current study, we use whole genome phage display library spanning the entire ZIKV genome (ZIKV-GFPDL) for in-depth immune profiling of IgG and IgM antibody repertoires in serum and urine longitudinal samples from individuals acutely infected with ZIKV. We observe a very diverse IgM immune repertoire encompassing the entire ZIKV polyprotein on day 0 in both serum and urine. ZIKV-specific IgG antibodies increase 10-fold between day 0 and day 7 in serum, but not in urine; these are highly focused on prM/E, NS1 and NS2B. Differential antibody affinity maturation is observed against ZIKV structural E protein compared with nonstructural protein NS1. Serum antibody affinity to ZIKV-E protein inversely correlates with ZIKV disease symptoms. Our study provides insight into unlinked evolution of immune response to ZIKV infection and identified unique targets for ZIKV serodiagnostics.

Asunto(s)

Anticuerpos Antivirales/sangre , Infección por el Virus Zika/inmunología , Anticuerpos Antivirales/inmunología , Afinidad de Anticuerpos/fisiología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/sangre , Proteínas no Estructurales Virales/metabolismo , Vacunas Virales/inmunología , Virus Zika/inmunología , Infección por el Virus Zika/diagnóstico

RESUMEN

Several Ebola vaccines and therapeutics are under clinical development. However, limited knowledge exists on the quality of antibody response generated by different Ebola vaccines. In this study, antibody repertoire induced by vaccination of transchromosomal bovine (TcB) with Ebola virus (EBOV) glycoprotein ([GP]; recombinant GP [rGP]) encoded by either deoxyribonucleic acid (DNA) or nanoparticle-based vaccine platform was analyzed using EBOV genome fragment phage display library and surface plasmon resonance (SPR)-based real-time kinetics assay to measure antibody affinity maturation to both native and partially denatured Ebola GP as well as GP containing the receptor binding domain but lacking the mucin-like domain. Immunoglobulin (IgG) obtained from rGP nanoparticle-vaccinated TcB demonstrated ~4-fold higher binding affinity compared with DNA-vaccinated TcB-induced IgG against the native rGP's. The rGP nanoparticle vaccine generated a more robust and diverse antibody immune response to the native EBOV-GP compared with the DNA vaccine, which may explain the protective efficacy observed for these antibody preparations.

Asunto(s)

Anticuerpos Antivirales/inmunología , Vacunas contra el Virus del Ébola/inmunología , Ebolavirus/inmunología , Glicoproteínas/inmunología , Vacunación , Vacunas de ADN/inmunología , Animales , Bovinos , Humanos , Nanopartículas , Vacunas Sintéticas/inmunología

RESUMEN

We report on the effect of surface charge and the ligand coating composition of CdSe/ZnS core/shell quantum dot (QD) nanoparticles on human keratinocyte toxicity using fluorescent microscopy, flow cytometry, transmission electron microscopy. Two commonly reported positive charged (cysteamine, polyethylenimine) and two negative charged (glutathione, dihydrolipoic acid) ligands were studied. The QDs were fully characterized by UV-vis absorption spectroscopy, fluorescence emission spectroscopy, dynamic light scattering and zeta potential. Differences in surface coatings and charges were evaluated against cellular uptake, ROS generation, cytotoxicity, and mitochondrial targeting. Results show that the negative charged QDs coated with GSH exhibit excellent water solubility, high quantum yield and low cytotoxicity. Ligand composition is more important in ROS generation than surface charge whereas surface charge is an important driver of cytotoxicity. Most importantly we observe the selective accumulation of glutathione coated QDs in vesicles in the mitochondria matrix. This observation suggests a new strategy for developing mitochondria-targeted nanomaterials for drug/gene delivery.

Asunto(s)

Membrana Celular/metabolismo , Mitocondrias/metabolismo , Puntos Cuánticos , Compuestos de Cadmio/química , Compuestos de Cadmio/farmacocinética , Compuestos de Cadmio/toxicidad , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Glutatión , Humanos , Puntos Cuánticos/química , Puntos Cuánticos/metabolismo , Puntos Cuánticos/toxicidad , Compuestos de Selenio/química , Compuestos de Selenio/farmacocinética , Compuestos de Selenio/toxicidad , Solubilidad , Sulfuros/química , Sulfuros/farmacocinética , Sulfuros/toxicidad , Propiedades de Superficie , Compuestos de Zinc/química , Compuestos de Zinc/farmacocinética , Compuestos de Zinc/toxicidad

RESUMEN

Development of an effective vaccine against Ebola virus is of high priority. However, knowledge about potential correlates of protection and the durability of immune response after vaccination is limited. Here, we elucidate the human antibody repertoire after administration of vesicular stomatitis virus (VSV)-Ebola vaccine at 3 million, 20 million and 100 million plaque-forming units (PFU) and homologous VSV-Ebola vaccine boost in healthy adult volunteers. Whole genome-fragment phage display libraries, expressing linear and conformational epitopes of Ebola glycoprotein (GP), showed higher diversity of antibody epitopes in individuals vaccinated with 20 million PFU than in those vaccinated with 3 million or 100 million PFU. Surface plasmon resonance kinetics showed higher levels of GP-binding antibodies after a single vaccination with 20 million or 100 million PFU than with 3 million PFU, and these correlated strongly with neutralization titers. A second vaccination did not boost antibody or virus neutralization titers, which declined rapidly, and induced only minimal antibody affinity maturation. Isotype analysis revealed a predominant IgM response even after the second vaccination, which contributed substantially to virus neutralization in vitro. These findings may help identify new vaccine targets and aid development and evaluation of effective countermeasures against Ebola.

Asunto(s)

Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Vacunas contra el Virus del Ébola/administración & dosificación , Ebolavirus/inmunología , Inmunoglobulina M/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Epítopos/inmunología , Femenino , Glicoproteínas/inmunología , Voluntarios Sanos , Humanos , Inmunogenicidad Vacunal , Masculino , Vacunas Atenuadas/administración & dosificación

RESUMEN

The increasing use of nanoparticles (NPs) in technological applications and in commercial products has escalated environmental health and safety concerns. The detection of NPs in the environment and in biological systems is challenged by limitations associated with commonly used analytical techniques. In this paper we report on the development and characterization of NP binding antibodies, termed NProbes. Phage display methodology was used to discover antibodies that bind NPs dispersed in solution. We present a proof-of-concept for the generation of NProbes and their use for detecting quantum dots and titanium dioxide NPs in vitro and in an ex vivo human skin model. Continued development and refinement of NProbes to detect NPs that vary in composition, shape, size, and surface coating will comprise a powerful tool kit that can be used to advance nanotechnology research particularly in the nanotoxicology and nanotherapeutics fields.

Asunto(s)

Indicadores y Reactivos/química , Nanopartículas/química , Anticuerpos de Cadena Única/química , Albúminas/química , Humanos , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Nanomedicina/métodos , Biblioteca de Péptidos , Puntos Cuánticos/química , Piel/efectos de los fármacos , Piel/patología , Titanio/química

RESUMEN

Studies have shown that UVB can slightly increase the penetration of nanoparticles through skin and significantly alter skin cell biology, thus it is important to understand if and how UVB may impact subsequent nanoparticle skin cell interactions. The research presented herein evaluates the effect of UVB on quantum dot (QD) uptake and reactive oxygen species (ROS) generation in primary keratinocytes, primary melanocytes, and related cell lines. QD exposure induced cell type dependent ROS responses increased by pre-exposing cells to UVB and correlated with the level of QD uptake. Our results suggest that keratinocytes may be at greater risk for QD induced ROS generation than melanocytes, and raise awareness about the differential cellular effects that topically applied nanomaterials may have on UVB exposed skin.

Asunto(s)

Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Puntos Cuánticos , Especies Reactivas de Oxígeno/metabolismo , Absorción Cutánea/fisiología , Piel/metabolismo , Línea Celular , Humanos , Ensayo de Materiales , Dosis de Radiación , Piel/efectos de la radiación , Absorción Cutánea/efectos de la radiación , Rayos Ultravioleta