RESUMEN
Rapidly evolving RNA viruses, such as the GII.4 strain of human norovirus (HuNoV), and their vaccines elicit complex serological responses associated with previous exposure. Specific correlates of protection, moreover, remain poorly understood. Here, we report the GII.4-serological antibody repertoire-pre- and post-vaccination-and select several antibody clonotypes for epitope and structural analysis. The humoral response was dominated by GII.4-specific antibodies that blocked ancestral strains or by antibodies that bound to divergent genotypes and did not block viral-entry-ligand interactions. However, one antibody, A1431, showed broad blockade toward tested GII.4 strains and neutralized the pandemic GII.P16-GII.4 Sydney strain. Structural mapping revealed conserved epitopes, which were occluded on the virion or partially exposed, allowing for broad blockade with neutralizing activity. Overall, our results provide high-resolution molecular information on humoral immune responses after HuNoV vaccination and demonstrate that infection-derived and vaccine-elicited antibodies can exhibit broad blockade and neutralization against this prevalent human pathogen.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Infecciones por Caliciviridae/inmunología , Infecciones por Caliciviridae/prevención & control , Norovirus/inmunología , Vacunas Virales/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/química , Infecciones por Caliciviridae/epidemiología , Infecciones por Caliciviridae/virología , Línea Celular , Secuencia Conservada , Epítopos/química , Epítopos/inmunología , Humanos , Inmunoglobulina G/inmunología , Modelos Moleculares , Norovirus/clasificación , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/inmunología , VacunaciónRESUMEN
The ongoing evolution of Ebolaviruses poses significant challenges to the development of immunodiagnostics for detecting emergent viral variants. There is a critical need for the discovery of monoclonal antibodies with distinct affinities and specificities for different Ebolaviruses. We developed an efficient technology for the rapid discovery of a plethora of antigen-specific monoclonal antibodies from immunized animals by mining the VH:VL paired antibody repertoire encoded by highly expanded B cells in the draining popliteal lymph node (PLN). This approach requires neither screening nor selection for antigen-binding. Specifically we show that mouse immunization with Ebola VLPs gives rise to a highly polarized antibody repertoire in CD138(+) antibody-secreting cells within the PLN. All highly expanded antibody clones (7/7 distinct clones/animal) were expressed recombinantly, and shown to recognize the VLPs used for immunization. Using this approach we obtained diverse panels of antibodies including: (i) antibodies with high affinity towards GP; (ii) antibodies which bound Ebola VLP Kissidougou-C15, the strain circulating in the recent West African outbreak; (iii) non-GP binding antibodies that recognize wild type Sudan or Bundibugyo viruses that have 39% and 37% sequence divergence from Ebola virus, respectively and (iv) antibodies to the Reston virus GP for which no antibodies have been reported.
Asunto(s)
Anticuerpos Antivirales/inmunología , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/inmunología , Animales , Anticuerpos Antivirales/genética , Formación de Anticuerpos/genética , Formación de Anticuerpos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Reacciones Cruzadas , Modelos Animales de Enfermedad , Epítopos/genética , Epítopos/inmunología , Fiebre Hemorrágica Ebola/genética , Humanos , Inmunización , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Ganglios Linfáticos/inmunología , Ratones , Fenotipo , Unión Proteica/inmunologíaRESUMEN
UNLABELLED: Animal viruses frequently cause zoonotic disease in humans. As these viruses are highly diverse, evaluating the threat that they pose remains a major challenge, and efficient approaches are needed to rapidly predict virus-host compatibility. Here, we develop a combined computational and experimental approach to assess the compatibility of New World arenaviruses, endemic in rodents, with the host TfR1 entry receptors of different potential new host species. Using signatures of positive selection, we identify a small motif on rodent TfR1 that conveys species specificity to the entry of viruses into cells. However, we show that mutations in this region affect the entry of each arenavirus differently. For example, a human single nucleotide polymorphism (SNP) in this region, L212V, makes human TfR1 a weaker receptor for one arenavirus, Machupo virus, but a stronger receptor for two other arenaviruses, Junin and Sabia viruses. Collectively, these findings set the stage for potential evolutionary trade-offs, where natural selection for resistance to one virus may make humans or rodents susceptible to other arenavirus species. Given the complexity of this host-virus interplay, we propose a computational method to predict these interactions, based on homology modeling and computational docking of the virus-receptor protein-protein interaction. We demonstrate the utility of this model for Machupo virus, for which a suitable cocrystal structural template exists. Our model effectively predicts whether the TfR1 receptors of different species will be functional receptors for Machupo virus entry. Approaches such at this could provide a first step toward computationally predicting the "host jumping" potential of a virus into a new host species. IMPORTANCE: We demonstrate how evolutionary trade-offs may exist in the dynamic evolutionary interplay between viruses and their hosts, where natural selection for resistance to one virus could make humans or rodents susceptible to other virus species. We present an algorithm that predicts which species have cell surface receptors that make them susceptible to Machupo virus, based on computational docking of protein structures. Few molecular models exist for predicting the risk of spillover of a particular animal virus into humans or new animal populations. Our results suggest that a combination of evolutionary analysis, structural modeling, and experimental verification may provide an efficient approach for screening and assessing the potential spillover risks of viruses circulating in animal populations.
Asunto(s)
Antígenos CD/genética , Arenavirus del Nuevo Mundo/fisiología , Especificidad del Huésped , Receptores de Transferrina/genética , Receptores Virales/metabolismo , Acoplamiento Viral , Algoritmos , Animales , Línea Celular Tumoral , Biología Computacional/métodos , Resistencia a la Enfermedad/genética , Perros , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Receptores de Transferrina/metabolismo , Receptores Virales/ultraestructura , Internalización del VirusRESUMEN
Epithelial tubes are the infrastructure for organs and tissues, and tube morphogenesis requires precise orchestration of cell signaling, shape, migration, and adhesion. Follicle cells in the Drosophila ovary form a pair of epithelial tubes whose lumens act as molds for the eggshell respiratory filaments, or dorsal appendages (DAs). DA formation is a robust and accessible model for studying the patterning, formation, and expansion of epithelial tubes. Tramtrack69 (TTK69), a transcription factor that exhibits a variable embryonic DNA-binding preference, controls DA lumen volume and shape by promoting tube expansion; the tramtrack mutation twin peaks (ttk(twk)) reduces TTK69 levels late in oogenesis, inhibiting this expansion. Microarray analysis of wild-type and ttk(twk) ovaries, followed by in situ hybridization and RNAi of candidate genes, identified the Phospholipase B-like protein Lamina ancestor (LAMA), the scaffold protein Paxillin, the endocytotic regulator Shibire (Dynamin), and the homeodomain transcription factor Mirror, as TTK69 effectors of DA-tube expansion. These genes displayed enriched expression in DA-tube cells, except lama, which was expressed in all follicle cells. All four genes showed reduced expression in ttk(twk) mutants and exhibited RNAi phenotypes that were enhanced in a ttk(twk)/+ background, indicating ttk(twk) genetic interactions. Although previous studies show that Mirror patterns the follicular epithelium prior to DA tubulogenesis, we show that Mirror has an independent, novel role in tube expansion, involving positive regulation of Paxillin. Thus, characterization of ttk(twk)-differentially expressed genes expands the network of TTK69 effectors, identifies novel epithelial tube-expansion regulators, and significantly advances our understanding of this vital developmental process.