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1.
Cell ; 183(5): 1367-1382.e17, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33160446

RESUMO

A safe, effective, and scalable vaccine is needed to halt the ongoing SARS-CoV-2 pandemic. We describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 SARS-CoV-2 spike receptor-binding domains (RBDs) in a highly immunogenic array and induce neutralizing antibody titers 10-fold higher than the prefusion-stabilized spike despite a 5-fold lower dose. Antibodies elicited by the RBD nanoparticles target multiple distinct epitopes, suggesting they may not be easily susceptible to escape mutations, and exhibit a lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the assembled nanoparticles suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms and have launched cGMP manufacturing efforts to advance the SARS-CoV-2-RBD nanoparticle vaccine into the clinic.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Nanopartículas/química , Domínios Proteicos/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Vacinação , Adolescente , Adulto , Idoso , Animais , COVID-19/virologia , Chlorocebus aethiops , Estudos de Coortes , Epitopos/imunologia , Feminino , Células HEK293 , Humanos , Macaca nemestrina , Masculino , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero , Adulto Jovem
2.
bioRxiv ; 2020 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-32817941

RESUMO

A safe, effective, and scalable vaccine is urgently needed to halt the ongoing SARS-CoV-2 pandemic. Here, we describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 copies of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) in a highly immunogenic array and induce neutralizing antibody titers roughly ten-fold higher than the prefusion-stabilized S ectodomain trimer despite a more than five-fold lower dose. Antibodies elicited by the nanoparticle immunogens target multiple distinct epitopes on the RBD, suggesting that they may not be easily susceptible to escape mutations, and exhibit a significantly lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the protein components and assembled nanoparticles, especially compared to the SARS-CoV-2 prefusion-stabilized S trimer, suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms for inducing potent neutralizing antibody responses and have launched cGMP manufacturing efforts to advance the lead RBD nanoparticle vaccine into the clinic.

3.
Nat Biotechnol ; 32(4): 356-63, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24633243

RESUMO

If immunized with an antigen of interest, transgenic mice with large portions of unrearranged human immunoglobulin loci can produce fully human antigen-specific antibodies; several such antibodies are in clinical use. However, technical limitations inherent to conventional transgenic technology and sequence divergence between the human and mouse immunoglobulin constant regions limit the utility of these mice. Here, using repetitive cycles of genome engineering in embryonic stem cells, we have inserted the entire human immunoglobulin variable-gene repertoire (2.7 Mb) into the mouse genome, leaving the mouse constant regions intact. These transgenic mice are viable and fertile, with an immune system resembling that of wild-type mice. Antigen immunization results in production of high-affinity antibodies with long human-like complementarity-determining region 3 (CDR3H), broad epitope coverage and strong signatures of somatic hypermutation. These mice provide a robust system for the discovery of therapeutic human monoclonal antibodies; as a surrogate readout of the human antibody response, they may also aid vaccine design efforts.


Assuntos
Anticorpos Monoclonais/genética , Engenharia Genética/métodos , Região Variável de Imunoglobulina/genética , Transgenes/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Linfócitos B/fisiologia , Cromossomos Artificiais Bacterianos/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular
4.
Hum Mol Genet ; 17(5): 631-41, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17635842

RESUMO

Spondylocarpotarsal synostosis syndrome (SCT) is an autosomal recessive disease that is characterized by short stature, and fusions of the vertebrae and carpal and tarsal bones. SCT results from homozygosity or compound heterozygosity for nonsense mutations in FLNB. FLNB encodes filamin B, a multifunctional cytoplasmic protein that plays a critical role in skeletal development. Protein extracts derived from cells of SCT patients with nonsense mutations in FLNB did not contain filamin B, demonstrating that SCT results from absence of filamin B. To understand the role of filamin B in skeletal development, an Flnb-/- mouse model was generated. The Flnb-/- mice were phenotypically similar to individuals with SCT as they exhibited short stature and similar skeletal abnormalities. Newborn Flnb-/- mice had fusions between the neural arches of the vertebrae in the cervical and thoracic spine. At postnatal day 60, the vertebral fusions were more widespread and involved the vertebral bodies as well as the neural arches. In addition, fusions were seen in sternum and carpal bones. Analysis of the Flnb-/- mice phenotype showed that an absence of filamin B causes progressive vertebral fusions, which is contrary to the previous hypothesis that SCT results from failure of normal spinal segmentation. These findings suggest that spinal segmentation can occur normally in the absence of filamin B, but the protein is required for maintenance of intervertebral, carpal and sternal joints, and the joint fusion process commences antenatally.


Assuntos
Anormalidades Múltiplas/genética , Proteínas Contráteis/genética , Proteínas dos Microfilamentos/genética , Mutação , Osteocondrodisplasias/genética , Sinostose/genética , Animais , Animais Recém-Nascidos , Tornozelo/anormalidades , Códon sem Sentido , Proteínas Contráteis/química , Proteínas Contráteis/deficiência , Cruzamentos Genéticos , Dimerização , Modelos Animais de Doenças , Embrião de Mamíferos , Filaminas , Regulação da Expressão Gênica no Desenvolvimento , Genes Recessivos , Heterozigoto , Homozigoto , Humanos , Metacarpo/anormalidades , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/deficiência , Modelos Biológicos , Modelos Genéticos , Peso Molecular , Fenótipo , Estrutura Terciária de Proteína , Coluna Vertebral/anormalidades , Síndrome
5.
Mol Endocrinol ; 17(12): 2418-35, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14500757

RESUMO

Recent work indicates that thyroid hormone receptor-associated protein 220 (TRAP220), a subunit of the multiprotein TRAP coactivator complex, is essential for embryonic survival. We have generated TRAP220 conditional null mice that are hypomorphic and express the gene at reduced levels. In contrast to TRAP220 null mice, which die at embryonic d 11.5 (E11.5), hypomorphic mice survive until E13.5. The reduced expression in hypomorphs results in hepatic necrosis, defects in hematopoiesis, and hypoplasia of the ventricular myocardium, similar to that observed in TRAP220 null embryos at an earlier stage. The embryonic lethality of null embryos at E11.5 is due to placental insufficiency. Tetraploid aggregation assays partially rescues embryonic development until E13.5, when embryonic loss occurs due to hepatic necrosis coupled with poor myocardial development as observed in hypomorphs. These findings demonstrate that, for normal placental function, there is an absolute requirement for TRAP220 in extraembryonic tissues at E11.5, with an additional requirement in embryonic tissues for hepatic and cardiovascular development thereafter.


Assuntos
Coração/embriologia , Fígado/embriologia , Placenta/fisiologia , Fatores de Transcrição/fisiologia , Animais , Feminino , Morte Fetal , Coração/fisiologia , Heterozigoto , Homozigoto , Hibridização In Situ , Fígado/patologia , Subunidade 1 do Complexo Mediador , Camundongos , Camundongos Knockout , Miocárdio/patologia , Poliploidia , Gravidez , Receptores dos Hormônios Tireóideos/deficiência , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/fisiologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
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