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1.
Nature ; 592(7853): 283-289, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33524990

RESUMO

A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates (BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD-foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD-foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD 'down', one-RBD 'up' state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4+ and IFNγ+CD8+ T cell responses. Prime-boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2-18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA1-3, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728).


Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Modelos Animais de Doenças , SARS-CoV-2/imunologia , Envelhecimento/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Vacina BNT162 , COVID-19/sangue , COVID-19/terapia , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/química , Vacinas contra COVID-19/genética , Linhagem Celular , Ensaios Clínicos como Assunto , Feminino , Humanos , Imunização Passiva , Internacionalidade , Macaca mulatta/imunologia , Macaca mulatta/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Multimerização Proteica , RNA Viral/análise , Sistema Respiratório/imunologia , Sistema Respiratório/virologia , SARS-CoV-2/química , SARS-CoV-2/genética , Solubilidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T/imunologia , Vacinação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Soroterapia para COVID-19 , Vacinas de mRNA
2.
Angew Chem Int Ed Engl ; 53(35): 9198-202, 2014 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-25045108

RESUMO

It is becoming increasingly clear that nature uses RNAs extensively for regulating vital functions of the cell, and short sequences are frequently used to suppress gene expression. However, controlling the concentration of small molecules intracellularly through designed RNA sequences that fold into ligand-binding structures is difficult. The development of "endless", a triplex-based folding motif that can be expressed in mammalian cells and binds the second messenger 3',5'-cyclic guanosine monophosphate (cGMP), is described. In vitro, DNA or RNA versions of endless show low micromolar to nanomolar dissociation constants for cGMP. To test its functionality in vivo, four endless RNA motifs arranged in tandem were co-expressed with a fluorescent cGMP sensor protein in murine vascular smooth muscle cells. Nitric oxide induced endogenous cGMP signals were suppressed in endless-expressing cells compared to cells expressing a control motif, which suggests that endless can act as a genetically encoded cGMP sink to modulate signal transduction in cells.


Assuntos
Músculo Liso Vascular/metabolismo , Motivos de Nucleotídeos/genética , Purinas/metabolismo , Animais , Expressão Gênica , Camundongos , Músculo Liso Vascular/citologia , Purinas/química
3.
Chemistry ; 19(47): 15879-87, 2013 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-24194407

RESUMO

Cofactors are pivotal compounds for the cell and many biotechnological processes. It is therefore interesting to ask how well cofactors can be bound by oligonucleotides designed not to convert but to store and release these biomolecules. Here we show that triplex-based DNA binding motifs can be used to bind nucleotides and cofactors, including NADH, FAD, SAM, acetyl CoA, and tetrahydrofolate (THF). Dissociation constants between 0.1 µM for SAM and 35 µM for THF were measured. A two-nucleotide gap still binds NADH. The selectivity for one ligand over the others can be changed by changing the sequence of the binding pocket. For example, a mismatch placed in one of the two triplets adjacent to the base-pairing site changes the selectivity, favoring the binding of FAD over that of ATP. Further, changing one of the two thymines of an A-binding motif to cytosine gives significant affinity for G, whereas changing the other does not. Immobilization of DNA motifs gives beads that store NADH. Exploratory experiments show that the beads release the cofactor upon warming to body temperature.


Assuntos
DNA/metabolismo , Coenzimas/química , Coenzimas/metabolismo , DNA/química , Ácidos Nucleicos Imobilizados/química , Motivos de Nucleotídeos , Transição de Fase , Raios Ultravioleta
4.
J Org Chem ; 76(8): 2933-6, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21413744

RESUMO

Gaps in the central strand of oligonucleotide triplexes bind nucleoside phosphates tightly. Watson-Crick and Hoogsteen base pairing as design principle yield motifs with high affinity for nucleoside phosphates with A or G as nucleobase, including ATP. The second messenger 3',5'-cAMP is bound with nanomolar affinity. A designed DNA motif accommodates seven nucleotides at a time. The design was implemented for both DNA and RNA.


Assuntos
Pareamento de Bases , AMP Cíclico/metabolismo , DNA/metabolismo , Nucleotídeos/metabolismo , Oligonucleotídeos/metabolismo , RNA/metabolismo , Sequência de Bases , Sítios de Ligação , Conservação de Recursos Energéticos/métodos , DNA/química , Ligação de Hidrogênio , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Nucleotídeos/química , Oligonucleotídeos/química , Espectroscopia Fotoeletrônica , RNA/química , Sistemas do Segundo Mensageiro , Termodinâmica
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