RESUMO
BNT162b2, a nucleoside-modified mRNA formulated in lipid nanoparticles that encodes the SARS-CoV-2 spike glycoprotein (S) stabilized in its prefusion conformation, has demonstrated 95% efficacy in preventing COVID-191. Here we extend a previous phase-I/II trial report2 by presenting data on the immune response induced by BNT162b2 prime-boost vaccination from an additional phase-I/II trial in healthy adults (18-55 years old). BNT162b2 elicited strong antibody responses: at one week after the boost, SARS-CoV-2 serum geometric mean 50% neutralizing titres were up to 3.3-fold above those observed in samples from individuals who had recovered from COVID-19. Sera elicited by BNT162b2 neutralized 22 pseudoviruses bearing the S of different SARS-CoV-2 variants. Most participants had a strong response of IFNγ+ or IL-2+ CD8+ and CD4+ T helper type 1 cells, which was detectable throughout the full observation period of nine weeks following the boost. Using peptide-MHC multimer technology, we identified several BNT162b2-induced epitopes that were presented by frequent MHC alleles and conserved in mutant strains. One week after the boost, epitope-specific CD8+ T cells of the early-differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating CD8+ T cells and were detectable (0.01-0.28%) eight weeks later. In summary, BNT162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes that are conserved in a broad range of variants, at well-tolerated doses.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Vacina BNT162 , Linfócitos T CD8-Positivos/imunologia , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/efeitos adversos , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Memória Imunológica , Interferon gama/imunologia , Interleucina-2/imunologia , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Células Th1/imunologia , Adulto JovemRESUMO
An effective vaccine is needed to halt the spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. Recently, we reported safety, tolerability and antibody response data from an ongoing placebo-controlled, observer-blinded phase I/II coronavirus disease 2019 (COVID-19) vaccine trial with BNT162b1, a lipid nanoparticle-formulated nucleoside-modified mRNA that encodes the receptor binding domain (RBD) of the SARS-CoV-2 spike protein1. Here we present antibody and T cell responses after vaccination with BNT162b1 from a second, non-randomized open-label phase I/II trial in healthy adults, 18-55 years of age. Two doses of 1-50 µg of BNT162b1 elicited robust CD4+ and CD8+ T cell responses and strong antibody responses, with RBD-binding IgG concentrations clearly above those seen in serum from a cohort of individuals who had recovered from COVID-19. Geometric mean titres of SARS-CoV-2 serum-neutralizing antibodies on day 43 were 0.7-fold (1-µg dose) to 3.5-fold (50-µg dose) those of the recovered individuals. Immune sera broadly neutralized pseudoviruses with diverse SARS-CoV-2 spike variants. Most participants had T helper type 1 (TH1)-skewed T cell immune responses with RBD-specific CD8+ and CD4+ T cell expansion. Interferon-γ was produced by a large fraction of RBD-specific CD8+ and CD4+ T cells. The robust RBD-specific antibody, T cell and favourable cytokine responses induced by the BNT162b1 mRNA vaccine suggest that it has the potential to protect against COVID-19 through multiple beneficial mechanisms.
Assuntos
Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Células Th1/imunologia , Vacinas Virais/imunologia , Adulto , Anticorpos Neutralizantes/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , COVID-19 , Vacinas contra COVID-19 , Infecções por Coronavirus/prevenção & controle , Citocinas/imunologia , Feminino , Alemanha , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Pandemias , Células Th1/citologia , Vacinas Virais/administração & dosagem , Vacinas Virais/efeitos adversos , Adulto JovemRESUMO
Exon definition is the predominant initial spliceosome assembly pathway in higher eukaryotes, but it remains much less well-characterized compared to the intron-defined assembly pathway. Addition in trans of an excess of 5'ss containing RNA to a splicing reaction converts a 37S exon-defined complex, formed on a single exon RNA substrate, into a 45S B-like spliceosomal complex with stably integrated U4/U6.U5 tri-snRNP. This 45S complex is compositonally and structurally highly similar to an intron-defined spliceosomal B complex. Stable tri-snRNP integration during B-like complex formation is accompanied by a major structural change as visualized by electron microscopy. The changes in structure and stability during transition from a 37S to 45S complex can be induced in affinity-purified cross-exon complexes by adding solely the 5'ss RNA oligonucleotide. This conformational change does not require the B-specific proteins, which are recruited during this stabilization process, or site-specific phosphorylation of hPrp31. Instead it is triggered by the interaction of U4/U6.U5 tri-snRNP components with the 5'ss sequence, most importantly between Prp8 and nucleotides at the exon-intron junction. These studies provide novel insights into the conversion of a cross-exon to cross-intron organized spliceosome and also shed light on the requirements for stable tri-snRNP integration during B complex formation.
Assuntos
Sítios de Splice de RNA/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Spliceossomos/genética , Linhagem Celular Tumoral , Éxons/genética , Proteínas do Olho/genética , Células HeLa , Humanos , Íntrons/genética , Fosforilação/genética , Splicing de RNA/genéticaRESUMO
The amoeba Dictyostelium discoideum is a well established model organism for studying numerous aspects of cellular and developmental functions. Its ribosomal RNA (rRNA) is encoded in an extrachromosomal palindrome that exists in â¼100 copies in the cell. In this study, we have set out to investigate the sequence of the expressed rRNA. For this, we have ligated the rRNA ends and performed RT-PCR on these circular RNAs. Sequencing revealed that the mature 26 S, 17 S, 5.8 S, and 5 S rRNAs have sizes of 3741, 1871, 162, and 112 nucleotides, respectively. Unlike the published data, all mature rRNAs of the same type uniformly display the same start and end nucleotides in the analyzed AX2 strain. We show the existence of a short lived primary transcript covering the rRNA transcription unit of 17 S, 5.8 S, and 26 S rRNA. Northern blots and RT-PCR reveal that from this primary transcript two precursor molecules of the 17 S and two precursors of the 26 S rRNA are generated. We have also determined the sequences of these precursor molecules, and based on these data, we propose a model for the maturation of the rRNAs in Dictyostelium discoideum that we compare with the processing of the rRNA transcription unit of Saccharomyces cerevisiae.
Assuntos
Dictyostelium/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , RNA Ribossômico/biossíntese , Sequência de Bases , Dictyostelium/genética , Dados de Sequência Molecular , RNA Ribossômico/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein acquired a D614G mutation early in the pandemic that confers greater infectivity and is now the globally dominant form. To determine whether D614G might also mediate neutralization escape that could compromise vaccine efficacy, sera from spike-immunized mice, nonhuman primates, and humans were evaluated for neutralization of pseudoviruses bearing either D614 or G614 spike. In all cases, the G614 pseudovirus was moderately more susceptible to neutralization. The G614 pseudovirus also was more susceptible to neutralization by receptor-binding domain (RBD) monoclonal antibodies and convalescent sera from people infected with either form of the virus. Negative stain electron microscopy revealed a higher percentage of the 1-RBD "up" conformation in the G614 spike, suggesting increased epitope exposure as a mechanism of enhanced vulnerability to neutralization. Based on these findings, the D614G mutation is not expected to be an obstacle for current vaccine development.
Assuntos
COVID-19/terapia , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Adolescente , Adulto , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Feminino , Células HEK293 , Humanos , Imunização Passiva/métodos , Macaca mulatta , Masculino , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Testes de Neutralização , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , Adulto Jovem , Soroterapia para COVID-19RESUMO
The precise role of the spliceosomal DEAD-box protein Prp28 in higher eukaryotes remains unclear. We show that stable tri-snRNP association during pre-catalytic spliceosomal B complex formation is blocked by a dominant-negative hPrp28 mutant lacking ATPase activity. Complexes formed in the presence of ATPase-deficient hPrp28 represent a novel assembly intermediate, the pre-B complex, that contains U1, U2 and loosely associated tri-snRNP and is stalled before disruption of the U1/5'ss base pairing interaction, consistent with a role for hPrp28 in the latter. Pre-B and B complexes differ structurally, indicating that stable tri-snRNP integration is accompanied by substantial rearrangements in the spliceosome. Disruption of the U1/5'ss interaction alone is not sufficient to bypass the block by ATPase-deficient hPrp28, suggesting hPrp28 has an additional function at this stage of splicing. Our data provide new insights into the function of Prp28 in higher eukaryotes, and the requirements for stable tri-snRNP binding during B complex formation.