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
Structural and biochemical features suggest that the almost ubiquitous bacterial YbeY protein may serve catalytic and/or Hfq-like protective functions central to small RNA (sRNA)-mediated regulation and RNA metabolism. We have biochemically and genetically characterized the YbeY ortholog of the legume symbiont Sinorhizobium meliloti (SmYbeY). Co-immunoprecipitation (CoIP) with a FLAG-tagged SmYbeY yielded a poor enrichment in RNA species, compared to Hfq CoIP-RNA uncovered previously by a similar experimental setup. Purified SmYbeY behaved as a monomer that indistinctly cleaved single- and double-stranded RNA substrates, a unique ability among bacterial endoribonucleases. SmYbeY-mediated catalysis was supported by the divalent metal ions Mg2+, Mn2+ and Ca2+, which influenced in a different manner cleavage efficiency and reactivity patterns, with Ca2+ specifically blocking activity on double-stranded and some structured RNA molecules. SmYbeY loss-of-function compromised expression of core energy and RNA metabolism genes, whilst promoting accumulation of motility, late symbiotic and transport mRNAs. Some of the latter transcripts are known Hfq-binding sRNA targets and might be SmYbeY substrates. Genetic reporter and in vitro assays confirmed that SmYbeY is required for sRNA-mediated down-regulation of the amino acid ABC transporter prbA mRNA. We have thus discovered a bacterial endoribonuclease with unprecedented catalytic features, acting also as gene silencing enzyme.
Assuntos
Proteínas de Bactérias/metabolismo , Endorribonucleases/metabolismo , Sinorhizobium meliloti/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Catálise , Cromossomos Bacterianos/genética , Endorribonucleases/química , Endorribonucleases/genética , Deleção de Genes , Perfilação da Expressão Gênica , Inativação Gênica , Genes Bacterianos , Genes Reporter , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Metaloproteínas/química , Metaloproteínas/genética , Metaloproteínas/metabolismo , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/genética , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sinorhizobium meliloti/genética , Especificidade por Substrato , Simbiose/genéticaRESUMO
MOTIVATION: The vast amount of already available and currently generated read mapping data requires comprehensive visualization, and should benefit from bioinformatics tools offering a wide spectrum of analysis functionality from just one source. Appropriate handling of multiple mapped reads during mapping analyses remains an issue that demands improvement. RESULTS: The capabilities of the read mapping analysis and visualization tool ReadXplorer were vastly enhanced. Here, we present an even finer granulated read mapping classification, improving the level of detail for analyses and visualizations. The spectrum of automatic analysis functions has been broadened to include genome rearrangement detection as well as correlation analysis between two mapping data sets. Existing functions were refined and enhanced, namely the computation of differentially expressed genes, the read count and normalization analysis and the transcription start site detection. Additionally, ReadXplorer 2 features a highly improved support for large eukaryotic data sets and a command line version, enabling its integration into workflows. Finally, the new version is now able to display any kind of tabular results from other bioinformatics tools. AVAILABILITY AND IMPLEMENTATION: http://www.readxplorer.org CONTACT: readxplorer@computational.bio.uni-giessen.deSupplementary information: Supplementary data are available at Bioinformatics online.
Assuntos
Biologia Computacional/métodos , Variação Estrutural do Genoma , Análise de Sequência de RNA/métodos , Software , Sítio de Iniciação de Transcrição , Arabidopsis/genética , Expressão Gênica , Genoma , RNA de Plantas/genéticaRESUMO
UNLABELLED: Sinorhizobium meliloti undergoes major lifestyle changes between planktonic states, biofilm formation, and symbiosis with leguminous plant hosts. In many bacteria, the second messenger 3',5'-cyclic di-GMP (c-di-GMP, or cdG) promotes a sessile lifestyle by regulating a plethora of processes involved in biofilm formation, including motility and biosynthesis of exopolysaccharides (EPS). Here, we systematically investigated the role of cdG in S. meliloti Rm2011 encoding 22 proteins putatively associated with cdG synthesis, degradation, or binding. Single mutations in 21 of these genes did not cause evident changes in biofilm formation, motility, or EPS biosynthesis. In contrast, manipulation of cdG levels by overproducing endogenous or heterologous diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) affected these processes and accumulation of N-Acyl-homoserine lactones in the culture supernatant. Specifically, individual overexpression of the S. meliloti genes pleD, SMb20523, SMb20447, SMc01464, and SMc03178 encoding putative DGCs and of SMb21517 encoding a single-domain PDE protein had an impact and resulted in increased levels of cdG. Compared to the wild type, an S. meliloti strain that did not produce detectable levels of cdG (cdG(0)) was more sensitive to acid stress. However, it was symbiotically potent, unaffected in motility, and only slightly reduced in biofilm formation. The SMc01790-SMc01796 locus, homologous to the Agrobacterium tumefaciens uppABCDEF cluster governing biosynthesis of a unipolarly localized polysaccharide, was found to be required for cdG-stimulated biofilm formation, while the single-domain PilZ protein McrA was identified as a cdG receptor protein involved in regulation of motility. IMPORTANCE: We present the first systematic genome-wide investigation of the role of 3',5'-cyclic di-GMP (c-di-GMP, or cdG) in regulation of motility, biosynthesis of exopolysaccharides, biofilm formation, quorum sensing, and symbiosis in a symbiotic alpha-rhizobial species. Phenotypes of an S. meliloti strain unable to produce cdG (cdG(0)) demonstrated that this second messenger is not essential for root nodule symbiosis but may contribute to acid tolerance. Our data further suggest that enhanced levels of cdG promote sessility of S. meliloti and uncovered a single-domain PilZ protein as regulator of motility.
Assuntos
GMP Cíclico/análogos & derivados , Regulação Bacteriana da Expressão Gênica/fisiologia , Sinorhizobium meliloti/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , GMP Cíclico/genética , GMP Cíclico/metabolismo , Mutação , Polissacarídeos Bacterianos/biossíntese , Sinorhizobium meliloti/genéticaRESUMO
BACKGROUND: Differential RNA-Seq (dRNA-Seq) is a recently developed method of performing primary transcriptome analyses that allows for the genome-wide mapping of transcriptional start sites (TSSs) and the identification of novel transcripts. Although the transcriptomes of diverse bacterial species have been characterized by dRNA-Seq, the transcriptome analysis of archaeal species is still rather limited. Therefore, we used dRNA-Seq to characterize the primary transcriptome of the model archaeon Haloferax volcanii. RESULTS: Three independent cultures of Hfx. volcanii grown under optimal conditions to the mid-exponential growth phase were used to determine the primary transcriptome and map the 5'-ends of the transcripts. In total, 4749 potential TSSs were detected. A position weight matrix (PWM) was derived for the promoter predictions, and the results showed that 64 % of the TSSs were preceded by stringent or relaxed basal promoters. Of the identified TSSs, 1851 belonged to protein-coding genes. Thus, fewer than half (46 %) of the 4040 protein-coding genes were expressed under optimal growth conditions. Seventy-two percent of all protein-coding transcripts were leaderless, which emphasized that this pathway is the major pathway for translation initiation in haloarchaea. A total of 2898 of the TSSs belonged to potential non-coding RNAs, which accounted for an unexpectedly high fraction (61 %) of all transcripts. Most of the non-coding TSSs had not been previously described (2792) and represented novel sequences (59 % of all TSSs). A large fraction of the potential novel non-coding transcripts were cis-antisense RNAs (1244 aTSSs). A strong negative correlation between the levels of antisense transcripts and cognate sense mRNAs was found, which suggested that the negative regulation of gene expression via antisense RNAs may play an important role in haloarchaea. The other types of novel non-coding transcripts corresponded to internal transcripts overlapping with mRNAs (1153 iTSSs) and intergenic small RNA (sRNA) candidates (395 TSSs). CONCLUSION: This study provides a comprehensive map of the primary transcriptome of Hfx. volcanii grown under optimal conditions. Fewer than half of all protein-coding genes have been transcribed under these conditions. Unexpectedly, more than half of the detected TSSs belonged to several classes of non-coding RNAs. Thus, RNA-based regulation appears to play a more important role in haloarchaea than previously anticipated.
Assuntos
Genoma Arqueal , Haloferax volcanii/genética , RNA Arqueal/metabolismo , Regiões 5' não Traduzidas , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas , RNA Antissenso/metabolismo , RNA Arqueal/química , RNA Arqueal/isolamento & purificação , RNA não Traduzido/metabolismo , Análise de Sequência de RNA , Sítio de Iniciação de Transcrição , TranscriptomaRESUMO
The population genomics of Pseudomonas aeruginosa was analysed by genome sequencing of representative strains of the 15 most frequent clonal complexes in the P. aeruginosa population and of the five most common clones from the environment of which so far no isolate from a human infection has been detected. Gene annotation identified 5892-7187 open reading frame (ORFs; median 6381 ORFs) in the 20 6.4-7.4 Mbp large genomes. The P. aeruginosa pangenome consists of a conserved core of at least 4000 genes, a combinatorial accessory genome of a further 10 000 genes and 30 000 or more rare genes that are present in only a few strains or clonal complexes. Whole genome comparisons of single nucleotide polymorphism synteny indicated unrestricted gene flow between clonal complexes by recombination. Using standardized acute lettuce, Galleria mellonella and murine airway infection models the full spectrum of possible host responses to P. aeruginosa was observed with the 20 strains ranging from unimpaired health following infection to 100% lethality. Genome comparisons indicate that the differential genetic repertoire of clones maintains a habitat-independent gradient of virulence in the P. aeruginosa population.
Assuntos
Genoma Bacteriano , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Animais , Microbiologia Ambiental , Feminino , Variação Genética , Humanos , Pneumopatias/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mariposas/microbiologia , Fases de Leitura Aberta , Doenças das Plantas/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , Virulência/genéticaRESUMO
MOTIVATION: Fast algorithms and well-arranged visualizations are required for the comprehensive analysis of the ever-growing size of genomic and transcriptomic next-generation sequencing data. RESULTS: ReadXplorer is a software offering straightforward visualization and extensive analysis functions for genomic and transcriptomic DNA sequences mapped on a reference. A unique specialty of ReadXplorer is the quality classification of the read mappings. It is incorporated in all analysis functions and displayed in ReadXplorer's various synchronized data viewers for (i) the reference sequence, its base coverage as (ii) normalizable plot and (iii) histogram, (iv) read alignments and (v) read pairs. ReadXplorer's analysis capability covers RNA secondary structure prediction, single nucleotide polymorphism and deletion-insertion polymorphism detection, genomic feature and general coverage analysis. Especially for RNA-Seq data, it offers differential gene expression analysis, transcription start site and operon detection as well as RPKM value and read count calculations. Furthermore, ReadXplorer can combine or superimpose coverage of different datasets. AVAILABILITY AND IMPLEMENTATION: ReadXplorer is available as open-source software at http://www.readxplorer.org along with a detailed manual.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Alinhamento de Sequência/métodos , Análise de Sequência de DNA/métodos , Software , Gráficos por Computador , Perfilação da Expressão Gênica , Genômica/métodos , Óperon , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , RNA/química , Sítio de Iniciação de TranscriçãoRESUMO
SUMMARY: UniMoG is a software combining five genome rearrangement models: double cut and join (DCJ), restricted DCJ, Hannenhalli and Pevzner (HP), inversion and translocation. It can compute the pairwise genomic distances and a corresponding optimal sorting scenario for an arbitrary number of genomes. All five models can be unified through the DCJ model, thus the implementation is based on DCJ and, where reasonable, uses the most efficient existing algorithms for each distance and sorting problem. Both textual and graphical output is possible for visualizing the operations. AVAILABILITY AND IMPLEMENTATION: The software is available through the Bielefeld University Bioinformatics Web Server at http://bibiserv.techfak.uni-bielefeld.de/dcj with instructions and example data. CONTACT: rhilker@cebitec.uni-bielefeld.de.
Assuntos
Algoritmos , Biologia Computacional/métodos , Genômica/métodos , Software , Internet , Modelos Genéticos , Interface Usuário-ComputadorAssuntos
Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Transferência Genética Horizontal/genética , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Genoma Bacteriano/genética , Humanos , Plasmídeos/genética , Virulência/genética , Sequenciamento Completo do GenomaRESUMO
Bacterial populations differentiate at the subspecies level into clonal complexes. Intraclonal genome diversity was studied in 100 isolates of the two dominant Pseudomonas aeruginosa clones C and PA14 collected from the inanimate environment, acute and chronic infections. The core genome was highly conserved among clone members with a median pairwise within-clone single nucleotide sequence diversity of 8 × 10(-6) for clone C and 2 × 10(-5) for clone PA14. The composition of the accessory genome was, on the other hand, as variable within the clone as between unrelated clones. Each strain carried a large cargo of unique genes. The two dominant worldwide distributed P. aeruginosa clones combine an almost invariant core with the flexible gain and loss of genetic elements that spread by horizontal transfer.
Assuntos
Variação Genética , Genótipo , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/genética , Sequência Conservada , Microbiologia Ambiental , Transferência Genética Horizontal , Genoma Bacteriano , Humanos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificaçãoRESUMO
Members of the Sm protein family are important for the cellular RNA metabolism in all three domains of life. The family includes archaeal and eukaryotic Lsm proteins, eukaryotic Sm proteins and archaeal and bacterial Hfq proteins. While several studies concerning the bacterial and eukaryotic family members have been published, little is known about the archaeal Lsm proteins. Although structures for several archaeal Lsm proteins have been solved already more than ten years ago, we still do not know much about their biological function, however one can confidently propose that the archaeal Lsm proteins will also be involved in RNA metabolism. Therefore, we investigated this protein in the halophilic archaeon Haloferax volcanii. The Haloferax genome encodes a single Lsm protein, the lsm gene overlaps and is co-transcribed with the gene for the ribosomal L37.eR protein. Here, we show that the reading frame of the lsm gene contains a promoter which regulates expression of the overlapping rpl37R gene. This rpl37R specific promoter ensures high expression of the rpl37R gene in exponential growth phase. To investigate the biological function of the Lsm protein we generated a lsm deletion mutant that had the coding sequence for the Sm1 motif removed but still contained the internal promoter for the downstream rpl37R gene. The transcriptome of this deletion mutant was compared to the wild type transcriptome, revealing that several genes are down-regulated and many genes are up-regulated in the deletion strain. Northern blot analyses confirmed down-regulation of two genes. In addition, the deletion strain showed a gain of function in swarming, in congruence with the up-regulation of transcripts encoding proteins required for motility.
Assuntos
Proteínas Arqueais/genética , Deleção de Genes , Haloferax volcanii/genética , Motivos de Nucleotídeos/genética , Transcriptoma/genética , Proteínas Arqueais/metabolismo , Sequência de Bases , Northern Blotting , Regulação para Baixo , Regulação da Expressão Gênica em Archaea , Genoma Arqueal/genética , Haloferax volcanii/metabolismo , Haloferax volcanii/fisiologia , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas/genética , RNA Arqueal/genética , RNA Arqueal/metabolismo , Proteínas Ribossômicas/genética , Regulação para CimaRESUMO
Listeria monocytogenes is a bacterial pathogen and causative agent for the foodborne infection listeriosis, which is mainly a threat for pregnant, elderly, or immunocompromised individuals. Due to its ability to invade and colonize diverse eukaryotic cell types including cells from invertebrates, L. monocytogenes has become a well-established model organism for intracellular growth. Almost 10 years ago, we and others presented the first whole-genome microarray-based intracellular transcriptome of L. monocytogenes. With the advent of newer technologies addressing transcriptomes in greater detail, we revisit this work, and analyze the intracellular transcriptome of L. monocytogenes during growth in murine macrophages using a deep sequencing based approach. We detected 656 differentially expressed genes of which 367 were upregulated during intracellular growth in macrophages compared to extracellular growth in Brain Heart Infusion broth. This study confirmed â¼64% of all regulated genes previously identified by microarray analysis. Many of the regulated genes that were detected in the current study involve transporters for various metals, ions as well as complex sugars such as mannose. We also report changes in antisense transcription, especially upregulations during intracellular bacterial survival. A notable finding was the detection of regulatory changes for a subset of temperate A118-like prophage genes, thereby shedding light on the transcriptional profile of this bacteriophage during intracellular growth. In total, our study provides an updated genome-wide view of the transcriptional landscape of L. monocytogenes during intracellular growth and represents a rich resource for future detailed analysis.
RESUMO
Streptococcus pneumoniae is a major cause of pneumonia, sepsis and meningitis. The pore-forming toxin pneumolysin is a key virulence factor of S. pneumoniae, which can be sensed by the NLRP3 inflammasome. Among the over 90 serotypes, serotype 1 pneumococci (particularly MLST306) have emerged across the globe as a major cause of invasive disease. The cause for its particularity is, however, incompletely understood. We therefore examined pneumococcal infection in human cells and a human lung organ culture system mimicking infection of the lower respiratory tract. We demonstrate that different pneumococcal serotypes differentially activate inflammasome-dependent IL-1ß production in human lung tissue and cells. Whereas serotype 2, 3, 6B, 9N pneumococci expressing fully haemolytic pneumolysins activate NLRP3 inflammasome-dependent responses, serotype 1 and 8 strains expressing non-haemolytic toxins are poor activators of IL-1ß production. Accordingly, purified haemolytic pneumolysin but not serotype 1-associated non-haemolytic toxin activates strong IL-1ß production in human lungs. Our data suggest that the evasion of inflammasome-dependent innate immune responses by serotype 1 pneumococci might contribute to their ability to cause invasive diseases in humans.