Background The ChAdOx1 nCoV-19 (AZD1222) vaccine has been approved for emergency use by the UK regulatory authority, Medicines and Healthcare products Regulatory Agency, with a regimen of two standard doses given with an interval of 4–12 weeks. The planned roll-out in the UK will involve vaccinating people in high-risk categories with their first dose immediately, and delivering the second dose 12 weeks later. Here, we provide both a further prespecified pooled analysis of trials of ChAdOx1 nCoV-19 and exploratory analyses of the impact on immunogenicity and efficacy of extending the interval between priming and booster doses. In addition, we show the immunogenicity and protection afforded by the first dose, before a booster dose has been offered. Methods We present data from three single-blind randomised controlled trials—one phase 1/2 study in the UK (COV001), one phase 2/3 study in the UK (COV002), and a phase 3 study in Brazil (COV003)—and one double-blind phase 1/2 study in South Africa (COV005). As previously described, individuals 18 years and older were randomly assigned 1:1 to receive two standard doses of ChAdOx1 nCoV-19 (5 × 10 10 viral particles) or a control vaccine or saline placebo. In the UK trial, a subset of participants received a lower dose (2·2 × 10 10 viral particles) of the ChAdOx1 nCoV-19 for the first dose. The primary outcome was virologically confirmed symptomatic COVID-19 disease, defined as a nucleic acid amplification test (NAAT)-positive swab combined with at least one qualifying symptom (fever ≥37·8°C, cough, shortness of breath, or anosmia or ageusia) more than 14 days after the second dose. Secondary efficacy analyses included cases occuring at least 22 days after the first dose. Antibody responses measured by immunoassay and by pseudovirus neutralisation were exploratory outcomes. All cases of COVID-19 with a NAAT-positive swab were adjudicated for inclusion in the analysis by a masked independent endpoint review committee. The primary analysis included all participants who were SARS-CoV-2 N protein seronegative at baseline, had had at least 14 days of follow-up after the second dose, and had no evidence of previous SARS-CoV-2 infection from NAAT swabs. Safety was assessed in all participants who received at least one dose. The four trials are registered at ISRCTN89951424 (COV003) and ClinicalTrials.gov , NCT04324606 (COV001), NCT04400838 (COV002), and NCT04444674 (COV005). Findings Between April 23 and Dec 6, 2020, 24 422 participants were recruited and vaccinated across the four studies, of whom 17 178 were included in the primary analysis (8597 receiving ChAdOx1 nCoV-19 and 8581 receiving control vaccine). The data cutoff for these analyses was Dec 7, 2020. 332 NAAT-positive infections met the primary endpoint of symptomatic infection more t...

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We have isolated artificial ligands or aptamers for infectious prions in order to investigate conformational aspects of prion pathogenesis. The aptamers are 2 -fluoro-modified RNA produced by in vitro selection from a large, randomized library. One of these ligands (aptamer SAF-93) had more than 10-fold higher affinity for PrP Sc than for recombinant PrP C and inhibited the accumulation of PrP res in near physiological cell-free conversion assay. To understand the molecular basis of these properties and to distinguish specific from nonspecific aptamer-PrP interactions, we studied deletion mutants of bovine PrP in denatured, ␣-helix-rich and ␤-sheet-rich forms. We provide evidence that, like scrapie-associated fibrils (SAF), the ␤-oligomer of PrP bound to SAF-93 with at least 10-fold higher affinity than did the ␣-form. This differential affinity could be explained by the existence of two binding sites within the PrP molecule. Site 1 lies within residues 23-110 in the unstructured N terminus and is a nonspecific RNA binding site found in all forms of PrP. The region between residue 90 and 110 forms a hinge region that is occluded in the ␣-rich form of PrP but becomes exposed in the denatured form of PrP. Site 2 lies in the region C-terminal of residue 110. This site is ␤-sheet conformation-specific and is not recognized by control RNAs. Taken together, these data provide for the first time a specific ligand for a disease conformation-associated site in a region of PrP critical for conformational conversion. This aptamer could provide tools for the further analysis of the processes of PrP misfolding during prion disease and leads for the development of diagnostic and therapeutic approaches to TSEs.

The conformational transition of the human prion protein from an ␣-helical to a ␤-sheet-rich structure is believed to be the critical event in prion pathogenesis. The molecular mechanism of misfolding and the role of intermediate states during this transition remain poorly understood. To overcome the obstacle of insolubility of amyloid fibrils, we have studied a ␤-sheet-rich misfolded isoform of the prion protein, the ␤-oligomer, which shares some structural properties with amyloid, including partial proteinase resistance. We demonstrate here that the ␤-oligomer can be studied by solution-state NMR spectroscopy and obtain insights into the misfolding mechanism via its transient monomeric precursor. It is often assumed that misfolding into ␤-sheet-rich isoforms proceeds via a compatible precursor with a ␤-sheet subunit structure. We show here, on the contrary, evidence for an almost natively ␣-helix-rich monomeric precursor state with molten globule characteristics, converting in vitro into the ␤-oligomer. We propose a possible mechanism for the formation of the ␤-oligomer, triggered by intermolecular contacts between constantly rearranging structures. It is concluded that the ␤-oligomer is not preceded by precursors with ␤-sheet structure but by a partially unfolded clearly distinguishable ␣-helical state.The misfolding of the prion protein is the cause of several fatal neurodegenerative diseases in humans and animals (1, 2). Among these are scrapie in sheep, bovine spongiform encephalopathy in cattle and, in humans, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, fatal familial insomnia, and kuru. These diseases are associated with the misfolding of the ␣-helix-rich cellular prion PrP C 3 protein to the ␤-sheetrich, PrP Sc form (1, 3). The native state of PrP C is represented by that of the 19.8-kDa protein (residues 89 -231), which consists of three ␣-helices and a short anti-parallel ␤-sheet (4) and will be denoted here as ␣ N . The mechanism of conversion of ␣ N to the pathogenic PrP Sc form remains unclear, although it is now known that it occurs post-translationally without detectable covalent modifications (5). The transformation requires a substantial change of conformation from an ␣-helix-rich monomer to a ␤-sheet-rich amyloid structure.In vitro, ␣ N can be converted into a variety of stable nonnative structures with high ␤-sheet content (6, 7). Different solution conditions can induce different structures, one of which is the ␤-oligomer, ␤ ANS). In contrast to PrPSc , ␤ O is soluble, monodisperse, and does not bind thioflavin T (6). Importantly, ␤ O is not a template for the formation of PrP Sc ; on the contrary, it is a very stable long-lived macromolecular assembly, the formation of which proceeds through a pathway that competes with amyloid formation (6). In recent publications (9, 10), the polymorphism at codon 129 was shown to exhibit a measurable effect on the kinetics of formation of ␤ O . It was further shown that ␤ O formed from an equimolar mixture of valine 129 and methio...

The human PrP gene (PRNP) has two common alleles that encode either methionine or valine at codon 129. This polymorphism modulates disease susceptibility and phenotype of human transmissible spongiform encyphalopathies, but the molecular mechanism by which these effects are mediated remains unclear. Here, we compared the misfolding pathway that leads to the formation of ␤-sheet-rich oligomeric isoforms of the methionine 129 variant of PrP to that of the valine 129 variant. We provide evidence for differences in the folding behavior between the two variants at the early stages of oligomer formation. We show that Met 129 has a higher propensity to form ␤-sheet-rich oligomers, whereas Val 129 has a higher tendency to fold into ␣-helical-rich monomers. An equimolar mixture of both variants displayed an intermidate folding behavior. We show that the oligomers of both variants are initially a mixture of ␣-and ␤-rich conformers that evolve with time to an increasingly homogeneous ␤-rich form. This maturation process, which involves no further change in proteinase K resistance, occurs more rapidly in the Met 129 form than the Val 129 form. Although the involvement of such ␤-rich oligomers in prion pathogenesis is speculative, the misfolding behavior could, in part, explain the higher susceptibility of individuals that are methionine homozygote to both sporadic and variant CreutzfeldtJakob disease.

New evidence indicates that termination of transcription is an important regulatory step, closely related to transcriptional interference and even transcriptional initiation. However, how this occurs is poorly understood. Recently, in vivo analysis of transcriptional termination for the human beta-globin gene revealed a new phenomenon--co-transcriptional cleavage (CoTC). This primary cleavage event within beta-globin pre-messenger RNA, downstream of the poly(A) site, is critical for efficient transcriptional termination by RNA polymerase II. Here we show that the CoTC process in the human beta-globin gene involves an RNA self-cleaving activity. We characterize the autocatalytic core of the CoTC ribozyme and show its functional role in efficient termination in vivo. The identified core CoTC is highly conserved in the 3' flanking regions of other primate beta-globin genes. Functionally, it resembles the 3' processive, self-cleaving ribozymes described for the protein-encoding genes from the myxomycetes Didymium iridis and Physarum polycephalum, indicating evolutionary conservation of this molecular process. We predict that regulated autocatalytic cleavage elements within pre-mRNAs may be a general phenomenon and that functionally it may provide the entry point for exonucleases involved in mRNA maturation, turnover and, in particular, transcriptional termination.

We have recently described the isolation of 2-fluoropyrimidine-substituted RNA aptamers that bind selectively to disease-associated ␤-sheet-rich forms of the prion protein, PrP, from a number of mammalian species. These aptamers inhibit the accumulation of protease-resistant forms of PrP in a prion-seeded, in vitro conversion assay. Here we identify the minimal portions of two of these aptamers that retain binding specificity. We determine their secondary structures by a combination of modeling and solution probing. Finally, we identify an internal site for biotinylation of a minimized, synthetic aptamer and use the resultant reagent in the detection of abnormal forms of PrP in vitro.