Comprehensive assessment of sequence variation within the copy number variable defensin cluster on 8p23 by target enriched in-depth 454 sequencing

<p>Abstract</p> <p>Background</p> <p>In highly copy number variable (CNV) regions such as the human defensin gene locus, comprehensive assessment of sequence variations is challenging. PCR approaches are practically restricted to tiny fractions, and next-generation sequencing (NGS) approaches of whole individual genomes e.g. by the 1000 Genomes Project is confined by an affordable sequence depth. Combining target enrichment with NGS may represent a feasible approach.</p> <p>Results</p> <p>As a proof of principle, we enriched a ~850 kb section comprising the CNV defensin gene cluster DEFB, the invariable DEFA part and 11 control regions from two genomes by sequence capture and sequenced it by 454 technology. 6,651 differences to the human reference genome were found. Comparison to HapMap genotypes revealed sensitivities and specificities in the range of 94% to 99% for the identification of variations.</p> <p>Using error probabilities for rigorous filtering revealed 2,886 unique single nucleotide variations (SNVs) including 358 putative novel ones. DEFB CN determinations by haplotype ratios were in agreement with alternative methods.</p> <p>Conclusion</p> <p>Although currently labor extensive and having high costs, target enriched NGS provides a powerful tool for the comprehensive assessment of SNVs in highly polymorphic CNV regions of individual genomes. Furthermore, it reveals considerable amounts of putative novel variations and simultaneously allows CN estimation.</p

Single-dose administration and the influence of the timing of the booster dose on immunogenicity and efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine: a pooled analysis of four randomised trials.

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 × 1010 viral particles) or a control vaccine or saline placebo. In the UK trial, a subset of participants received a lower dose (2·2 × 1010 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 than 14 days after the second dose. Overall vaccine efficacy more than 14 days after the second dose was 66·7% (95% CI 57·4-74·0), with 84 (1·0%) cases in the 8597 participants in the ChAdOx1 nCoV-19 group and 248 (2·9%) in the 8581 participants in the control group. There were no hospital admissions for COVID-19 in the ChAdOx1 nCoV-19 group after the initial 21-day exclusion period, and 15 in the control group. 108 (0·9%) of 12 282 participants in the ChAdOx1 nCoV-19 group and 127 (1·1%) of 11 962 participants in the control group had serious adverse events. There were seven deaths considered unrelated to vaccination (two in the ChAdOx1 nCov-19 group and five in the control group), including one COVID-19-related death in one participant in the control group. Exploratory analyses showed that vaccine efficacy after a single standard dose of vaccine from day 22 to day 90 after vaccination was 76·0% (59·3-85·9). Our modelling analysis indicated that protection did not wane during this initial 3-month period. Similarly, antibody levels were maintained during this period with minimal waning by day 90 (geometric mean ratio [GMR] 0·66 [95% CI 0·59-0·74]). In the participants who received two standard doses, after the second dose, efficacy was higher in those with a longer prime-boost interval (vaccine efficacy 81·3% [95% CI 60·3-91·2] at ≥12 weeks) than in those with a short interval (vaccine efficacy 55·1% [33·0-69·9] at <6 weeks). These observations are supported by immunogenicity data that showed binding antibody responses more than two-fold higher after an interval of 12 or more weeks compared with an interval of less than 6 weeks in those who were aged 18-55 years (GMR 2·32 [2·01-2·68]). INTERPRETATION: The results of this primary analysis of two doses of ChAdOx1 nCoV-19 were consistent with those seen in the interim analysis of the trials and confirm that the vaccine is efficacious, with results varying by dose interval in exploratory analyses. A 3-month dose interval might have advantages over a programme with a short dose interval for roll-out of a pandemic vaccine to protect the largest number of individuals in the population as early as possible when supplies are scarce, while also improving protection after receiving a second dose. FUNDING: UK Research and Innovation, National Institutes of Health Research (NIHR), The Coalition for Epidemic Preparedness Innovations, the Bill & Melinda Gates Foundation, the Lemann Foundation, Rede D'Or, the Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca

Proof-of-possession for KEM certificates using verifiable generation

Certificate authorities in public key infrastructures typically require entities to prove possession of the secret key corresponding to the public key they want certified. While this is straightforward for digital signature schemes, the most efficient solution for public key encryption and key encapsulation mechanisms (KEMs) requires an interactive challenge-response protocol, requiring a departure from current issuance processes. In this work we investigate how to non-interactively prove possession of a KEM secret key, specifically for lattice-based KEMs, motivated by the recently proposed KEMTLS protocol which replaces signature-based authentication in TLS 1.3 with KEM-based authentication. Although there are various zero-knowledge (ZK) techniques that can be used to prove possession of a lattice key, they yield large proofs or are inefficient to generate. We propose a technique called verifiable generation, in which a proof of possession is generated at the same time as the key itself is generated. Our technique is inspired by the Picnic signature scheme and uses the multi-party-computation-in-the-head (MPCitH) paradigm; this similarity to a signature scheme allows us to bind attribute data to the proof of possession, as required by certificate issuance protocols. We show how to instantiate this approach for two lattice-based KEMs in Round 3 of the NIST post-quantum cryptography standardization project, Kyber and FrodoKEM, and achieve reasonable proof sizes and performance. Our proofs of possession are faster and an order of magnitude smaller than the previous best MPCitH technique for knowledge of a lattice key, and in size-optimized cases can be comparable to even state-of-the-art direct lattice-based ZK proofs for Kyber. Our approach relies on a new result showing the uniqueness of Kyber and FrodoKEM secret keys, even if the requirement that all secret key components are small is partially relaxed, which may be of independent interest for improving efficiency of zero-knowledge proofs for other lattice-based statements

Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease.

We identified rare coding variants associated with Alzheimer's disease in a three-stage case-control study of 85,133 subjects. In stage 1, we genotyped 34,174 samples using a whole-exome microarray. In stage 2, we tested associated variants (P < 1 × 10-4) in 35,962 independent samples using de novo genotyping and imputed genotypes. In stage 3, we used an additional 14,997 samples to test the most significant stage 2 associations (P < 5 × 10-8) using imputed genotypes. We observed three new genome-wide significant nonsynonymous variants associated with Alzheimer's disease: a protective variant in PLCG2 (rs72824905: p.Pro522Arg, P = 5.38 × 10-10, odds ratio (OR) = 0.68, minor allele frequency (MAF)cases = 0.0059, MAFcontrols = 0.0093), a risk variant in ABI3 (rs616338: p.Ser209Phe, P = 4.56 × 10-10, OR = 1.43, MAFcases = 0.011, MAFcontrols = 0.008), and a new genome-wide significant variant in TREM2 (rs143332484: p.Arg62His, P = 1.55 × 10-14, OR = 1.67, MAFcases = 0.0143, MAFcontrols = 0.0089), a known susceptibility gene for Alzheimer's disease. These protein-altering changes are in genes highly expressed in microglia and highlight an immune-related protein-protein interaction network enriched for previously identified risk genes in Alzheimer's disease. These genetic findings provide additional evidence that the microglia-mediated innate immune response contributes directly to the development of Alzheimer's disease