Publications by authors named "David I Stuart"

248 Publications

Quantitative SARS-CoV-2 anti-spike responses to Pfizer-BioNTech and Oxford-AstraZeneca vaccines by previous infection status.

Clin Microbiol Infect 2021 Jun 7. Epub 2021 Jun 7.

Oxford University Hospitals NHS Foundation Trust, Oxford, UK.

Objectives: We investigate determinants of SARS-CoV-2 anti-spike IgG responses in healthcare workers (HCWs) following one or two doses of Pfizer-BioNTech or Oxford-AstraZeneca vaccines.

Methods: HCWs participating in regular SARS-CoV-2 PCR and antibody testing were invited for serological testing prior to first and second vaccination, and 4 weeks post-vaccination if receiving a 12-week dosing interval. Quantitative post-vaccination anti-spike antibody responses were measured using the Abbott SARS-CoV-2 IgG II Quant assay (detection threshold: ≥50 AU/ml). We used multivariable logistic regression to identify predictors of seropositivity and generalised additive models to track antibody responses over time.

Results: 3570/3610(98.9%) HCWs were seropositive >14 days post-first vaccination and prior to second vaccination, 2706/2720(99.5%) after Pfizer-BioNTech and 864/890(97.1%) following Oxford-AstraZeneca vaccines. Previously infected and younger HCWs were more likely to test seropositive post-first vaccination, with no evidence of differences by sex or ethnicity. All 470 HCWs tested >14 days after second vaccine were seropositive. Quantitative antibody responses were higher after previous infection: median(IQR) >21 days post-first Pfizer-BioNTech 14,604(7644-22,291) AU/ml vs. 1028(564-1985) AU/ml without prior infection (p<0.001). Oxford-AstraZeneca vaccine recipients had lower readings post-first dose compared to Pfizer-BioNTech, with and without previous infection, 10,095(5354-17,096) and 435(203-962) AU/ml respectively (both p<0.001 vs. Pfizer-BioNTech). Antibody responses >21 days post-second Pfizer vaccination in those not previously infected, 10,058 (6408-15,582) AU/ml, were similar to those after prior infection and one vaccine dose.

Conclusions: SARS-CoV-2 vaccination leads to detectable anti-spike antibodies in nearly all adult HCWs. Whether differences in response impact vaccine efficacy needs further study.
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http://dx.doi.org/10.1016/j.cmi.2021.05.041DOI Listing
June 2021

Antibody evasion by the P.1 strain of SARS-CoV-2.

Cell 2021 05 30;184(11):2939-2954.e9. Epub 2021 Mar 30.

Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.

Terminating the SARS-CoV-2 pandemic relies upon pan-global vaccination. Current vaccines elicit neutralizing antibody responses to the virus spike derived from early isolates. However, new strains have emerged with multiple mutations, including P.1 from Brazil, B.1.351 from South Africa, and B.1.1.7 from the UK (12, 10, and 9 changes in the spike, respectively). All have mutations in the ACE2 binding site, with P.1 and B.1.351 having a virtually identical triplet (E484K, K417N/T, and N501Y), which we show confer similar increased affinity for ACE2. We show that, surprisingly, P.1 is significantly less resistant to naturally acquired or vaccine-induced antibody responses than B.1.351, suggesting that changes outside the receptor-binding domain (RBD) impact neutralization. Monoclonal antibody (mAb) 222 neutralizes all three variants despite interacting with two of the ACE2-binding site mutations. We explain this through structural analysis and use the 222 light chain to largely restore neutralization potency to a major class of public antibodies.
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http://dx.doi.org/10.1016/j.cell.2021.03.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008340PMC
May 2021

Site-specific steric control of SARS-CoV-2 spike glycosylation.

bioRxiv 2021 Mar 9. Epub 2021 Mar 9.

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity between the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against infectious virus S protein. We find patterns which are conserved across all samples and this can be associated with site-specific stalling of glycan maturation which act as a highly sensitive reporter of protein structure. Molecular dynamics (MD) simulations of a fully glycosylated spike support s a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.
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http://dx.doi.org/10.1101/2021.03.08.433764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986994PMC
March 2021

The antigenic anatomy of SARS-CoV-2 receptor binding domain.

Cell 2021 04 18;184(8):2183-2200.e22. Epub 2021 Feb 18.

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, UK.

Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC < 0.1 μg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.
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http://dx.doi.org/10.1016/j.cell.2021.02.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891125PMC
April 2021

Antibody testing for COVID-19: A report from the National COVID Scientific Advisory Panel.

Wellcome Open Res 2020 11;5:139. Epub 2020 Jun 11.

Oxford University Hospitals NHS Foundation Trust, Oxford, OX3 9DU, UK.

The COVID-19 pandemic caused >1 million infections during January-March 2020. There is an urgent need for reliable antibody detection approaches to support diagnosis, vaccine development, safe release of individuals from quarantine, and population lock-down exit strategies. We set out to evaluate the performance of ELISA and lateral flow immunoassay (LFIA) devices. We tested plasma for COVID (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) IgM and IgG antibodies by ELISA and using nine different LFIA devices. We used a panel of plasma samples from individuals who have had confirmed COVID infection based on a PCR result (n=40), and pre-pandemic negative control samples banked in the UK prior to December-2019 (n=142). ELISA detected IgM or IgG in 34/40 individuals with a confirmed history of COVID infection (sensitivity 85%, 95%CI 70-94%), vs. 0/50 pre-pandemic controls (specificity 100% [95%CI 93-100%]). IgG levels were detected in 31/31 COVID-positive individuals tested ≥10 days after symptom onset (sensitivity 100%, 95%CI 89-100%). IgG titres rose during the 3 weeks post symptom onset and began to fall by 8 weeks, but remained above the detection threshold. Point estimates for the sensitivity of LFIA devices ranged from 55-70% versus RT-PCR and 65-85% versus ELISA, with specificity 95-100% and 93-100% respectively. Within the limits of the study size, the performance of most LFIA devices was similar. Currently available commercial LFIA devices do not perform sufficiently well for individual patient applications. However, ELISA can be calibrated to be specific for detecting and quantifying SARS-CoV-2 IgM and IgG and is highly sensitive for IgG from 10 days following first symptoms.
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http://dx.doi.org/10.12688/wellcomeopenres.15927.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941096PMC
June 2020

Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera.

Cell 2021 04 18;184(8):2201-2211.e7. Epub 2021 Feb 18.

NIHR Oxford Biomedical Research Centre, Oxford, UK; Oxford Vaccine Group, Department of Paediatrics, University of Oxford, Oxford, UK.

SARS-CoV-2 has caused over 2 million deaths in little over a year. Vaccines are being deployed at scale, aiming to generate responses against the virus spike. The scale of the pandemic and error-prone virus replication is leading to the appearance of mutant viruses and potentially escape from antibody responses. Variant B.1.1.7, now dominant in the UK, with increased transmission, harbors 9 amino acid changes in the spike, including N501Y in the ACE2 interacting surface. We examine the ability of B.1.1.7 to evade antibody responses elicited by natural SARS-CoV-2 infection or vaccination. We map the impact of N501Y by structure/function analysis of a large panel of well-characterized monoclonal antibodies. B.1.1.7 is harder to neutralize than parental virus, compromising neutralization by some members of a major class of public antibodies through light-chain contacts with residue 501. However, widespread escape from monoclonal antibodies or antibody responses generated by natural infection or vaccination was not observed.
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http://dx.doi.org/10.1016/j.cell.2021.02.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891044PMC
April 2021

Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera.

Cell 2021 04 23;184(9):2348-2361.e6. Epub 2021 Feb 23.

NIHR Oxford Biomedical Research Centre, Oxford, UK; Oxford Vaccine Group, Department of Paediatrics, University of Oxford, Oxford, UK.

The race to produce vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began when the first sequence was published, and this forms the basis for vaccines currently deployed globally. Independent lineages of SARS-CoV-2 have recently been reported: UK, B.1.1.7; South Africa, B.1.351; and Brazil, P.1. These variants have multiple changes in the immunodominant spike protein that facilitates viral cell entry via the angiotensin-converting enzyme-2 (ACE2) receptor. Mutations in the receptor recognition site on the spike are of great concern for their potential for immune escape. Here, we describe a structure-function analysis of B.1.351 using a large cohort of convalescent and vaccinee serum samples. The receptor-binding domain mutations provide tighter ACE2 binding and widespread escape from monoclonal antibody neutralization largely driven by E484K, although K417N and N501Y act together against some important antibody classes. In a number of cases, it would appear that convalescent and some vaccine serum offers limited protection against this variant.
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http://dx.doi.org/10.1016/j.cell.2021.02.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901269PMC
April 2021

Stringent thresholds in SARS-CoV-2 IgG assays lead to under-detection of mild infections.

BMC Infect Dis 2021 Feb 18;21(1):187. Epub 2021 Feb 18.

Oxford University Hospitals NHS Foundation Trust, Oxford, UK.

Background: Thresholds for SARS-CoV-2 antibody assays have typically been determined using samples from symptomatic, often hospitalised, patients. In this setting the sensitivity and specificity of the best performing assays can both exceed 98%. However, antibody assay performance following mild infection is less clear.

Methods: We assessed quantitative IgG responses in a cohort of healthcare workers in Oxford, UK, with a high pre-test probability of Covid-19, in particular the 991/11,475(8.6%) who reported loss of smell/taste. We use anosmia/ageusia and other risk factors as probes for Covid-19 infection potentially undiagnosed by immunoassays by investigating their relationship with antibody readings either side of assay thresholds.

Results: The proportion of healthcare workers reporting anosmia/ageusia increased at antibody readings below diagnostic thresholds using an in-house ELISA (n = 9324) and the Abbott Architect chemiluminescent microparticle immunoassay (CMIA; n = 11,324): 426/906 (47%) reported anosmia/ageusia with a positive ELISA, 59/449 (13.1%) with high-negative and 326/7969 (4.1%) with low-negative readings. Similarly, by CMIA, 518/1093 (47.4%) with a positive result reported anosmia/ageusia, 106/686 (15.5%) with a high-negative and 358/9563 (3.7%) with a low-negative result. Adjusting for the proportion of staff reporting anosmia/ageusia suggests the sensitivity of both assays in mild infection is lower than previously reported: Oxford ELISA 89.8% (95%CI 86.6-92.8%) and Abbott CMIA 79.3% (75.9-82.7%).

Conclusion: Following mild SARS-CoV-2 infection 10-30% of individuals may have negative immunoassay results. While lowered diagnostic thresholds may result in unacceptable specificity, our findings have implications for epidemiological analyses and result interpretation in individuals with a high pre-test probability. Samples from mild PCR-confirmed infections should be included in SARS-CoV-2 immunoassay evaluations.
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http://dx.doi.org/10.1186/s12879-021-05878-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889711PMC
February 2021

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses.

Nat Commun 2021 01 22;12(1):542. Epub 2021 Jan 22.

The Pirbright Institute, Ash Road, Pirbright, GU24 0NF, UK.

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.
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http://dx.doi.org/10.1038/s41467-020-20654-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822889PMC
January 2021

Mammalian expression of virus-like particles as a proof of principle for next generation polio vaccines.

NPJ Vaccines 2021 Jan 8;6(1). Epub 2021 Jan 8.

Division of Structural Biology, University of Oxford, The Henry Wellcome Building for Genomic Medicine, Headington, Oxford, OX3 7BN, UK.

Global vaccination programs using live-attenuated oral and inactivated polio vaccine (OPV and IPV) have almost eradicated poliovirus (PV) but these vaccines or their production pose significant risk in a polio-free world. Recombinant PV virus-like particles (VLPs), lacking the viral genome, represent safe next-generation vaccines, however their production requires optimisation. Here we present an efficient mammalian expression strategy producing good yields of wild-type PV VLPs for all three serotypes and a thermostabilised variant for PV3. Whilst the wild-type VLPs were predominantly in the non-native C-antigenic form, the thermostabilised PV3 VLPs adopted the native D-antigenic conformation eliciting neutralising antibody titres equivalent to the current IPV and were indistinguishable from natural empty particles by cryo-electron microscopy with a similar stabilising lipidic pocket-factor in the VP1 β-barrel. This factor may not be available in alternative expression systems, which may require synthetic pocket-binding factors. VLPs equivalent to these mammalian expressed thermostabilized particles, represent safer non-infectious vaccine candidates for the post-eradication era.
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http://dx.doi.org/10.1038/s41541-020-00267-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794334PMC
January 2021

The duration, dynamics and determinants of SARS-CoV-2 antibody responses in individual healthcare workers.

Clin Infect Dis 2021 Jan 6. Epub 2021 Jan 6.

Big Data Institute, University of Oxford, Oxford, UK.

Background: SARS-CoV-2 IgG antibody measurements can be used to estimate the proportion of a population exposed or infected and may be informative about the risk of future infection. Previous estimates of the duration of antibody responses vary.

Methods: We present 6 months of data from a longitudinal seroprevalence study of 3276 UK healthcare workers (HCWs). Serial measurements of SARS-CoV-2 anti-nucleocapsid and anti-spike IgG were obtained. Interval censored survival analysis was used to investigate the duration of detectable responses. Additionally, Bayesian mixed linear models were used to investigate anti-nucleocapsid waning.

Results: Anti-spike IgG levels remained stably detected after a positive result, e.g., in 94% (95% credibility interval, CrI, 91-96%) of HCWs at 180 days. Anti-nucleocapsid IgG levels rose to a peak at 24 (95% credibility interval, CrI 19-31) days post first PCR-positive test, before beginning to fall. Considering 452 anti-nucleocapsid seropositive HCWs over a median of 121 days from their maximum positive IgG titre, the mean estimated antibody half-life was 85 (95%CrI, 81-90) days. Higher maximum observed anti-nucleocapsid titres were associated with longer estimated antibody half-lives. Increasing age, Asian ethnicity and prior self-reported symptoms were independently associated with higher maximum anti-nucleocapsid levels and increasing age and a positive PCR test undertaken for symptoms with longer anti-nucleocapsid half-lives.

Conclusion: SARS-CoV-2 anti-nucleocapsid antibodies wane within months, and faster in younger adults and those without symptoms. However, anti-spike IgG remains stably detected. Ongoing longitudinal studies are required to track the long-term duration of antibody levels and their association with immunity to SARS-CoV-2 reinfection.
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http://dx.doi.org/10.1093/cid/ciab004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929225PMC
January 2021

Antibody Status and Incidence of SARS-CoV-2 Infection in Health Care Workers.

N Engl J Med 2021 02 23;384(6):533-540. Epub 2020 Dec 23.

From Oxford University Hospitals NHS Foundation Trust (S.F.L., N.E.S., P.C.M., S.C., T.J., F.W., L.W., D.A., A.-M.O., K.J.), Nuffield Department of Medicine (S.F.L., D.O., N.E.S., P.C.M., A.H., S.B.H., B.D.M., R.J.C., E.Y.J., D.I.S., G.S., D.E., S. Hoosdally, D.W.C., C.P.C., A.S.W., T.E.A.P., T.M.W.), the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (N.E.S., P.C.M., S. Hoosdally, D.W.C., A.S.W., T.E.A.P., D.W.E.), the Kennedy Institute of Rheumatology Research (B.D.M.), the Medical School, University of Oxford (L.J.P., T.G.R., Z.T.), Target Discovery Institute (D.E.), Nuffield Department of Population Health (A.-M.O., K.B.P., D.W.E.), and the Big Data Institute (D.W.E.), University of Oxford, and the NIHR Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at University of Oxford in partnership with Public Health England (N.E.S., P.C.M., S. Hoosdally, D.W.C., K.B.P., A.S.W., T.E.A.P., D.W.E.), Oxford, and the National Infection Service, Public Health England at Colindale, London (M.C., S. Hopkins) - all in the United Kingdom; and the Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam (T.M.W.).

Background: The relationship between the presence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the risk of subsequent reinfection remains unclear.

Methods: We investigated the incidence of SARS-CoV-2 infection confirmed by polymerase chain reaction (PCR) in seropositive and seronegative health care workers attending testing of asymptomatic and symptomatic staff at Oxford University Hospitals in the United Kingdom. Baseline antibody status was determined by anti-spike (primary analysis) and anti-nucleocapsid IgG assays, and staff members were followed for up to 31 weeks. We estimated the relative incidence of PCR-positive test results and new symptomatic infection according to antibody status, adjusting for age, participant-reported gender, and changes in incidence over time.

Results: A total of 12,541 health care workers participated and had anti-spike IgG measured; 11,364 were followed up after negative antibody results and 1265 after positive results, including 88 in whom seroconversion occurred during follow-up. A total of 223 anti-spike-seronegative health care workers had a positive PCR test (1.09 per 10,000 days at risk), 100 during screening while they were asymptomatic and 123 while symptomatic, whereas 2 anti-spike-seropositive health care workers had a positive PCR test (0.13 per 10,000 days at risk), and both workers were asymptomatic when tested (adjusted incidence rate ratio, 0.11; 95% confidence interval, 0.03 to 0.44; P = 0.002). There were no symptomatic infections in workers with anti-spike antibodies. Rate ratios were similar when the anti-nucleocapsid IgG assay was used alone or in combination with the anti-spike IgG assay to determine baseline status.

Conclusions: The presence of anti-spike or anti-nucleocapsid IgG antibodies was associated with a substantially reduced risk of SARS-CoV-2 reinfection in the ensuing 6 months. (Funded by the U.K. Government Department of Health and Social Care and others.).
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http://dx.doi.org/10.1056/NEJMoa2034545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781098PMC
February 2021

Diamond Light Source: contributions to SARS-CoV-2 biology and therapeutics.

Biochem Biophys Res Commun 2021 01 19;538:40-46. Epub 2020 Nov 19.

Diamond Light Source Ltd., Harwell Science and Innovation Campus, Didcot, OX11 0DE, UK; Division of Structural Biology, The Nuffield Department of Medicine, University of Oxford, Headington, Oxford, OX3 7BN, UK; Instruct-ERIC, Oxford House, Parkway Court, John Smith Drive, Oxford, OX4 2JY, UK. Electronic address:

The impact of COVID-19 on public health and the global economy has led to an unprecedented research response, with a major emphasis on the development of safe vaccines and drugs. However, effective, safe treatments typically take over a decade to develop and there are still no clinically approved therapies to treat highly pathogenic coronaviruses. Repurposing of known drugs can speed up development and this strategy, along with the use of biologicals (notably monoclonal antibody therapy) and vaccine development programmes remain the principal routes to dealing with the immediate impact of COVID-19. Nevertheless, the development of broadly-effective highly potent antivirals should be a major longer term goal. Structural biology has been applied with enormous effect, with key proteins structurally characterised only weeks after the SARS-CoV-2 sequence was released. Open-access to advanced infrastructure for structural biology techniques at synchrotrons and high-end cryo-EM and NMR centres has brought these technologies centre-stage in drug discovery. We summarise the role of Diamond Light Source in responses to the pandemic and note the impact of the immediate release of results in fuelling an open-science approach to early-stage drug discovery.
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http://dx.doi.org/10.1016/j.bbrc.2020.11.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676326PMC
January 2021

SARS-CoV-2 antibody prevalence, titres and neutralising activity in an antenatal cohort, United Kingdom, 14 April to 15 June 2020.

Euro Surveill 2020 10;25(42)

The Division of Structural Biology, Nuffield Department of Medicine, University of Oxford, The Henry Wellcome Building, Roosevelt Dr, Headington, Oxford, United Kingdom.

SARS-CoV-2 IgG screening of 1,000 antenatal serum samples in the Oxford area, United Kingdom, between 14 April and 15 June 2020, yielded a 5.3% seroprevalence, mirroring contemporaneous regional data. Among the 53 positive samples, 39 showed in vitro neutralisation activity, correlating with IgG titre (Pearson's correlation p<0.0001). While SARS-CoV-2 seroprevalence in pregnancy cohorts could potentially inform population surveillance, clinical correlates of infection and immunity in pregnancy, and antenatal epidemiology evolution over time need further study.
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http://dx.doi.org/10.2807/1560-7917.ES.2020.25.41.2001721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7651878PMC
October 2020

Structural and functional analysis of protective antibodies targeting the threefold plateau of enterovirus 71.

Nat Commun 2020 10 16;11(1):5253. Epub 2020 Oct 16.

Division of Structural Biology, The Wellcome Centre for Human Genetics, University of Oxford, Headington, Oxford, OX3 7BN, UK.

Enterovirus 71 (EV71)-neutralizing antibodies correlate with protection and have potential as therapeutic agents. We isolate and characterize a panel of plasmablast-derived monoclonal antibodies from an infected child whose antibody response focuses on the plateau epitope near the icosahedral 3-fold axes. Eight of a total of 19 antibodies target this epitope and three of these potently neutralize the virus. Representative neutralizing antibodies 38-1-10A and 38-3-11A both confer effective protection against lethal EV71 challenge in hSCARB2-transgenic mice. The cryo-electron microscopy structures of the EV71 virion in complex with Fab fragments of these potent and protective antibodies reveal the details of a conserved epitope formed by residues in the BC and HI loops of VP2 and the BC and HI loops of VP3 spanning the region around the 3-fold axis. Remarkably, the two antibodies interact with the epitope in quite distinct ways. These plateau-binding antibodies provide templates for promising candidate therapeutics.
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http://dx.doi.org/10.1038/s41467-020-19013-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567869PMC
October 2020

Symmetrical arrangement of positively charged residues around the 5-fold axes of SAT type foot-and-mouth disease virus enhances cell culture of field viruses.

PLoS Pathog 2020 09 29;16(9):e1008828. Epub 2020 Sep 29.

Vaccine and Diagnostic Development Programme, Onderstepoort Veterinary Institute, Agricultural Research Council, Onderstepoort, South Africa.

Field isolates of foot-and-mouth disease viruses (FMDVs) utilize integrin-mediated cell entry but many, including Southern African Territories (SAT) viruses, are difficult to adapt to BHK-21 cells, thus hampering large-scale propagation of vaccine antigen. However, FMDVs acquire the ability to bind to cell surface heparan sulphate proteoglycans, following serial cytolytic infections in cell culture, likely by the selection of rapidly replicating FMDV variants. In this study, fourteen SAT1 and SAT2 viruses, serially passaged in BHK-21 cells, were virulent in CHO-K1 cells and displayed enhanced affinity for heparan, as opposed to their low-passage counterparts. Comparative sequence analysis revealed the fixation of positively charged residues clustered close to the icosahedral 5-fold axes of the virus, at amino acid positions 83-85 in the βD-βE loop and 110-112 in the βF-βG loop of VP1 upon adaptation to cultured cells. Molecular docking simulations confirmed enhanced binding of heparan sulphate to a model of the adapted SAT1 virus, with the region around VP1 arginine 112 contributing the most to binding. Using this information, eight chimeric field strain mutant viruses were constructed with additional positive charges in repeated clusters on the virion surface. Five of these bound heparan sulphate with expanded cell tropism, which should facilitate large-scale propagation. However, only positively charged residues at position 110-112 of VP1 enhanced infectivity of BHK-21 cells. The symmetrical arrangement of even a single amino acid residue in the FMD virion is a powerful strategy enabling the virus to generate novel receptor binding and alternative host-cell interactions.
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http://dx.doi.org/10.1371/journal.ppat.1008828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7577442PMC
September 2020

Assembly intermediates of orthoreovirus captured in the cell.

Nat Commun 2020 09 7;11(1):4445. Epub 2020 Sep 7.

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK.

Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Here, we directly visualise an unsuspected 'single shelled' intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is 'collapsed' compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses.
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http://dx.doi.org/10.1038/s41467-020-18243-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477198PMC
September 2020

Broad and strong memory CD4 and CD8 T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19.

Nat Immunol 2020 11 4;21(11):1336-1345. Epub 2020 Sep 4.

Sequencing and Flow Cytometry Facility, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4 and/or CD8 epitopes, including six immunodominant regions. Six optimized CD8 epitopes were defined, with peptide-MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8 T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.
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http://dx.doi.org/10.1038/s41590-020-0782-6DOI Listing
November 2020

Differential occupational risks to healthcare workers from SARS-CoV-2 observed during a prospective observational study.

Elife 2020 08 21;9. Epub 2020 Aug 21.

Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom.

We conducted voluntary Covid-19 testing programmes for symptomatic and asymptomatic staff at a UK teaching hospital using naso-/oro-pharyngeal PCR testing and immunoassays for IgG antibodies. 1128/10,034 (11.2%) staff had evidence of Covid-19 at some time. Using questionnaire data provided on potential risk-factors, staff with a confirmed household contact were at greatest risk (adjusted odds ratio [aOR] 4.82 [95%CI 3.45-6.72]). Higher rates of Covid-19 were seen in staff working in Covid-19-facing areas (22.6% vs. 8.6% elsewhere) (aOR 2.47 [1.99-3.08]). Controlling for Covid-19-facing status, risks were heterogenous across the hospital, with higher rates in acute medicine (1.52 [1.07-2.16]) and sporadic outbreaks in areas with few or no Covid-19 patients. Covid-19 intensive care unit staff were relatively protected (0.44 [0.28-0.69]), likely by a bundle of PPE-related measures. Positive results were more likely in Black (1.66 [1.25-2.21]) and Asian (1.51 [1.28-1.77]) staff, independent of role or working location, and in porters and cleaners (2.06 [1.34-3.15]).
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http://dx.doi.org/10.7554/eLife.60675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486122PMC
August 2020

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient.

Nat Struct Mol Biol 2020 10 31;27(10):950-958. Epub 2020 Jul 31.

William Dunn School of Pathology, University of Oxford, Oxford, UK.

The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. We have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (K of 2 nM), and a 2.6-Å-resolution crystal structure of an RBD-EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19.
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http://dx.doi.org/10.1038/s41594-020-0480-yDOI Listing
October 2020

Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2.

Nat Struct Mol Biol 2020 09 13;27(9):846-854. Epub 2020 Jul 13.

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (K of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.
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http://dx.doi.org/10.1038/s41594-020-0469-6DOI Listing
September 2020

3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway.

Cell 2020 07 30;182(2):515-530.e17. Epub 2020 Jun 30.

Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK. Electronic address:

Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.
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http://dx.doi.org/10.1016/j.cell.2020.05.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7391008PMC
July 2020

Neutralization of SARS-CoV-2 by Destruction of the Prefusion Spike.

Cell Host Microbe 2020 09 19;28(3):445-454.e6. Epub 2020 Jun 19.

Division of Structural Biology, University of Oxford, The Wellcome Centre for Human Genetics, Headington, Oxford, OX3 7BN, UK; Diamond Light Source Ltd, Harwell Science & Innovation Campus, Didcot, OX11 0DE, UK; Instruct-ERIC, Oxford House, Parkway Court, John Smith Drive, Oxford, OX4 2JY, UK. Electronic address:

There are as yet no licensed therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2, initiating conformational changes that drive membrane fusion. We find that the monoclonal antibody CR3022 binds the RBD tightly, neutralizing SARS-CoV-2, and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilizing CR3022 epitope is inaccessible in the prefusion spike, suggesting that CR3022 binding facilitates conversion to the fusion-incompetent post-fusion state. Cryogenic electron microscopy (cryo-EM) analysis confirms that incubation of spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope could be useful therapeutically, possibly in synergy with an antibody that blocks receptor attachment.
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http://dx.doi.org/10.1016/j.chom.2020.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7303615PMC
September 2020

Hand-foot-and-mouth disease virus receptor KREMEN1 binds the canyon of Coxsackie Virus A10.

Nat Commun 2020 01 7;11(1):38. Epub 2020 Jan 7.

Division of Structural Biology, The Wellcome Centre for Human Genetics, University of Oxford, Headington, Oxford, OX3 7BN, UK.

Coxsackievirus A10 (CV-A10) is responsible for an escalating number of severe infections in children, but no prophylactics or therapeutics are currently available. KREMEN1 (KRM1) is the entry receptor for the largest receptor-group of hand-foot-and-mouth disease causing viruses, which includes CV-A10. We report here structures of CV-A10 mature virus alone and in complex with KRM1 as well as of the CV-A10 A-particle. The receptor spans the viral canyon with a large footprint on the virus surface. The footprint has some overlap with that seen for the neonatal Fc receptor complexed with enterovirus E6 but is larger and distinct from that of another enterovirus receptor SCARB2. Reduced occupancy of a particle-stabilising pocket factor in the complexed virus and the presence of both unbound and expanded virus particles suggests receptor binding initiates a cascade of conformational changes that produces expanded particles primed for viral uncoating.
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http://dx.doi.org/10.1038/s41467-019-13936-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6946704PMC
January 2020

Glutathione facilitates enterovirus assembly by binding at a druggable pocket.

Commun Biol 2020 3;3. Epub 2020 Jan 3.

1Division of Structural Biology, University of Oxford, The Henry Wellcome Building for Genomic Medicine, Headington, Oxford OX3 7BN UK.

Enteroviruses cause a range of human and animal diseases, some life-threatening, but there remain no licenced anti-enterovirus drugs. However, a benzene-sulfonamide derivative and related compounds have been shown recently to block infection of a range of enteroviruses by binding the capsid at a positively-charged surface depression conserved across many enteroviruses. It has also been established that glutathione is essential for the assembly of many enteroviruses, interacting with the capsid proteins to facilitate the formation of the pentameric assembly intermediate, although the mechanism is unknown. Here we show, by high resolution structure analyses of enterovirus F3, that reduced glutathione binds to the same interprotomer pocket as the benzene-sulfonamide derivative. Bound glutathione makes strong interactions with adjacent protomers, thereby explaining the underlying biological role of this druggable binding pocket and delineating the pharmacophore for potential antivirals.
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http://dx.doi.org/10.1038/s42003-019-0722-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941975PMC
January 2020

Atomic structure of the Epstein-Barr virus portal.

Nat Commun 2019 08 29;10(1):3891. Epub 2019 Aug 29.

Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 10-12, 08028, Barcelona, Spain.

Herpesviridae is a vast family of enveloped DNA viruses that includes eight distinct human pathogens, responsible for diseases that range from almost asymptomatic to severe and life-threatening. Epstein-Barr virus infects B-cells and epithelial cells, causing infectious mononucleosis, as well as a number of cancers. Epstein-Barr infection cannot be cured since neither vaccine nor antiviral drug treatments are available. All herpesviruses contain a linear double-stranded DNA genome, enclosed within an icosahedral capsid. Viral portal protein plays a key role in the procapsid assembly and DNA packaging. The portal is the entrance and exit pore for the viral genome, making it an attractive pharmacological target for the development of new antivirals. Here we present the atomic structure of the portal protein of Epstein-Barr virus, solved by cryo-electron microscopy at 3.5 Å resolution. The detailed architecture of this protein suggests that it plays a functional role in DNA retention during packaging.
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http://dx.doi.org/10.1038/s41467-019-11706-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715670PMC
August 2019