Genome-first detection of emerging resistance to novel therapeutic agents for SARS-CoV-2

Some COVID-19 patients are unable to clear their infection or are at risk of severe disease, requiring treatment with neutralising monoclonal antibodies (nmAb) and/or antivirals. The rapid roll-out of novel therapeutics means there is limited understanding of the likely genetic barrier to drug resistance. Unprecedented genomic surveillance of SARS-CoV-2 in the UK has enabled a genome-first approach to the detection of emerging drug resistance. Here we report the accrual of mutations in Delta and Omicron cases treated with casirivimab+imdevimab and sotrovimab respectively. Mutations occur within the epitopes of the respective nmAbs. For casirivimab+imdevimab these are present on contiguous raw reads, simultaneously affecting both components. Using surface plasmon resonance and pseudoviral neutralisation assays we demonstrate these mutations reduce or completely abrogate antibody affinity and neutralising activity, suggesting they are driven by immune evasion. In addition, we show that some mutations also reduce the neutralising activity of vaccine-induced serum.

Further antibody escape by Omicron BA.4 and BA.5 from vaccine and BA.1 serum

The Omicron lineage of SARS-CoV-2, first described in November 2021, spread rapidly to become globally dominant and has split into a number of sub-lineages. BA.1 dominated the initial wave but has been replaced by BA.2 in many countries. Recent sequencing from South Africas Gauteng region uncovered two new sub-lineages, BA.4 and BA.5 which are taking over locally, driving a new wave. BA.4 and BA.5 contain identical spike sequences and, although closely related to BA.2, contain further mutations in the receptor binding domain of spike. Here, we study the neutralization of BA.4/5 using a range of vaccine and naturally immune serum and panels of monoclonal antibodies. BA.4/5 shows reduced neutralization by serum from triple AstraZeneca or Pfizer vaccinated individuals compared to BA.1 and BA.2. Furthermore, using serum from BA.1 vaccine breakthrough infections there are likewise, significant reductions in the neutralization of BA.4/5, raising the possibility of repeat Omicron infections.

Receptor binding and escape from Beta antibody responses drive Omicron-B.1.1.529 evolution

On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titres of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic as well as Alpha, Beta, Gamma, Delta are substantially reduced or fail to neutralize. Titres against Omicron are boosted by third vaccine doses and are high in cases both vaccinated and infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of a large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses, combining mutations conferring tight binding to ACE2 to unleash evolution driven by immune escape, leading to a large number of mutations in the ACE2 binding site which rebalance receptor affinity to that of early pandemic viruses.

SARS-CoV-2 anti-spike IgG antibody responses after second dose of ChAdOx1 or BNT162b2 in the UK general population

We investigated anti-spike IgG antibody responses and correlates of protection following second doses of ChAdOx1 or BNT162b2 SARS-CoV-2 vaccines in the UK general population. In 222,493 individuals, we found significant boosting of anti-spike IgG by second doses of both vaccines in all ages and using different dosing intervals, including the 3-week interval for BNT162b2. After second vaccination, BNT162b2 generated higher peak levels than ChAdOX1. Older individuals and males had lower peak levels with BNT162b2 but not ChAdOx1, while declines were similar across ages and sexes with ChAdOX1 or BNT162b2. Prior infection significantly increased antibody peak level and half-life with both vaccines. Anti-spike IgG levels were associated with protection from infection after vaccination and, to an even greater degree, after prior infection. At least 67% protection against infection was estimated to last for 2-3 months after two ChAdOx1 doses and 5-8 months after two BNT162b2 doses in those without prior infection, and 1-2 years for those unvaccinated after natural infection. A third booster dose may be needed, prioritised to ChAdOx1 recipients and those more clinically vulnerable.

Anti-spike antibody response to natural SARS-CoV-2 infection in the general population

We estimated the duration and determinants of antibody response after SARS-CoV-2 infection in the general population using representative data from 7,256 United Kingdom COVID-19 infection survey participants who had positive swab SARS-CoV-2 PCR tests from 26-April-2020 to 14-June-2021. A latent class model classified 24% of participants as non-responders not developing anti-spike antibodies. These seronegative non-responders were older, had higher SARS-CoV-2 cycle threshold values during infection (i.e. lower viral burden), and less frequently reported any symptoms. Among those who seroconverted, using Bayesian linear mixed models, the estimated anti-spike IgG peak level was 7.3-fold higher than the level previously associated with 50% protection against reinfection, with higher peak levels in older participants and those of non-white ethnicity. The estimated anti-spike IgG half-life was 184 days, being longer in females and those of white ethnicity. We estimated antibody levels associated with protection against reinfection likely last 1.5-2 years on average, with levels associated with protection from severe infection present for several years. These estimates could inform planning for vaccination booster strategies.

The impact of SARS-CoV-2 vaccines on antibody responses in the general population in the United Kingdom

Real-world data on antibody response post-vaccination in the general population are limited. 45,965 adults in the UKs national COVID-19 Infection Survey receiving Pfizer-BioNTech or Oxford-AstraZeneca vaccines had 111,360 anti-spike IgG measurements. Without prior infection, seroconversion rates and quantitative antibody levels post single dose were lower in older individuals, especially >60y. Two doses achieved high responses across all ages, particularly increasing seroconversion in older people, to similar levels to those achieved after prior infection followed by a single dose. Antibody levels rose more slowly and to lower levels with Oxford-AstraZeneca vs Pfizer-BioNTech, but waned following a single Pfizer-BioNTech dose. Latent class models identified four responder phenotypes: older people, males, and those having long-term health conditions were more commonly low responders. Where supplies are limited, vaccines should be prioritised for those not previously infected, and second doses to individuals >60y. Further data on the relationship between vaccine-mediated protection and antibody responses are needed.

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

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: 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 RBD impact neutralisation. Monoclonal antibody 222 neutralises 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.

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

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.

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

ObjectivesWe 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. MethodsHCWs 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. ResultsVaccine uptake was 80%, but less in lower-paid roles and Black, south Asian and minority ethnic groups. 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 post-second vaccination were similar to those after prior infection and one vaccine dose. ConclusionsVaccination leads to detectable anti-spike antibodies in nearly all adult HCWs. Whether differences in response impact vaccine efficacy needs further study.

An observational cohort study on the incidence of SARS-CoV-2 infection and B.1.1.7 variant infection in healthcare workers by antibody and vaccination status

BackgroundNatural and vaccine-induced immunity will play a key role in controlling the SARS-CoV-2 pandemic. SARS-CoV-2 variants have the potential to evade natural and vaccine-induced immunity. MethodsIn a longitudinal cohort study of healthcare workers (HCWs) in Oxfordshire, UK, we investigated the protection from symptomatic and asymptomatic PCR-confirmed SARS-CoV-2 infection conferred by vaccination (Pfizer-BioNTech BNT162b2, Oxford-AstraZeneca ChAdOx1 nCOV-19) and prior infection (determined using anti-spike antibody status), using Poisson regression adjusted for age, sex, temporal changes in incidence and role. We estimated protection conferred after one versus two vaccinations and from infections with the B.1.1.7 variant identified using whole genome sequencing. Results13,109 HCWs participated; 8285 received the Pfizer-BioNTech vaccine (1407 two doses) and 2738 the Oxford-AstraZeneca vaccine (49 two doses). Compared to unvaccinated seronegative HCWs, natural immunity and two vaccination doses provided similar protection against symptomatic infection: no HCW vaccinated twice had symptomatic infection, and incidence was 98% lower in seropositive HCWs (adjusted incidence rate ratio 0.02 [95%CI <0.01-0.18]). Two vaccine doses or seropositivity reduced the incidence of any PCR-positive result with or without symptoms by 90% (0.10 [0.02-0.38]) and 85% (0.15 [0.08-0.26]) respectively. Single-dose vaccination reduced the incidence of symptomatic infection by 67% (0.33 [0.21-0.52]) and any PCR-positive result by 64% (0.36 [0.26-0.50]). There was no evidence of differences in immunity induced by natural infection and vaccination for infections with S-gene target failure and B.1.1.7. ConclusionNatural infection resulting in detectable anti-spike antibodies and two vaccine doses both provide robust protection against SARS-CoV-2 infection, including against the B.1.1.7 variant. SummaryNatural infection resulting in detectable anti-spike antibodies and two vaccine doses both provided [≥] 85% protection against symptomatic and asymptomatic SARS-CoV-2 infection in healthcare workers, including against the B.1.1.7 variant. Single dose vaccination reduced symptomatic infection by 67%.

Antibodies to SARS-CoV-2 are associated with protection against reinfection

BackgroundIt is critical to understand whether infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) protects from subsequent reinfection. MethodsWe investigated the incidence of SARS-CoV-2 PCR-positive results in seropositive and seronegative healthcare workers (HCWs) attending asymptomatic and symptomatic staff testing at Oxford University Hospitals, UK. Baseline antibody status was determined using anti-spike and/or anti-nucleocapsid IgG assays and staff followed for up to 30 weeks. We used Poisson regression to estimate the relative incidence of PCR-positive results and new symptomatic infection by antibody status, accounting for age, gender and changes in incidence over time. ResultsA total of 12219 HCWs participated and had anti-spike IgG measured, 11052 were followed up after negative and 1246 after positive antibody results including 79 who seroconverted during follow up. 89 PCR-confirmed symptomatic infections occurred in seronegative individuals (0.46 cases per 10,000 days at risk) and no symptomatic infections in those with anti-spike antibodies. Additionally, 76 (0.40/10,000 days at risk) anti-spike IgG seronegative individuals had PCR-positive tests in asymptomatic screening, compared to 3 (0.21/10,000 days at risk) seropositive individuals. Overall, positive baseline anti-spike antibodies were associated with lower rates of PCR-positivity (with or without symptoms) (adjusted rate ratio 0.24 [95%CI 0.08-0.76, p=0.015]). Rate ratios were similar using anti-nucleocapsid IgG alone or combined with anti-spike IgG to determine baseline status. ConclusionsPrior SARS-CoV-2 infection that generated antibody responses offered protection from reinfection for most people in the six months following infection. Further work is required to determine the long-term duration and correlates of post-infection immunity.

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

BackgroundSARS-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. MethodsWe present 6 months of data from a longitudinal seroprevalence study of 3217 UK healthcare workers (HCWs). Serial measurements of IgG antibodies to SARS-CoV-2 nucleocapsid were obtained. Bayesian mixed linear models were used to investigate antibody waning and associations with age, gender, ethnicity, previous symptoms and PCR results. ResultsIn this cohort of working age HCWs, antibody levels rose to a peak at 24 (95% credibility interval, CrI 19-31) days post-first positive PCR test, before beginning to fall. Considering 452 IgG seropositive HCWs over a median of 121 days (maximum 171 days) from their maximum positive IgG titre, the mean estimated antibody half-life was 85 (95%CrI, 81-90) days. The estimated mean time to loss of a positive antibody result was 137 (95%CrI 127-148) days. We observed variation between individuals; higher maximum observed IgG titres were associated with longer estimated antibody half-lives. Increasing age, Asian ethnicity and prior self-reported symptoms were independently associated with higher maximum antibody levels, and increasing age and a positive PCR test undertaken for symptoms with longer antibody half-lives. ConclusionIgG antibody levels to SARS-CoV-2 nucleocapsid wane within months, and faster in younger adults and those without symptoms. Ongoing longitudinal studies are required to track the long-term duration of antibody levels and their association with immunity to SARS-CoV-2 reinfection. SummarySerially measured SARS-CoV-2 anti-nucleocapsid IgG titres from 452 seropositive healthcare workers demonstrate levels fall by half in 85 days. From a peak result, detectable antibodies last a mean 137 days. Levels fall faster in younger adults and following asymptomatic infection.

The SARS-CoV-2 Spike harbours a lipid binding pocket which modulates stability of the prefusion trimer

Large trimeric Spikes decorate SARS-CoV-2 and bind host cells via receptor binding domains (RBDs). We report a conformation in which the trimer is locked into a compact well-ordered form. This differs from previous structures where the RBD can flip up to recognise the receptor. In the locked form regions associated with fusion transitions are stabilised and the RBD harbours curved lipids. The acyl chains bind a hydrophobic pocket in one RBD whilst the polar headgroups attach to an adjacent RBD of the trimer. By functional analogy with enteroviral pocket factors loss of the lipid would destabilise the locked form facilitating receptor attachment, conversion to the postfusion state and virus infection. The nature of lipids available at the site of infection might affect the antigenicity/pathogenicity of released virus. These results reveal a potentially druggable pocket and suggest that the natural prefusion state occludes neutralising RBD epitopes, achieving conformational shielding from antibodies. HighlightsO_LISARS-CoV-2 Spike can adopt a locked conformation with all receptor binding domains (RBDs) down, likely to represent the prefusion resting state C_LIO_LIThis locked conformation is compact and stable, braced by lipid bound within a potentially druggable pocket C_LIO_LIKey neutralization epitopes are shielded in the locked form C_LIO_LILoss of lipid may trigger a cascade of events that lead to cell entry analogous to the role of lipids in enterovirus cell entry C_LI

Stringent thresholds for SARS-CoV-2 IgG assays result in under-detection of cases reporting loss of taste/smell

Thresholds for SARS-CoV-2 antibody assays have typically been determined using samples from symptomatic, often hospitalised, patients. Assay performance following mild/asymptomatic infection is unclear. We assessed IgG responses in asymptomatic healthcare workers with a high pre-test probability of Covid-19, e.g. 807/9292(8.9%) reported loss of smell/taste. The proportion reporting anosmia/ageusia increased at antibody titres below diagnostic thresholds for both an in-house ELISA and the Abbott Architect chemiluminescent microparticle immunoassay (CMIA): 424/903(47%) reported anosmia/ageusia with a positive ELISA, 59/387(13.2%) with high-negative titres, and 324/7943(4.1%) with low-negative results. Adjusting for the proportion of staff reporting anosmia/ageusia suggests the sensitivity of both assays is lower than previously reported: Oxford ELISA 90.8% (95%CI 86.1-92.1%) and Abbott CMIA 80.9% (77.5-84.3%). However, the sensitivity may be lower if some anosmia/ageusia in those with low-negative titres is Covid-19-associated. Samples from individuals with mild/asymptomatic infection should be included in SARS-CoV-2 immunoassay evaluations. Reporting equivocal SARS-CoV-2 antibody results should be considered.

Population-wide evolution of SARS-CoV-2 immunity tracked by a ternary immunoassay

Effective public-health measures and vaccination campaigns against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against the ectodomain and the receptor-binding domain of the spike protein as well as the nucleocapsid protein of SARS-CoV-2. We used TRABI for continuous seromonitoring of hospital patients and healthy blood donors (n=72222) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). Seroprevalence peaked in May 2020 and rose again in November 2020 in both cohorts. Validations of results included antibody diffusional sizing and Western Blotting. Using an extended Susceptible-Exposed-Infectious-Removed model, we found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020 in the population of the canton of Zurich. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19 and up to the timepoint of survey participation. Crucially, we found no evidence for a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2- infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.

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

The COVID-19 pandemic has had unprecedented health and economic impact, but currently there are no approved therapies. We have isolated an antibody, EY6A, from a late-stage COVID-19 patient and show it neutralises SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds tightly (KD of 2 nM) the receptor binding domain (RBD) of the viral Spike glycoprotein and a 2.6[A] crystal structure of an RBD/EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues of this epitope are key to stabilising the pre-fusion Spike. Cryo-EM analyses of the pre-fusion Spike incubated with EY6A Fab reveal a complex of the intact trimer with three Fabs bound and two further multimeric forms comprising destabilized Spike attached to Fab. EY6A binds what is probably a major neutralising epitope, making it a candidate therapeutic for COVID-19.

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

COVID-19 is an ongoing global crisis in which the development of effective vaccines and therapeutics will depend critically on understanding the natural immunity to the virus, including the role of SARS-CoV-2-specific T cells. We have conducted a study of 42 patients following recovery from COVID-19, including 28 mild and 14 severe cases, comparing their T cell responses to those of 16 control donors. We assessed the immune memory of T cell responses using IFN{gamma} based assays with overlapping peptides spanning SARS-CoV-2 apart from ORF1. We found the breadth, magnitude and frequency of memory T cell responses from COVID-19 were significantly higher in severe compared to mild COVID-19 cases, and this effect was most marked in response to spike, membrane, and ORF3a proteins. Total and spike-specific T cell responses correlated with the anti-Spike, anti-Receptor Binding Domain (RBD) as well as anti-Nucleoprotein (NP) endpoint antibody titre (p<0.001, <0.001 and =0.002). We identified 39 separate peptides containing CD4+ and/or CD8+ epitopes, which strikingly included six immunodominant epitope clusters targeted by T cells in many donors, including 3 clusters in spike (recognised by 29%, 24%, 18% donors), two in the membrane protein (M, 32%, 47%) and one in the nucleoprotein (Np, 35%). CD8+ responses were further defined for their HLA restriction, including B*4001-restricted T cells showing central memory and effector memory phenotype. In mild cases, higher frequencies of multi-cytokine producing M- and NP-specific CD8+ T cells than spike-specific CD8+ T cells were observed. They furthermore showed a higher ratio of SARS-CoV-2-specific CD8+ to CD4+ T cell responses. Immunodominant epitope clusters and peptides containing T cell epitopes identified in this study will provide critical tools to study the role of virus-specific T cells in control and resolution of SARS-CoV-2 infections. The identification of T cell specificity and functionality associated with milder disease, highlights the potential importance of including non-spike proteins within future COVID-19 vaccine design.

Neutralization of SARS-CoV-2 by destruction of the prefusion Spike

There are as yet no licenced 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 (ACE2), initiating conformational changes that drive membrane fusion. We find that monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 [A] of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding would facilitate conversion to the fusion-incompetent post-fusion state. 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 may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment. HighlightsO_LICR3022 neutralises SARS-CoV-2 C_LIO_LINeutralisation is by destroying the prefusion SPIKE conformation C_LIO_LIThis antibody may have therapeutic potential alone or with one blocking receptor attachment C_LI