Comparative Analysis of SARS-CoV-2 Antigenicity across Assays and in Human and Animal Model Sera (preprint)

The antigenic evolution of SARS-CoV-2 requires ongoing monitoring to judge the immune escape of newly arising variants. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal sera. We compared 18 datasets generated using human, hamster, and mouse sera, and six different neutralization assays. Titer magnitude was lowest in human, intermediate in hamster, and highest in mouse sera. Fold change, immunodominance patterns and antigenic maps were similar among sera. Most assays yielded similar results, except for differences in fold change in cytopathic effect assays. Not enough data was available for conclusively judging mouse sera, but hamster sera were a consistent surrogate for human first-infection sera.

Cooperativity and induced oligomerisation control the interaction of SARS- CoV-2 with its cellular receptor and patient-derived antibodies (preprint)

Viral entry is mediated by oligomeric proteins on the virus and cell surfaces. The association is therefore open to multivalent interactions between these proteins, yet such recognition is typically rationalised as affinity between monomeric equivalents. As a result, assessment of the thermodynamic mechanisms that control viral entry has been limited. Here, we use mass photometry to overcome the analytical challenges consequent to multivalency. Examining the interaction between the spike protein of SARS-CoV-2 and the ACE2 receptor, we find that ACE2 induces oligomerisation of spike in a variant- dependent fashion. We also demonstrate that patient-derived antibodies use induced-oligomerisation as a primary inhibition mechanism or to enhance the effects of receptor-site blocking. Our results reveal that naive affinity measurements are poor predictors of potency, and introduce a novel antibody-based inhibition mechanism for oligomeric targets.

The molecular reach of antibodies determines their SARS-CoV-2 neutralisation potency (preprint)

Antibodies play crucial roles in health and disease and are invaluable tools for diagnostics, research, and therapy. Although antibodies bind bivalently, we lack methods to analyse bivalent binding. Here, we introduce a particle-based model and use it to analyse bivalent binding of SARS-CoV-2 RBD-specific antibodies in surface plasmon resonance assays. The method reproduces the monovalent on/off-rates and enables measurements of new parameters, including the molecular reach, which is the maximum antigen separation that supports bivalent binding. We show that the molecular reach (22-46 nm) exceeds the physical size of an antibody (15 nm) and that the variation in reach across 45 patient-isolated antibodies is the best correlate of viral neutralisation. Using the complete set of fitted parameters, the model predicts an emergent antibody binding potency that equals the neutralisation potency. This novel analytical method should improve our understanding and exploitation of antibodies and other bivalent molecules.

Evolution of long-term hybrid immunity in healthcare workers after different COVID-19 vaccination regimens: a longitudinal observational cohort study (preprint)

Both infection and vaccination, alone or in combination, generate antibody and T cell responses against SARS-CoV-2. However, the maintenance of such responses - and hence protection from disease - requires careful characterisation. In a large prospective study of UK healthcare workers (PITCH, within the larger SIREN study) we previously observed that prior infection impacted strongly on subsequent cellular and humoral immunity induced after long and short dosing intervals of BNT162b2 (Pfizer/BioNTech) vaccination. Here, we report longer follow up of 684 HCWs in this cohort over 6-9 months following two doses of BNT162b2 or AZ1222 (Oxford/AstraZeneca) vaccination and following a subsequent BNT162b2 booster vaccination. We make three important observations: Firstly, the dynamics of humoral and cellular responses differ; binding and neutralising antibodies declined whereas T and B cell responses were better maintained after the second vaccine dose. Secondly, vaccine boosting restored IgG levels to post second dose levels and broadened neutralising activity against variants of concern including omicron BA.1, alongside further boosting of T cell responses. Thirdly, prior infection maintained its impact driving larger T cell responses compared to never infected people, including after the third dose. In conclusion, the maintenance of T cell responses in time and against variants of concern may account for continued protection against severe disease.

Accelerating Inhibitor Discovery for Multiple SARS-CoV-2 Targets with a Single, Sequence-Guided Deep Generative Framework (preprint)

The COVID-19 pandemic has highlighted the urgency for developing more efficient molecular discovery pathways. As exhaustive exploration of the vast chemical space is infeasible, discovering novel inhibitor molecules for emerging drug-target proteins is challenging, particularly for targets with unknown structure or ligands. We demonstrate the broad utility of a single deep generative framework toward discovering novel drug-like inhibitor molecules against two distinct SARS-CoV-2 targets -- the main protease (Mpro) and the receptor binding domain (RBD) of the spike protein. To perform target-aware design, the framework employs a target sequence-conditioned sampling of novel molecules from a generative model. Micromolar-level in vitro inhibition was observed for two candidates (out of four synthesized) for each target. The most potent spike RBD inhibitor also emerged as a rare non-covalent antiviral with broad-spectrum activity against several SARS-CoV-2 variants in live virus neutralization assays. These results show that a broadly deployable machine intelligence framework can accelerate hit discovery across different emerging drug-targets.

Memory cytotoxic SARS-CoV-2 spike protein-specific CD4+ T cells associate with viral control (preprint)

Little is known of the role of cytotoxic CD4+ T-cells in the control of viral replication. Here, we investigate CD4+ T-cell responses to three dominant SARS-CoV-2 epitopes and evaluate antiviral activity, including cytotoxicity and antiviral cytokine production. Diverse T cell receptor (TCR) usage including public TCRs were identified; surprisingly, cytotoxic CD4+ T-cells were found to have signalling and cytotoxic pathways distinct from classical CD8+ T-cells, with increased expression of chemokines and tissue homing receptors promoting migration. We show the presence of cytolytic CD4+ T-cells during primary infection associates with COVID-19 disease severity. Robust immune memory 6-9 months post-infection or vaccination provides CD4+ T-cells with potent antiviral activity. Our data support a model where CD4+ killer cells drive immunopathogenesis during primary infection and CD4+ memory responses are protective during secondary infection. Our study highlights the unique features of cytotoxic CD4+ T-cells that use distinct functional pathways, providing preventative and therapeutic opportunities.

Reduced neutralisation of SARS-COV-2 Omicron-B.1.1.529 variant by post-immunisation serum (preprint)

In this report, we present live neutralisation titres against SARS-CoV-2 Omicron variant, compared with neutralisation against Victoria, Beta and Delta variants. Sera from day-28 post second-dose were obtained from participants in the Com-COV2 study who had received a two-dose COVID-19 vaccination schedule with either AstraZeneca (AZD1222) or Pfizer (BNT162b2) vaccines. There was a substantial fall in neutralisation titres in recipients of both AZD1222 and BNT16b2 primary courses, with evidence of some recipients failing to neutralise at all. This will likely lead to increased breakthrough infections in previously infected or double vaccinated individuals, which could drive a further wave of infection, although there is currently no evidence of increased potential to cause severe disease, hospitalization or death.

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

On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced. Omicron contains a total of 30 substitutions plus deletions and an insertion in Spike, far more than any previously reported variant. The mutations include those previously identified by In-vitro evolution to contribute to high-affinity binding to ACE2, including mutations Q498R and N501Y critical in forming additional interactions in the interface. Together with increased charge complementarity between the RBD and ACE2, these substantially increase affinity and potentially virus transmissibility through increased syncytia formation. Further mutations promote immune evasion. We have studied the binding of a large panel of potent monoclonal antibodies generated from early pandemic or Beta infected cases. Mutations in Omicron will likely compromise the binding of many of these and additionally, the binding of antibodies under commercial development, however residual binding should provide protection from severe disease.

Durability of ChAdOx1 nCov-19 (AZD1222) vaccination in people living with HIV - responses to SARS-CoV-2, variants of concern and circulating coronaviruses (preprint)

Duration of protection from SARS-CoV-2 infection in people with HIV (PWH) following vaccination is unclear. In a sub-study of the phase 2/3 the COV002 trial (NCT04400838), 54 HIV positive male participants on antiretroviral therapy (undetectable viral loads, CD4+ T cells >350 cells/ul) received two doses of ChAdOx1 nCoV-19 (AZD1222) 4-6 weeks apart and were followed for 6 months. Responses to vaccination were determined by serology (IgG ELISA and MesoScale Discovery (MSD)), neutralisation, ACE-2 inhibition, gamma interferon ELISpot, activation-induced marker (AIM) assay and T cell proliferation. We show that 6 months after vaccination the majority of measurable immune responses were greater than pre-vaccination baseline, but with evidence of a decline in both humoral and cell mediated immunity. There was, however, no significant difference compared to a cohort of HIV-uninfected individuals vaccinated with the same regimen. Responses to the variants of concern were detectable, although were lower than wild type. Pre-existing cross-reactive T cell responses to SARS-CoV-2 spike were associated with greater post-vaccine immunity and correlated with prior exposure to beta coronaviruses. These data support the on-going policy to vaccinate PWH against SARS-CoV-2, and underpin the need for long-term monitoring of responses after vaccination.

Immunodominant NP105-113-B*0702 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease (preprint)

NP 105-113 -B*07:02 specific CD8 + T-cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP 105-113 -B*07:02 specific T-cell clones and single cell sequencing were performed concurrently, with functional avidity and anti-viral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with TCR usage, transcriptome signature, and disease severity (acute N=77, convalescent N=52). We demonstrated a beneficial association of NP 105-113 -B*07:02 specific T-cells in COVID-19 disease progression, linked with expansion of T-cell precursors, high functional avidity and anti-viral effector function. Broad immune memory pools were narrowed post-infection but NP 105-113 -B*07:02 specific T-cells were maintained 6 months after infection with preserved anti-viral efficacy to the SARS-CoV-2 Victoria strain, as well as new Alpha, Beta and Gamma variants. Our data shows that NP 105-113 -B*07:02 specific T-cell responses associate with mild disease and high anti-viral efficacy, pointing to inclusion for future vaccine design.

Tolerability and Immunogenicity After a Late Second Dose or a Third Dose of ChAdOx1 nCoV-19 (AZD1222) (preprint)

Background: COVID-19 vaccine supply shortages are causing concerns about compromised immunity in some countries as the interval between first and second dose extends. Conversely, countries with no supply constraints are considering administering a third dose. We assessed the persistence of immunogenicity after a single dose, the immunity after an extended interval between the first and second dose of ChAdOx1 nCoV-19(AZD1222), and the response to a third dose as a late booster. Methods: Volunteers aged 18-55 years who were enrolled in a Phase 1/2 or Phase 2/3 clinical trial of ChAdOx1 nCoV-19 and had received either a single dose or two doses of 5×10 10 viral particles were invited back for vaccination. Reactogenicity and immunogenicity of a delayed second dose or a third dose are reported here.Findings: Antibody titres after a single dose and measured on d362 remain higher than the titres measured on d0 (62.61 EU; 95% CI 47.43-82.64 vs 1 EU 95% CI 1-16). 30 participants received a late second dose of ChAdOx1 nCoV-19 (median 44 weeks after first dose), antibody titres were higher in those with a longer interval between first and second dose (median EU for 8-12, 15-25, and 44-46 weeks were 923 [IQR 525-1764], 1860 [IQR 917-4934] and 3738 [IQR 1824-6625] respectively). 90 participants received a third dose and antibody titres were significantly higher following a third dose (FRNT50 612 [IQR 351-920]) when compared with the response 28 days after a second dose (FRNT 50 319 [IQR 176-591]. T-cell responses were also boosted after a third dose. Reactogenicity after a late second dose or a third dose was lower than reactogenicity after a first dose.Interpretation: A longer delay before the second dose of ChAdOx1 nCoV-19 leads to an increased antibody titre after the second dose. A third dose of ChAdOx1 nCoV-19 induces antibodies to a level that correlate with high efficacy after second dose and boosts T-cell responses.Funding: UK Research and Innovation (MC_PC_19055), Engineering and Physical Sciences Research Council (EP/R013756/1), National Institute for Health Research (COV19 OxfordVacc-01), Coalition for Epidemic Preparedness Innovations (Outbreak Response To Novel Coronavirus (COVID-19)), National Institute for Health Research Oxford Biomedical Research Centre (BRC4 Vaccines Theme), Thames Valley and South Midland’s NIHR Clinical Research Network, and AstraZeneca. The views expressed in this publication are those of the authors and not necessarily those of the NIHR or the UK Department of Health and Social Care.Declaration of Interest: Oxford University has entered into a partnership with AstraZeneca for further development of ChAdOx1 nCoV-19. AstraZeneca reviewed the data from the study and the final manuscript before submission, but the authors retained editorial control. SCG and AVSH are cofounders of and shareholders in Vaccitech (collaborators in the early development of this vaccine candidate) and named as inventors on a patent covering use of ChAdOx1-vectored vaccines (PCT/GB2012/000467) and a patent application covering this SARS-CoV-2 vaccine (SCG only). TL is named as an inventor on a patent covering use of ChAdOx1-vectored vaccines (PCT/GB2012/000467) and was a consultant to Vaccitech. PMF is a consultant to Vaccitech. AJP is Chair of the UK Department of Health and Social Care’s JCVI, but does not participate in policy advice on coronavirus vaccines, and is a member of the WHO Strategic Advisory Group of Experts (SAGE). AJP is a NIHR Senior Investigator.Ethical Approval: In the UK, the COV001 and COV002 studies were approved by the South Central Berkshire Research Ethics Committee (COV001 reference 20/SC/0145, March 23, 2020; and COV002 reference 20/SC/0179; conditional approval April 8, full approval April 19, 2020).

The ChAdOx1 vectored vaccine, AZD2816, induces strong immunogenicity against SARS-CoV-2 B.1.351 and other variants of concern in preclinical studies. (preprint)

There is an ongoing global effort, to design, manufacture, and clinically assess vaccines against SARS-CoV-2. Over the course of the ongoing pandemic a number of new SARS-CoV-2 virus isolates or variants of concern (VoC) have been identified containing mutations that negatively impact the role of neutralising antibodies. In this study we describe the generation and preclinical assessment of a ChAdOx1-vectored vaccine against the variant of concern B.1.351 (AZD2816). We demonstrate AZD2816 is immunogenic after a single dose and when used as a booster dose in animals primed with original vaccine AZD1222, we see no evidence of original antigenic sin but high titre antibodies against a number of variant spike proteins. In addition, neutralisation titres against B.1.351 (Beta), B.1.617.1 (Kappa) and B.1.617.2 (Delta), are induced in these boost regimens. These data support the ongoing clinical development and testing of this new variant vaccine.

Fatal COVID-19 outcomes are associated with an antibody response targeting epitopes shared with endemic coronaviruses (preprint)

It is unclear whether prior endemic coronavirus infections affect COVID-19 severity. Here, we show that in cases of fatal COVID-19, antibody responses to the SARS-COV-2 spike are directed against epitopes shared with endemic beta-coronaviruses in the S2 subunit of the SARS-CoV-2 spike protein. This immune response is associated with the compromised production of a de novo SARS-CoV-2 spike response among individuals with fatal COVID-19 outcomes.

Safety and Immunogenicity of the ChAdox1 nCoV-19 (AZD1222) Vaccine Against SARS-CoV-2 in HIV Infection (preprint)

Background: The ChAdOx1 nCoV-19 (AZD1222) vaccine is immunogenic and protects against COVID-19. However, data on vaccine immunogenicity are needed for the 40 million people living with HIV (PWH), who may have less functional immunity and more associated co-morbidities than the general population. Methods: Between the 5th and 24th November 2020, 54 adults with HIV, aged 18-55 years, were enrolled into a single arm open label vaccination study within the protocol of the larger phase 2/3 COV002 trial. A prime-boost regimen of ChAdOx1 nCoV-19, with two doses (5 × 1010 vp) was given 4-6 weeks apart. All participants were on antiretroviral therapy (ART) with undetectable plasma HIV viral loads and CD4+ T cell counts >350 cells/µl at enrolment. Data were captured on adverse events. Humoral responses were measured by anti-spike IgG ELISA and antibody-mediated live virus neutralisation. Cell-mediated immune responses were measured by ex-vivo interferon-γ enzyme-linked immunospot assay (ELISpot) and T cell proliferation. All outcomes were compared with a HIV uninfected group from the main COV002 study.Findings: 54 participants with HIV (median age 42.5 years (IQR 37.2-49.8)) received two doses of ChAdOx1 nCoV-19. Median CD4+ T cell count at enrolment was 694 cells/µl (IQR 562-864). Results are reported for 56 days of follow-up. Local and systemic reactions occurring during the first 7 days after prime vaccination included pain at the injection site (49%), fatigue (47%), headache (47%), malaise (34%), chills (23%), and muscle or (36%) joint pain (9%), the frequencies of which were similar to the HIV-negative participants. There were no serious adverse events. Anti-spike IgG responses by ELISA peaked at Day 42 (median 1440 ELISA units, IQR 704-2728) and were sustained out to Day 56. There was no correlation with CD4+ T cell count or age and the magnitude of the anti-spike IgG response at Day 56 (P>0.05 for both). ELISpot and T cell proliferative responses peaked between Day 14 and 28 after prime and were sustained through to Day 56. When compared to participants without HIV there was no statistical difference in magnitude or persistence of SARS-CoV-2 spike-specific humoral or cellular responses (P>0.05 for all analyses).Interpretation: In this study of PWH, vaccination with ChAdOx1 nCoV-19 was well tolerated and there was no difference in humoral and cell-mediated immune responses compared to an adult cohort without HIV who received the same vaccination regime. Trial Registration: Trial Registration number is NCT04400838. Funding: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midlands NIHR Clinical Research Network, and AstraZeneca. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care.Declaration of Interest: Oxford University has entered into a partnership with AstraZeneca for further development of ChAdOx1 nCoV-19 (AZD1222). AstraZeneca reviewed the data from the study and the final manuscript before 474 submission, but the authors retained editorial control. SCG is cofounder of Vaccitech (a collaborator in the early development of this vaccine candidate) and named as an inventor on a patent covering use of ChAdOx1-vectored vaccines (PCT/GB2012/000467) and a patent application covering this SARS-CoV-2 vaccine. TL is named as an inventor on a patent application covering this SARS-CoV-2 vaccine and was consultant to Vaccitech. PMF is a consultant to Vaccitech. AJP is Chair of the UK Department of Health and Social Care’s JCVI, but does not participate in policy advice on coronavirus vaccines, and is a member of the WHO Strategic Advisory Group of Experts (SAGE). AVSH is a cofounder of and consultant to Vaccitech and is named as an inventor on a patent covering design and use of ChAdOx1-vectored vaccines (PCT/GB2012/000467).Ethical Approval: Written informed consent was obtained from all participants, and the trial was done in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice. This study was approved in the UK by the Medicines and Healthcare products Regulatory Agency (reference 21584/0424/001-0001) and the South Central Berkshire Research Ethics Committee (reference 20/SC/0145). Vaccine use was authorised by Genetically Modified Organisms Safety Committees at each participating site.

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

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.

Native-like SARS-CoV-2 spike glycoprotein expressed by ChAdOx1 nCoV-19/AZD1222 vaccine (preprint)

Vaccine development against the SARS-CoV-2 virus focuses on the principal target of the neutralizing immune response, the spike (S) glycoprotein. Adenovirus-vectored vaccines offer an effective platform for the delivery of viral antigen, but it is important for the generation of neutralizing antibodies that they produce appropriately processed and assembled viral antigen that mimics that observed on the SARS-CoV-2 virus. Here, we describe the structure, conformation and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine. We demonstrate native-like post-translational processing and assembly, and reveal the expression of S proteins on the surface of cells adopting the trimeric prefusion conformation. The data presented here confirms the use of ChAdOx1 adenovirus vectors as a leading platform technology for SARS-CoV-2 vaccines.

Convalescent plasma therapy for the treatment of patients with COVID-19: Assessment of methods available for antibody detection and their correlation with neutralising antibody levels.

INTRODUCTION: The lack of approved specific therapeutic agents to treat coronavirus disease (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to the rapid implementation of convalescent plasma therapy (CPT) trials in many countries, including the United Kingdom. Effective CPT is likely to require high titres of neutralising antibody (nAb) in convalescent donations. Understanding the relationship between functional neutralising antibodies and antibody levels to specific SARS-CoV-2 proteins in scalable assays will be crucial for the success of a large-scale collection. We assessed whether neutralising antibody titres correlated with reactivity in a range of enzyme-linked immunosorbent assays (ELISA) targeting the spike (S) protein, the main target for human immune response. METHODS: Blood samples were collected from 52 individuals with a previous laboratory-confirmed SARS-CoV-2 infection. These were assayed for SARS-CoV-2 nAbs by microneutralisation and pseudo-type assays and for antibodies by four different ELISAs. Receiver operating characteristic (ROC) analysis was used to further identify sensitivity and specificity of selected assays to identify samples containing high nAb levels. RESULTS: All samples contained SARS-CoV-2 antibodies, whereas neutralising antibody titres of greater than 1:20 were detected in 43 samples (83% of those tested) and >1:100 in 22 samples (42%). The best correlations were observed with EUROimmun immunoglobulin G (IgG) reactivity (Spearman Rho correlation coefficient 0.88; p < 0.001). Based on ROC analysis, EUROimmun would detect 60% of samples with titres of >1:100 with 100% specificity using a reactivity index of 9.1 (13/22). DISCUSSION: Robust associations between nAb titres and reactivity in several ELISA-based antibody tests demonstrate their possible utility for scaled-up production of convalescent plasma containing potentially therapeutic levels of anti-SARS-CoV-2 nAbs.