SARS-CoV-2 Omicron spike mediated immune escape, infectivity and cell-cell fusion (preprint)

The Omicron variant emerged in southern Africa in late 2021 and is characterised by multiple spike mutations across all spike domains. Here we show that the Omicron spike confers very significant evasion of vaccine elicited neutralising antibodies that is more pronounced for ChAdOx-1 adenovirus vectored vaccine versus BNT162b2 mRNA vaccine. Indeed neutralisation of Omicron was not detectable for the majority of individuals who had received two doses of ChAdOx-1. Third dose mRNA vaccination rescues neutralisation in the short term. Despite three mutations predicted to favour spike S1/S2 cleavage, observed cleavage efficiency is lower than for wild type Wuhan-1 D614G and Delta. We demonstrate significantly lower infectivity of lung organoids and Calu-3 lung cells expressing endogenous levels of ACE2 and TMPRSS2 but similar infection as compared to Delta when using H1299 lung epithelial cells. Importantly, fusogenicity of the Omicron spike is impaired, leading to marked reduction in syncitia formation. These observations indicate that Omicron has gained immune evasion properties whilst possibly modulating properties associated with replication and pathogenicity.

Age-Related Heterogeneity in Neutralising Antibody Responses to SARS-CoV-2 Following BNT162b2 Vaccination (preprint)

Background: Vaccines remain the cornerstone for containing the SARS-CoV-2 pandemic. mRNA vaccines provide protection in clinical trials using a two-dose approach, separated by a three to four week gap. UK policy in 2021 is to extend the dosing interval from three to twelve weeks. There is a paucity of data in the elderly, even though these individuals are the first to receive vaccines due to risk of severe disease. Here we assessed real world immune responses following vaccination with mRNA-based vaccine BNT162b2.Methods: We did a prospective cohort study of individuals presenting for first dose vaccination. Following the first and second doses of the BNT162b2 vaccine, we measured IFNγ T cell responses, as well as binding antibody (IgA, IgG and IgG1-4) responses to Spike and Spike RBD. We also measured neutralising antibody responses to Spike in sera using a lentiviral pseudotyping system. We correlated age with immune responses and compared responses after the first and second doses.Findings: Median age was 63.5 years amongst 42 participants. Three weeks after the first dose a lower proportion of participants over 80 years old achieved adequate neutralisation titre of >1:20 for 50% neutralisation as compared to those under 80 (8/17 versus 19/24, p=0.03). Geometric mean neutralisation titres in this age group after the first dose were lower than in younger individuals (p<0.001). Binding IgA and IgG1 and 3 responses developed post vaccination, as observed in natural infection. T- cell responses were not different in those above or below 80 years. Following the second dose, 50% neutralising antibody titres were above 1:20 in all individuals and there was no longer a difference by age grouping.Interpretation: A high proportion of individuals above the age of 80 have suboptimal neutralising antibody responses following first dose vaccination with BNT162b2, cautioning against extending the dosing interval in this high risk population.Funding Statement: RKG is supported by a Wellcome Trust Senior Fellowship in Clinical Science (WT108082AIA). DAC is supported by a Wellcome Trust Clinical PhD Research Fellowship. KGCS is the recipient of a Wellcome Investigator Award (200871/Z/16/Z). This research was supported by the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre, the Cambridge Clinical Trials Unit (CCTU), the NIHR BioResource and Addenbrooke’s Charitable Trust. JAGB is supported by the Medical Research Council (MC_UP_1201/16). IATM is funded by a SANTHE award.Declaration of Interests: None to declare. Ethics Approval Statement: The study was approved by the East of England – Cambridge Central Research Ethics Committee (17/EE/0025).

Age-related heterogeneity in Neutralising antibody responses to SARS-CoV-2 following BNT162b2 vaccination (preprint)

Two dose mRNA vaccination provides excellent protection against SARS-CoV-2. However, there are few data on vaccine efficacy in elderly individuals above the age of 801. Additionally, new variants of concern (VOC) with reduced sensitivity to neutralising antibodies have raised fears for vulnerable groups. Here we assessed humoral and cellular immune responses following vaccination with mRNA vaccine BNT162b22 in elderly participants prospectively recruited from the community and younger health care workers. Median age was 72 years and 51% were females amongst 140 participants. Neutralising antibody responses after the first vaccine dose diminished with increasing age, with a marked drop in participants over 80 years old. Sera from participants below and above 80 showed significantly lower neutralisation potency against B.1.1.7, B.1.351 and P.1. variants of concern as compared to wild type. Those over 80 were more likely to lack any neutralisation against VOC compared to younger participants following first dose. The adjusted odds ratio for inadequate neutralisation activity against the B.1.1.7, P.1 and B.1.351 variant in the older versus younger age group was 4.3 (95% CI 2.0-9.3, p<0.001), 6.7 (95% CI 1.7-26.3, p=0.008) and 1.7 (95% CI 0.5-5.7, p=0.41). Binding IgG and IgA antibodies were lower in the elderly, as was the frequency of SARS-CoV-2 Spike specific B-memory cells. We observed a trend towards lower somatic hypermutation in participants with suboptimal neutralisation, and elderly participants demonstrated clear reduction in class switched somatic hypermutation, driven by the IgA1/2 isotype. SARS-CoV-2 Spike specific T-cell IFN{gamma} and IL-2 responses fell with increasing age, and both cytokines were secreted primarily by CD4 T cells. We conclude that the elderly are a high risk population that warrant specific measures in order to mitigate against vaccine failure, particularly where variants of concern are circulating.

SARS-CoV-2 B.1.1.7 escape from mRNA vaccine-elicited neutralizing antibodies (preprint)

SARS-CoV-2 transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 mutations found in the B.1.1.7 Spike protein. The vaccine sera exhibited a broad range of neutralizing titres against the wild-type pseudoviruses (<1:4 to 3449) that were reduced against B.1.1.7 variant by 3.85 fold (IQR 2.68-5.28). This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralization was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 outof 29), but not in neutralizing mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background led to a further loss of neutralizing activity by vaccine-elicited antibodies over that conferred by the B.1.1.7 mutations alone. Further work is needed to establish the impact of these observations on protective vaccine efficacy in the context of the evolving B.1.1.7 lineage that will likely acquire E484K.

Impact of SARS-CoV-2 B.1.1.7 Spike variant on neutralisation potency of sera from individuals vaccinated with Pfizer vaccine BNT162b2 (preprint)

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 amino acid mutations found in the B.1.1.7 spike protein. The vaccine sera exhibited a broad range of neutralising titres against the wild-type pseudoviruses that were modestly reduced against B.1.1.7 variant. This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralisation was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 out of 31), but not in neutralising mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background to reflect newly emerging viruses in the UK led to a more substantial loss of neutralising activity by vaccine-elicited antibodies and mAbs (19 out of 31) over that conferred by the B.1.1.7 mutations alone. E484K emergence on a B.1.1.7 background represents a threat to the vaccine BNT162b.

Recurrent emergence and transmission of a SARS-CoV-2 Spike deletion ΔH69/V70 (preprint)

SARS-CoV-2 amino acid replacements in the receptor binding domain (RBD) occur relatively frequently and some have a consequence for immune recognition. Here we report recurrent emergence and significant onward transmission of a six-nucleotide out of frame deletion in the S gene, which results in loss of two amino acids: H69 and V70. We report that in human infections {Delta}H69/V70 often co-occurs with the receptor binding motif amino acid replacements N501Y, N439K and Y453F, and in the latter two cases has followed the RBD mutation. One of the {Delta}H69/V70+ N501Y lineages, now known as B.1.1.7, has undergone rapid expansion and includes eight S gene mutations: RBD (N501Y and A570D), S1 ({Delta}H69/V70 and {Delta}144) and S2 (P681H, T716I, S982A and D1118H). In vitro, we show that {Delta}H69/V70 does not reduce serum neutralisation across multiple convalescent sera. However, {Delta}H69/V70 increases infectivity and is associated with increased incorporation of cleaved spike into virions. {Delta}H69/V70 is able to compensate for small infectivity defects induced by RBD mutations N501Y, N439K and Y453F. In addition, replacement of H69 and V70 residues in the B.1.1.7 spike reduces its infectivity and spike mediated cell-cell fusion. Based on our data {Delta}H69/V70 likely acts as a permissive mutation that allows acquisition of otherwise deleterious immune escape mutations. Enhanced surveillance for the {Delta}H69/V70 deletion with and without RBD mutations should be considered as a global priority not only as a marker for the B.1.1.7 variant, but potentially also for other emerging variants of concern. Vaccines designed to target the deleted spike protein could mitigate against its emergence as increased selective forces from immunity and vaccines increase globally. HighlightsO_LI{Delta}H69/V70 is present in at least 28 SARS-CoV-2 lineages C_LIO_LI{Delta}H69/V70 does not confer escape from convalescent sera C_LIO_LI{Delta}H69/V70 increases spike infectivity and compensates for RBD mutations C_LIO_LI{Delta}H69/V70 is associated with greater spike cleavage C_LIO_LIB.1.1.7 requires {Delta}H69/V70 for optimal spike cleavage and infectivity C_LI

Combined point of care nucleic acid and antibody testing for SARS-CoV-2: a prospective cohort study in suspected moderate to severe COVID-19 disease. (preprint)

BackgroundRapid COVID-19 diagnosis in hospital is essential for patient management and identification of infectious patients to limit the potential for nosocomial transmission. The diagnosis of infection is complicated by 30-50% of COVID-19 hospital admissions with nose/throat swabs testing negative for SARS-CoV-2 nucleic acid, frequently after the first week of illness when SARS-CoV-2 antibody responses become detectable. We assessed the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease in the emergency department. MethodsWe developed (i) an in vitro neutralization assay using a lentivirus expressing a genome encoding luciferase and pseudotyped with spike (S) protein and (ii) an ELISA test to detect IgG antibodies to nucleocapsid (N) and S proteins from SARS-CoV-2. We tested two lateral flow rapid fingerprick tests with bands for IgG and IgM. We then prospectively recruited participants with suspected moderate to severe COVID-19 and tested for SARS-CoV-2 nucleic acid in a combined nasal/throat swab using the standard laboratory RT-PCR and a validated rapid POC nucleic acid amplification (NAAT) test. Additionally, serum collected at admission was retrospectively tested by in vitro neutralisation, ELISA and the candidate POC antibody tests. We evaluated the performance of the individual and combined rapid POC diagnostic tests against a composite reference standard of neutralisation and standard laboratory based RT-PCR. Results45 participants had specimens tested for nucleic acid in nose/throat swabs as well as stored sera for antibodies. Using the composite reference standard, prevalence of COVID-19 disease was 53.3% (24/45). Median age was 73.5 (IQR 54.0-86.5) years in those with COVID-19 disease by our reference standard and 63.0 (IQR 41.0-72.0) years in those without disease. The overall detection rate by rapid NAAT was 79.2% (95CI 57.8-92.9%), decreasing from 100% (95% CI 65.3-98.6%) in days 1-4 to 50.0% (95% CI 11.8-88.2) for days 9-28 post symptom onset. Correct identification of COVID-19 with combined rapid POC diagnostic tests was 100% (95CI 85.8-100%) with a false positive rate of 5.3-14.3%, driven by POC LFA antibody tests. ConclusionsCombined POC tests have the potential to transform our management of COVID-19, including inflammatory manifestations later in disease where nucleic acid test results are negative. A rapid combined approach will also aid recruitment into clinical trials and in prescribing therapeutics, particularly where potentially harmful immune modulators (including steroids) are used.