Repeatability was 11

Repeatability was 11.1%, 7.9% and 11.8% and intermediate precision was 20.8%, 9.3% and 39% respectively. Cell based neutralisation assays involve actively replicating cells and virus which introduces inherent biological variation. E6/TMPRSS2 cells for 18h. Percentage infected cells was analysed by automated flow cytometry following trypsinisation, fixation and SARS-CoV-2 Nucleoprotein intracellular staining. Half-maximal Neutralisation Titres (NT50) were determined using non-linear regression. Our assay was compared to Plaque Reduction Neutralisation Test (PRNT) and validated against the First WHO International Standard for anti-SARS-CoV-2 immunoglobulin. Both Micro-NT and PRNT achieved comparable NT50 values. Further validation showed adequate correlation with PRNT using a panel of secondary standards of clinical convalescent and vaccinated plasma samples. We found the assay to be reproducible through measuring both repeatability and intermediate precision. Screening 190 convalescent samples and 11 COVID-19 naive controls (AIID cohort) we demonstrated that Micro-NT has broad dynamic range differentiating NT50s <1/20 to >1/5000. We could also characterise immune-escape VOC Beta and Omicron BA.5, achieving fold-reductions in neutralising capacity similar to those published. Our flow cytometry-based Micro-NT is a robust and reliable assay to quantify NAb titres, and has been selected as an endpoint in clinical trials. == Introduction == SARS-CoV-2 is the viral agent responsible for the Coronavirus Infectious Disease 2019 (COVID-19) pandemic [1]. The disease was given pandemic status by the World Health Organisation in March 2020, and as of September 2023, there has been 676 million cases, and 6.8 million deaths [2]. Both T-cell and humoral immune responses are required for protection from COVID-19. Humoral immunity relies on B cell exposure to SARS-CoV-2 antigens, which triggers their proliferation into antibody secreting plasma cells [3]. Following infection, antibodies are produced against SARS-CoV-2 viral proteins, predominantly the Spike (S) and the Nucleocapsid Protein (NP) [4,5]. Neutralising antibodies (NAbs) are a subset of SARS-CoV-2 antibodies that prevent viral entry, through either direct blocking of virus binding to the host cell receptor, or preventing conformational changes required for membrane fusion. SARS-CoV-2 NAbs target the S protein, making Cyclosporin H it the preferred COVID-19 vaccine candidate [6,7]. The S protein is comprised of trimeric S1/S2 heterodimers. S1, harbouring an N-terminal domain and a Receptor Binding Domain (RBD), interacts with the host cell through binding of the RBD to the angiotensin-converting enzyme 2 (ACE-2) receptor. Following S1/S2 cleavage by host cell proteases including furin, S2 is cleaved by TMPRSS2 or Cathepsin-L mediating membrane fusion and cell entry [8]. The most potent NAbs target the RBD as these directly compete with ACE-2 for binding. Mutations in this site are often associated with immune escape [9]. Non-RBD sites are more evolutionarily conserved, so NAbs targeting these sites can often maintain efficacy against SARS-CoV-2 variants as well as display Cyclosporin H cross-reactivity with other sarbecoviruses [10]. An effective antibody response provides protection against COVID-19. The Protective Neutralisation Classification Model described by Cyclosporin H Khoury and colleagues [6] suggests that the Cyclosporin H protective neutralisation titre (reducing risk of infection by 50%) is 20% of the mean neutralisation titre of a convalescent cohort. They have found this to strongly predict protective immunity (against symptomatic disease) elicited by COVID-19 vaccine trials, while achieving a titre of only 3% of the mean is sufficient to reduce risk of severe disease by 50%. Not all S-targeting antibodies are neutralising. Post-infection or post-vaccination, a polyclonal antibody population is produced, targeting sites along the S protein [11]. Some may only bind, but Sox18 not have any neutralising capacity due to their site of action. Others may offer protection against Wild-type (WT) SARS-CoV-2, the strain against which the vaccine S was originally modelled, but not against immune-escape Variants of Concern (VOC), including Beta which was the first SARS-CoV-2 Variant to display moderate immune escape, and Omicron, which far exceeded the escape capacity of previous variants, due to large numbers of amino acid mutations in key antibody binding sites in the RBD [12]. For this reason, an antibody titre, the measure of total anti-SARS-CoV-2 IgGs against a certain target present in a sample [13], is not sufficient to infer a protective immune response. Instead, the functional capacity of an antibody population can be determined using a neutralisation test. Neutralisation tests, used to measure the capacity of a monoclonal antibody or plasma/serum to inhibit viral infection of susceptible cells, have proved valuable in elucidating SARS-CoV-2 antibody responses over time [14], in convalescent versus vaccinated individuals [15], and against SARS-CoV-2 VOCs [16,17]. This information is critical to forming effective public health strategies, from understanding when vaccine-induced protection wanes in different cohorts to devise booster strategies [18,19], to identifying plasma donors for convalescent therapy [20,21], to rapid identification of new VOCs that escape pre-existing immunity [22,23]. The original gold standard viral.