TMPRSS2 activation of Omicron lineage Spike glycoproteins is regulated by TMPRSS2 cleavage of ACE2 (preprint)

Continued high levels spread of SARS-CoV-2 globally enabled accumulation of changes within the Spike glycoprotein, leading to resistance to neutralising antibodies and concomitant changes to entry requirements that increased viral transmission fitness. Herein, we demonstrate a significant change in ACE2 and TMPRSS2 use by primary SARS-CoV-2 isolates that occurred upon arrival of Omicron lineages. Mechanistically we show this shift to be a function of two distinct ACE2 pools based on cleavage or non-cleavage of ACE2 by TMPRSS2 activity. In engineered cells overexpressing ACE2 and TMPRSS2, ACE2 was cleaved by TMPRSS2 and this led to either augmentation or progressive attenuation of pre-Omicron and Omicron lineages, respectfully. In contrast, TMPRSS2 resistant ACE2 restored infectivity across all Omicron lineages through enabling ACE2 binding that facilitated TMPRSS2 spike activation. Therefore, our data support the tropism shift of Omicron lineages to be a function of evolution towards the use of uncleaved pools of ACE2 with the latter consistent with its role as a chaperone for many tissue specific amino acid transport proteins.

Improvement of immune dysregulation and health-related quality of life in individuals with long COVID at 24-months following SARS-CoV-2 infection (preprint)

This study investigated the humoral and cellular immune responses in individuals with long COVID (LC) compared to age and gender matched recovered COVID-19 controls (MC) over 24-months. LC participants showed elevated spike and nucleocapsid IgG levels, higher neutralizing capacity, and increased spike- and nucleocapsid-specific CD4+ T cells, PD-1, and TIM-3 expression on CD4+ and CD8+ T cells at 3- and 8-months, but these differences did not persist at 24-months. Some LC participants had detectable IFN-{beta} and IFN-{gamma} that was attributed to reinfection and antigen re-exposure. Single-cell RNA sequencing at 24-month timepoint revealed similar immune cell proportions and reconstitution of naive T and B cell subsets in LC. No significant differences in exhaustion scores or antigen-specific T cell clones were observed. These findings suggest resolution of immune activation in LC and return to comparable immune responses between LC and MC over time. Improvement in self-reported health-related quality of life at 24-months was also evident in the majority of LC (62%). PTX3, CRP levels and platelet count were associated with improvements in health-related quality of life.

Emergence and antibody evasion of BQ and BA.2.75 SARS-CoV-2 sublineages in the face of maturing antibody breadth at the population level (preprint)

The Omicron era of the COVID-19 pandemic commenced at the beginning of 2022 and whilst it started with primarily BA.1, it was latter dominated by BA.2 and related sub-lineages. Over the course of 2022, we monitored the potency and breadth of antibody neutralization responses to many emerging variants at two levels: (i) we tracked over 400,000 U.S. plasma donors over time through various vaccine booster roll outs and Omicron waves using antibody pools. (ii) we mapped the antibody response at the individual level using blood from strigently curated vaccine and convalescent cohorts. In pooled antibody samples, we observed the maturation of neutralization breadth to Omicron variants over time through continuing vaccine and infection waves. Importantly, in many cases we observed increased antibody breadth to variants that were yet to be in circulation. Resolution of viral neutralisation at the cohort level supported equivalent coverage across prior and emerging variants with emerging isolates BQ.1.1, XBB.1 and BR.2.1 the most evasive. Further, these emerging variants were resistant to Evusheld, whilst neutralization resistance to Sotrovimab was restricted to BQ.1.1 and further supported by lack of Spike glycoprotein binding to this variant. An outgrowth advantage through better utilization of TMPRSS2 was observed across BQ lineages and not those derived from BA.2.75. We conclude at this current point in time that variants derived from BQ lineages can evade antibodies at levels equivalent to their most evasive BA.2.75 counterparts but sustain an entry phenotype that would promote an additional outgrowth advantage.

High titre neutralizing antibodies in response to SARS-CoV-2 infection require RBD-specific CD4 T cells that include proliferative memory cells. (preprint)

Long-term immunity to SARS-CoV-2 infection, including neutralizing antibodies and T cell-mediated immunity, is required in a very large majority of the population in order to reduce ongoing disease burden. We have investigated the association between memory CD4 and CD8 T cells and levels of neutralizing antibodies in convalescent COVID-19 subjects. Higher titres of convalescent neutralizing antibodies were associated with significantly higher levels of RBD-specific CD4 T cells, including specific memory cells that proliferated vigorously in vitro. Conversely, up to half of convalescent individuals had low neutralizing antibody titres together with a lack of receptor binding domain (RBD)-specific memory CD4 T cells. These low antibody subjects had other, non-RBD, spike-specific CD4 T cells, but with more of an inhibitory Foxp3+ and CTLA-4+ cell phenotype, rather than the effector T-bet+, cytotoxic granzymes+ and perforin+ cells seen in high antibody subjects. Single cell transcriptomics of antigen-specific CD4+ T cells from high antibody subjects revealed heterogenous RBD-specific CD4+ T cells that comprised central memory, transitional memory and Tregs, as well as cytotoxic clusters containing diverse TCR repertoires, that were absent in individuals with low antibody levels. However, vaccination in low antibody convalescent individuals led to a slight but significant improvement in RBD-specific memory CD4 T cells and increased neutralizing antibody titres. Our results suggest that targeting CD4 T cell epitopes proximal to and within the RBD-region should be prioritized in booster vaccines.

SARS-CoV-2 Omicron BA.5: Evolving tropism and evasion of potent humoral responses and resistance to clinical immunotherapeutics relative to viral variants of concern. (preprint)

Genetically distinct viral variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recorded since January 2020. Over this time global vaccine programs have been introduced, contributing to lowered COVID-19 hospitalisation and mortality rates, particularly in developed countries. In late 2021, the Omicron BA.1 variant emerged, with significant genetic differences and clinical effects from other variants of concern (VOC). This variant demonstrated higher numbers of polymorphisms in the gene encoding the Spike (S) protein, and there has been displacement of the dominant Delta variant. Shortly after dominating global spread in early 2022, BA.1 was supplanted by the genetically distinct Omicron lineage BA.2. A sub-lineage of BA.2, designated BA.5 has now started to dominate globally, with the potential to supplant BA.2. To address the relative threat of BA.5, we determined infectivity to particle ratios in primary nasopharyngeal samples and expanded low passage isolates in a well characterised, genetically engineered ACE2/TMPRSS2 cell line. We then assessed the impact of BA.5 infection on humoral neutralisation in vitro, in vaccinated and convalescent cohorts, using concentrated human IgG pooled from thousands of plasma donors, and licensed monoclonal antibody therapies. The infectivity of virus in primary swabs and expanded isolates revealed whilst BA.1 and BA.2 are attenuated through ACE2/TMPRSS2, BA.5 infectivity is equivalent to that of an early 2020 circulating clade and has greater sensitivity to the TMPRSS2 inhibitor Nafamostat. As with BA.1, we observed BA.5 to significantly reduce neutralisation titres across all donors. Concentrated pooled human IgG from convalescent and vaccinated donors had greater breadth of neutralisation, although the potency was still reduced 7-fold with BA.5. Of all therapeutic antibodies tested, we observed a 14.3-fold reduction using Evusheld and 16.8 reduction using Sotrovimab when neutralising a Clade A versus BA.5 isolate. These results have implications for ongoing tracking and management of Omicron waves globally.

Clonal dynamics of SARS-CoV-2-specific T cells in children and adults with COVID-19 (preprint)

Children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop less severe coronavirus disease 2019 (COVID-19) than adults. The mechanisms for the age-specific differences and the implications for infection-induced immunity are beginning to be uncovered. We show by longitudinal multimodal analysis that SARS-CoV-2 leaves a small footprint in the circulating T cell compartment in children with mild/asymptomatic COVID-19 compared to adult household contacts with the same disease severity who had more evidence of systemic T cell interferon activation, cytotoxicity and exhaustion. Children harbored diverse polyclonal SARS-CoV-2-specific naive T cells whereas adults harbored clonally expanded SARS-CoV-2-specific memory T cells. More naive interferon-activated CD4+ T cells were recruited into the memory compartment and recovery was associated with the development of robust CD4+ memory T cell responses in adults but not children. These data suggest that rapid clearance of SARS-CoV-2 in children may compromise their cellular immunity and ability to resist reinfection.

Immunological dysfunction persists for 8 months following initial mild-moderate SARS-CoV-2 infection (preprint)

A proportion of patients surviving acute COVID-19 infection develop post-COVID syndrome (long COVID) encompassing physical and neuropsychiatric symptoms lasting longer than 12 weeks. Here we studied a prospective cohort of individuals with long COVID (the ADAPT study) compared to age/gender matched subjects without long COVID, healthy donors and individuals infected with other non-SARS CoV2 human coronaviruses (the ADAPT-C study). We found an elevated diffuse serum inflammatory cytokine profile in symptomatic long COVID subjects that was maintained at 8 months post-infection and was not observed in asymptomatic COVID-19 survivors. This inflammatory profile consisted of 15 cytokines that positively correlated; revealing an apparent diffuse, potentially coordinated, low level up regulation of a spectrum of immune and inflammatory mediators. In addition, we found an absence of subsets of un-activated naive T and B cells in peripheral blood of long COVID subjects, that did not reconstitute over time. In contrast, individual serum cytokines from the interferon I and III classes, T cell activation markers and plasma ACE2, while elevated in the serum of people previously infected with SARS-CoV-2 were not further elevated in subjects with long COVID symptoms. This work defines immunological parameters associated with long COVID and suggests future opportunities to prevention and treatment.

Limited recovery from post-acute sequelae of SARS-CoV-2 (PASC) at eight months of a prospective cohort (preprint)

There is increasing recognition of the prolonged illness following acute coronavirus disease 2019 (COVID-1). In a longitudinal cohort of 99 patients, 32% reported persistent symptoms and 19% had Long COVID (Defined as fatigue or dyspnoea or chest tightness) at median 240 days after initial infection. There was no significant improvement in symptoms or measures of health-related quality of life between 4 and 8-month assessments. In multivariable analysis, female gender (OR 3.2, 95%CI 1.3-7.8, p=0.01) and acute COVID-19 hospitalisation (OR 3.8, 95% 1.1-13.6, p=0.04) were independently associated with Long COVID at 8-months. Only 80% patients reported full recovery at 8 months. Further research is required to understand the immunologic correlates of abnormal recovery and the long-term significance.

SARS-CoV-2 neutralizing antibodies; longevity, breadth, and evasion by emerging viral variants (preprint)

The SARS-CoV-2 antibody neutralization response and its evasion by emerging viral variants are unknown. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus-cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 RT-PCR-confirmed COVID-19 individuals with detailed demographics and followed up to seven months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization were associated with COVID-19 severity. A subgroup of high responders maintained high neutralizing responses over time, representing ideal convalescent plasma therapy donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal plasma donors and vaccine monitoring and design.

SARS Coronavirus-2 microneutralisation and commercial serological assays correlated closely for some but not all enzyme immunoassays (preprint)

BackgroundSerological testing for SARS-CoV-2 specific antibodies provides important research and diagnostic information relating to COVID-19 prevalence, incidence, and host immune response. A greater understanding of the relationship between functionally neutralising antibodies detected using microneutralisation assays and binding antibodies detected using scalable enzyme immunoassays (EIA) is needed in order to address protective immunity post-infection or vaccination, and assess EIA suitability as a surrogate test for screening of convalescent plasma donors. We assessed whether neutralising antibody titres correlated with signal cut-off ratios in five commercially available EIAs, and one in-house assay based on expressed spike protein targets. MethodsSera from individuals recovered from patients or convalescent plasma donors who reported laboratory-confirmed SARS-CoV-2 infection (n=200), and negative control sera collected prior to the COVID-19 pandemic (n=100) were assessed in parallel. Performance was assessed by calculating EIA sensitivity and specificity with reference to microneutralisation. ResultsNeutralising antibodies were detected in 166 (83%) samples. Compared with this, the most sensitive EIAs were the Cobas Elecsys Anti-SARS-CoV-2 (98%) and Vitros Immunodiagnostic Anti-SARS-CoV-2 (100%), which detect total antibody targeting the N and S1 antigens, respectively. The assay with the best quantitative relationship with microneutralisation was the Euroimmun IgG. ConclusionsThese results suggest the marker used (total Ab vs IgG vs IgA), and the target antigen are important determinants of assay performance. The strong correlation between microneutralisation and some commercially available assays demonstrate their potential for clinical and research use in assessing protection following infection or vaccination, and use as a surrogate test to assess donor suitability for convalescent plasma donation.