Quantitative, multiplexed, targeted proteomics for ascertaining variant specific SARS-CoV-2 antibody response

Determining the protection an individual has to SARS-CoV-2 variants of concern (VoC) will be crucial for future immune surveillance and understanding the changing immune response. As further variants emerge, current serology tests are becoming less effective in reflecting neutralising capability of the immune system. A better measure of an evolving antigen-antibody immune response is needed. We describe a multiplexed, baited, targeted-proteomic assay for direct detection of multiple proteins in the SARS-CoV-2 anti-spike antibody immunocomplex. This enables a more sophisticated and informative characterisation of the antibody response to vaccination and infection against VoC. Using this assay, we detail different and specific responses to each variant by measuring several antibody classes, isotypes and associated complement binding. Furthermore, we describe how these proteins change using serum from individuals collected after infection, first and second dose vaccination. We show complete IgG1 test concordance with gold standard ELISA (r>0.8) and live virus neutralisation against Wuhan Hu-1, Alpha B.1.1.7, Beta B.1.351, and Delta B.1.617.1 variants (r>0.79). We also describe a wide degree of heterogeneity in the immunocomplex of individuals and a greater IgA response in those patients who had a previous infection. Significantly, our test points to an important role the complement system may play particularly against VoC. Where we observe altered Complement C1q association to the Delta VoC response and a stronger overall association with neutralising antibodies than IgG1. A detailed understanding of an individuals antibody response could benefit public health immunosurveillance, vaccine design and inform vaccination dosing using a personalised medicine approach.

Prior infection with SARS-CoV-2 boosts and broadens Ad26.COV2.S immunogenicity in a variant dependent manner

The Johnson and Johnson Ad26.COV2.S single dose vaccine represents an attractive option for COVID-19 vaccination in resource limited countries. We examined the effect of prior infection with different SARS-CoV-2 variants on Ad26.COV2.S immunogenicity. We compared participants who were SARS-CoV-2 naive with those either infected with the ancestral D614G virus, or infected in the second wave when Beta predominated. Prior infection significantly boosted spike binding antibodies, antibody-dependent cellular cytotoxicity and neutralizing antibodies against D614G, Beta and Delta, however neutralization cross-reactivity varied by wave. Robust CD4 and CD8 T cell responses were induced after vaccination, regardless of prior infection. T cell recognition of variants was largely preserved, apart from some reduction in CD8 recognition of Delta. Thus, Ad26.COV2.S vaccination following infection may result in enhanced protection against COVID-19. The impact of the infecting variant on neutralization breadth after vaccination has implications for the design of second-generation vaccines based on variants of concern.

Non-severe SARS-CoV-2 infection is characterised by very early T cell proliferation independent of type 1 interferon responses and distinct from other acute respiratory viruses.

The correlates of natural protective immunity to SARS-CoV-2 in the majority who experience asymptomatic infection or non-severe disease are not fully characterised, and remain important as new variants emerge. We addressed this question using blood transcriptomics, multiparameter flow cytometry and T cell receptor (TCR) sequencing spanning the time of incident infection. We identified a type 1 interferon (IFN) response common to other acute respiratory viruses, and a cell proliferation response that discriminated SARS-CoV-2 from other viruses. These responses peaked by the time the virus was first detected, and in some preceded virus detection. Cell proliferation was most evident in CD8 T cells and associated with rapid expansion of SARS-CoV-2 reactive TCRs. We found an equally rapid increase in immunoglobulin transcripts, but circulating virus-specific antibodies lagged by 1-2 weeks. Our data support a protective role for rapid induction of type 1 IFN and CD8 T cell responses to SARS-CoV-2.

Blood transcriptional biomarkers of acute viral infection for detection of pre-symptomatic SARS-CoV 2 infection

We hypothesised that host-response biomarkers of viral infections may contribute to early identification of SARS-CoV-2 infected individuals, critical to breaking chains of transmission. We identified 20 candidate blood transcriptomic signatures of viral infection by systematic review and evaluated their ability to detect SARS-CoV-2 infection, compared to the gold-standard of virus PCR tests, among a prospective cohort of 400 hospital staff subjected to weekly testing when fit to attend work. The transcriptional signatures had limited overlap, but were mostly co-correlated as components of type 1 interferon responses. We reconstructed each signature score in blood RNA sequencing data from 41 individuals over sequential weeks spanning a first positive SARS-CoV-2 PCR, and after 6-month convalescence. A single blood transcript for IFI27 provided the highest accuracy for discriminating individuals at the time of their first positive viral PCR result from uninfected controls, with area under the receiver operating characteristic curve (AUROC) of 0.95 (95% confidence interval 0.91-0.99), sensitivity 0.84 (0.7-0.93) and specificity 0.95 (0.85-0.98) at a predefined test threshold. The test performed equally well in individuals with and without symptoms, correlated with viral load, and identified incident infections one week before the first positive viral PCR with sensitivity 0.4 (0.17-0.69) and specificity 0.95 (0.85-0.98). Our findings strongly support further urgent evaluation and development of blood IFI27 transcripts as a biomarker for early phase SARS-CoV-2 infection, for screening individuals such as contacts of index cases, in order to facilitate early case isolation and early antiviral treatments as they emerge.

Healthcare workers with mild / asymptomatic SARS-CoV-2 infection show T cell responses and neutralising antibodies after the first wave

Studies of adaptive immunity to SARS-CoV-2 include characterisation of lethal, severe and mild cases1-8. Understanding how long immunity lasts in people who have had mild or asymptomatic infection is crucial. Healthcare worker (HCW) cohorts exposed to and infected by SARS-CoV-2 during the early stages of the pandemic are an invaluable resource to study this question9-14. The UK COVIDsortium is a longitudinal, London hospital HCW cohort, followed from the time of UK lockdown9,10 ; weekly PCR, serology and symptom diaries allowed capture of asymptomatic infection around the time of onset, so duration of immunity could be tracked. Here, we conduct a cross-sectional, case-control, sub-study of 136 HCW at 16-18 weeks after UK lockdown, with 76 having had laboratory-confirmed SARS-CoV-2 mild or asymptomatic infection. Neutralising antibodies (nAb) were present in 90% of infected HCW sampled after the first wave; titres, likely to correlate with functional protection, were present in 66% at 16-18 weeks. T cell responses tended to be lower in asymptomatic infected HCW than those reporting case-definition symptoms of COVID-19, while nAb titres were maintained irrespective of symptoms. T cell and antibody responses were discordant. HCW lacking nAb also showed undetectable T cells to Spike protein but had T cells of other specificities. Our findings suggest that the majority of HCW with mild or asymptomatic SARS-CoV-2 infection carry nAb complemented by multi-specific T cell responses for at least 4 months after mild or asymptomatic SARS-CoV-2 infection.

Analytical Performance and Reference Interval of a Microfluidic Viscometer, Viscore-300 for the Measurement of Whole Blood Viscosity

BACKGROUND: Whole blood viscosity (WBV) refers to the internal resistance that occurs when blood flows through blood vessels. WBV is known to be related to many diseases including cardiovascular and neurovascular diseases. We have investigated the analytical performance and established reference intervals for a newly developed microfluidic viscometer, Viscore-300 (NanoBiz, Korea), used for the measurement of WBV. METHODS: We performed a precision test of 240 measurements over 20 days using three control materials. For evaluation of repeatability, a total of 60 WBV measurements were made in 3 whole blood samples 20 times a day. A total of 100 whole blood samples were used to evaluate the accuracy of the Viscore-300 in comparison to a rotating viscometer, DV3T (Brookfield, USA), in accordance with the the Clinical and Laboratory Standards Institute's guidelines. To establish the reference intervals, 122 healthy individuals were enrolled in this study. RESULTS: The precision and repeatability results showed that the CV was less than 5% for three samples and two shear rates. In the accuracy test, the mean differences between two viscometers were 0.09 cP (0.9%) and −0.07 cP (−1.4%) at shear rates of 10 s−1 and 300 s−1, respectively. The reference intervals of WBV for men were 6.88–13.52 cP at 10 s−1 and 4.32–6.43 cP at 300 s−1; those of women were 5.74–13.29 cP at 10 s−1 and 3.60–6.12 cP at 300 s−1. CONCLUSIONS: Viscore-300 showed excellent precision and accuracy and it might be a good instrument for reporting WBV quickly and accurately.

Rejuvenating Aged Hematopoietic Stem Cells Through Improvement of Mitochondrial Function

Mitochondria are the powerhouses of the cell as well as the primary site of hematopoiesis, which also occurs in the cytoplasm. Hematopoietic stem cells (HSCs) are characterized by a very high turnover rate, and are thus considered to be relatively free from the age-related insults generated by mitochondria. However, HSCs are also subject to these age-related insults, including the incidence of myeloid proliferative diseases, marrow failure, hematopoietic neoplasms, and deterioration of the adaptive human immune system. Recently, NAD⁺ dietary supplements, known as niacin or vitamin B₃, including tryptophan, nicotinic acid, nicotinamide, and the newly identified NAD⁺ precursor nicotinamide riboside, have been shown to play a role in restoring adult stem cell function through the amelioration of mitochondrial dysfunction. This insight motivated a study that focused on reversing aging-related cellular dysfunction in adult mouse muscle stem cells by supplementing their diet with nicotinamide riboside. The remedial effect of nicotinamide riboside enhanced mitochondrial function in these muscle stem cells in a SIRT1-dependent manner, affecting cellular respiration, membrane potential, and production of ATP. Accordingly, numerous studies have demonstrated that sirtuins, under nuclear/mitochondrial control, have age-specific effects in determining HSC phenotypes. Based on the evidence accumulated thus far, we propose a clinical intervention for the restoration of aged HSC function by improving mitochondrial function through NAD⁺ precursor supplementation.