It is currently unknown how Post-COVID-19 Syndrome (PCS) may affect those infected with SARS-CoV-2. This longitudinal study reports on healthcare staff who tested positive for SARS-CoV-2 between March-April 2020 and follows their antibody titres and symptomatology. Over half (n=21/38) had PCS at 7-8 months. There was no statistically significant difference between initial RT-PCR viral titres or serial antibody levels between those who did and did not develop PCS. This study highlights the relative commonality of PCS in healthcare workers and this should be considered in vaccination scheduling and workforce planning to allow adequate frontline staffing numbers.

The COVID-19 pandemic is a serious healthcare challenge, leading to more than 5 million deaths worldwide so far. The rapid advent of modern messenger RNA-and adenovirus DNA vector-based anti-SARS-CoV-2 vaccines has already been associated with a significant reduction in the rate of COVID-19 deaths. 1 Haematological malignancies and/or their treatments are linked to impaired immunocompetence, which has been highlighted by recent reports of severe COVID-19 outcomes 2 and poor responses to SARS-CoV-2 vaccines. 3,4 Interestingly, patients with chronic myeloid leukaemia (CML) on BCR-ABL1 tyrosine kinase inhibitors (TKI) seem to have less severe COVID-19 compared to patients with other haematological cancers. 5 Also, although TKI have been reported to be immunosuppressive, 6,7 two recent prospective studies demonstrated that CML patients on TKI can mount an early immune response after the first dose of BNT162b2 (Pfizer-BioNTech). 8,9 We are conducting a prospective observational study (CML-Co-vax) at our centre, of which the primary objective is the evaluation of the seroconversion rate and the median anti-SARS-CoV-2 receptor binding domain (RBD) antibody level in CML patients on TKI compared to healthy subjects (HS), at early and late time points after the first and the second dose of SARS-CoV-2 vaccines. Patients and HS were assessed serially at baseline (T0), day +21 ± 7 (T1), day +49 ± 7 (T2) after the first dose, day +21 ± 7 (T3) and day +90 ± 20 (T4) after the second dose. Both groups received two doses of either BNT162b2 or ChAdOx1 nCov-19 (Oxford-AstraZeneca) vaccine 6-12 weeks apart (in accordance with UK government recommendations).Inclusion criteria were CML in chronic phase, current treatment with TKI and in at least complete cytogenetic remission. Exclusion from the study was determined by previous PCR-or serology-confirmed COVID-19 or by anti-SARS-CoV-2 immunisation with any of the available vaccines.Seroconversion for anti-RBD was confirmed using the Imperial double antigen-binding enzyme-linked immunosorbent assay (Imperial Hybrid DABA), which detects total anti-RBD with a specificity of 100% and a sensitivity of 98.9%. 10 All subjects gave their informed consent for participation in the study, which was approved by the Health Research Authority (IRAS ID: 289527) and by the Research Ethics

Accurate and sensitive detection of antibody to SARS-CoV-2 remains an essential component of the pandemic response. Measuring antibody that predicts neutralising activity and the vaccine response is an absolute requirement for laboratory-based confirmatory and reference activity. The viral receptor binding domain (RBD) constitutes the prime target antigen for neutralising antibody. A double antigen binding assay (DABA), providing the most sensitive format has been exploited in a novel hybrid manner employing a solid-phase S1 preferentially presenting RBD, coupled with a labelled RBD conjugate, used in a two-step sequential assay for detection and measurement of antibody to RBD (anti-RBD). This class and species neutral assay showed a specificity of 100% on 825 pre COVID-19 samples and a potential sensitivity of 99.6% on 276 recovery samples, predicting quantitatively the presence of neutralising antibody determined by pseudo-type neutralisation and by plaque reduction. Anti-RBD is also measurable in ferrets immunised with ChadOx1 nCoV-19 vaccine and in humans immunised with both AstraZeneca and Pfizer vaccines. This assay detects anti-RBD at presentation with illness, demonstrates its elevation with disease severity, its sequel to asymptomatic infection and its persistence after the loss of antibody to the nucleoprotein (anti-NP). It also provides serological confirmation of prior infection and offers a secure measure for seroprevalence and studies of vaccine immunisation in human and animal populations. The hybrid DABA also displays the attributes necessary for the detection and quantification of anti-RBD to be used in clinical practice. An absence of detectable anti-RBD by this assay predicates the need for passive immune prophylaxis in at-risk patients.

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Arginine methylation is a common protein post-translational modification (PTM) that plays a key role in eukaryotic cells. Three distinct types of this modification are found in mammals: asymmetric N η1 N η1 -dimethylarginine (aDMA), symmetric N η1 N η2 -dimethylarginine (sDMA), and an intermediate N η1 -monomethylarginine (MMA). Elucidation of regulatory mechanisms of arginine methylation in living organisms requires precise information on both the type of the modified residues and their location inside the protein amino acid sequences. Despite mass spectrometry (MS) being the method of choice for analysis of multiple protein PTMs, unambiguous characterization of protein arginine methylation may not be always straightforward. Indeed, frequent internal basic residues of Arg methylated tryptic peptides hamper their sequencing under positive ion mode collision-induced dissociation (CID), the standardly used tandem mass spectrometry method, while the relative stability of the aDMA and sDMA side chains under alternative non-ergodic electron-based fragmentation techniques, electron-capture and electron transfer dissociations (ECD and ETD), may impede differentiation between the isobaric residues. Here, for the first time, we demonstrate the potential of the negative ion mode collision-induced dissociation MS for analysis of protein arginine methylation and present data revealing that the negative polarity approach can deliver both an unambiguous identification of the arginine methylation type and extensive information on the modified peptide sequences.

The SARS-CoV-2 pandemic enables the analysis of immune responses induced against a novel coronavirus infecting immunologically naïve individuals. This provides an opportunity for analysis of immune responses and associations with age, sex and disease severity. Here we measured an array of solid-phase binding antibody and viral neutralising Ab (nAb) responses in participants (n=337) of the ISARIC4C cohort and characterised their correlation with peak disease severity during acute infection and early convalescence. Overall, the responses in a Double Antigen Binding Assay (DABA) for antibody to the receptor binding domain (anti-RBD) correlated well with IgM as well as IgG responses against viral spike, S1 and nucleocapsid protein (NP) antigens. DABA reactivity also correlated with nAb. As we and others reported previously, there is greater risk of severe disease and death in older men, whilst the sex ratio was found to be equal within each severity grouping in younger people. In older males with severe disease (mean age 68 years), peak antibody levels were found to be delayed by one to two weeks compared with women, and nAb responses were delayed further. Additionally, we demonstrated that solid-phase binding antibody responses reached higher levels in males as measured via DABA and IgM binding against Spike, NP and S1 antigens. In contrast, this was not observed for nAb responses. When measuring SARS-CoV-2 RNA transcripts (as a surrogate for viral shedding) in nasal swabs at recruitment, we saw no significant differences by sex or disease severity status. However, we have shown higher antibody levels associated with low nasal viral RNA indicating a role of antibody responses in controlling viral replication and shedding in the upper airway. In this study, we have shown discernible differences in the humoral immune responses between males and females and these differences associate with age as well as with resultant disease severity.

At-home sampling is key to large scale seroprevalence studies. Dried blood spot (DBS) self-sampling removes the need for medical personnel for specimen collection but facilitates specimen referral to an appropriately accredited laboratory for accurate sample analysis. To establish a highly sensitive and specific antibody assay that would facilitate self-sampling for prevalence and vaccine-response studies. Paired sera and DBS eluates collected from 439 sero-positive, 382 sero-negative individuals and DBS from 34 vaccine recipients were assayed by capture ELISAs for IgG and IgM antibody to SARS-CoV-2. IgG and IgM combined on DBS eluates achieved a diagnostic sensitivity of 97.9% (95%CI 96.6 to 99.3) and a specificity of 99.2% (95% CI 98.4 to 100) compared to serum, displaying limits of detection equivalent to 23 and 10 WHO IU/ml, respectively. A strong correlation (r = 0.81) was observed between serum and DBS reactivities. Reactivity remained stable with samples deliberately rendered inadequate, (p = 0.234) and when samples were accidentally damaged or ‘invalid’. All vaccine recipients were sero-positive. This assay provides a secure method for self-sampling by DBS with a sensitivity comparable to serum. The feasibility of DBS testing in sero-prevalence studies and in monitoring post-vaccine responses was confirmed, offering a robust and reliable tool for serological monitoring at a population level.