Persistent symptoms are frequently reported many months after SARS-CoV-2 infection. However, evidence of the association between long COVID features and initial Omicron infection severity is lacking. A prospective observational cohort study of n = 40 SARS-CoV-2 (Omicron B.1.1.529) infected individuals was performed, comprising n = 20 non-hospitalised and n = 20 hospitalised cases. ISARIC symptomology was recorded at baseline, 3-, 6- and 12-months post infection. Total and neutralising IgG antibody and inflammatory proteome concentrations were determined in plasma at baseline and 3 months. Pairwise comparisons were made between non-hospitalised and hospitalised subgroup biological and symptom data to investigate temporal changes. The non-hospitalised subgroup had higher median levels of Omicron variant specific ACE2 neutralising antibodies by 3 months, in particular females, though the difference was not statistically significant (BA.1 + R346K p = 0·06; BA.1 + L452R p = 0.08; BA.2 p = 0·09). Inflammatory proteins type 1 keratin, prostasin and leukocyte associated immunoglobulin receptor 1 were significantly elevated in hospitalised subgroup plasma 3 months post infection (padj < 0·05). Fatigue, muscle pain, headache, and cough symptoms subsided after 12 months in non-hospitalised, whilst significantly more hospitalised individuals reported continued muscle pain and shortness of breath (p < 0·05). This study provides the first evidence of the link between Omicron hospitalisation and long-term humoral immune response, inflammatory plasma proteome, and symptom persistence. ClinicalTrials.gov NCT05548829.

SARS-CoV-2 has claimed more than 7 million lives worldwide and has been associated with prolonged inflammation, immune dysregulation and persistence of symptoms following severe infection. Understanding the T cell mediated immune response and factors impacting development and continuity of SARS-CoV-2 specific memory T cells is pivotal for developing better therapeutic and monitoring strategies for those most at risk from COVID-19. Here we present a comprehensive analysis of memory T cells in a convalescent cohort (n=20), three months post Omicron infection. Utilising flow cytometry to investigate CD4+CD45RO+ and CD8+CD45RO+ memory T cell IL-2 expression following Omicron (B.1.1.529/BA.1) peptide pool stimulation, alongside T cell receptor repertoire profiling and RNA-Seq analysis, we have identified several immunological features associated with hospitalised status. We observed that while there was no significant difference in median CD4+CD45RO+ IL-2+ and CD8+ CD45RO+ IL-2+ memory T cell count between subgroups, the hospitalised subgroup expressed significantly more IL-2 per cell following Omicron peptide pool exposure in the CD8+CD45RO+ population (p <0.03) and trended towards significance in CD4+CD45RO+ cells (p <0.06). T cell receptor repertoire analysis found that the non-hospitalised subgroup had a much higher number of circulating clonotypes, targeting a wider range of predominantly MHC-I epitopes across the SARS-CoV-2 genome. Several immunodominant epitopes, conserved between both subgroups, were observed, however hospitalised individuals were less likely to express putative HLA alleles responsible for pMHC presentation which may impact TCR affinity. We observed a bias towards shorter CDR3 segments in TCRβ repertoire analysis within the hospitalised subgroup, alongside lower rates of repertoire overlap in CDR3 sequences compared to the non-hospitalised subgroup. We found a significant proportion of TCRs targeted epitopes along the SARS-CoV-2 genome including non-structural proteins, responsible for viral replication and immune evasion. These findings highlight how the continuity of T cell based protective immunity is impacted by both the viral replication cycle of SARS-CoV-2 upon intracellular and innate immune responses, and HLA-type upon TCR affinity and clonotype formation. Our novel Epitope Target Analysis Pipeline (Epi-TAP) could prove beneficial in development of new therapeutic strategies through rapid identification of shared immunodominant epitopes across non-hospitalised and hospitalised subgroups.

Background: With the spread of SARS-CoV-2 impacting upon public health directly and socioeconomically, further information was required to inform policy decisions designed to limit virus spread during the pandemic. This study sought to contribute to serosurveillance work within Northern Ireland to track SARS-CoV-2 progression and guide health strategy. Methods: Sera/plasma samples from clinical biochemistry laboratories were analysed for anti-SARS-CoV-2 antibodies. Samples were assessed using an Elecsys anti-SARS-CoV-2 or anti-SARS-CoV-2 S ECLIA (Roche) on an automated cobas e 801 analyser. Samples were also assessed via an anti-SARS-CoV-2 ELISA (Euroimmun). A subset of samples assessed via the Elecsys anti-SARS-CoV-2 ECLIA were subsequently analysed in an ACE2 pseudoneutralisation assay using a V-PLEX SARS-CoV-2 Panel 7 for IgG and ACE2 (Meso Scale Diagnostics). Results: Across three testing rounds (June–July 2020, November–December 2020 and June–July 2021 (rounds 1–3 respectively)), 4844 residual sera/plasma specimens were assayed for anti-SARS-CoV-2 antibodies. Seropositivity rates increased across the study, peaking at 11.6 % (95 % CI 10.4 %–13.0 %) during round 3. Varying trends in SARS-CoV-2 seropositivity were noted based on demographic factors. For instance, highest rates of seropositivity shifted from older to younger demographics across the study period. In round 3, Alpha (B.1.1.7) variant neutralising antibodies were most frequently detected across age groups, with median concentration of anti-spike protein antibodies elevated in 50–69 year olds and anti-S1 RBD antibodies elevated in 70+ year olds, relative to other age groups. Conclusions: With seropositivity rates of <15 % across the assessment period, it can be concluded that the significant proportion of the Northern Ireland population had not yet naturally contracted the virus by mid-2021.

Background: The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has posed unprecedented challenges to healthcare systems worldwide. Here, we have identified proteomic and genetic signatures for improved prognosis which is vital for COVID-19 research. Methods: We investigated the proteomic and genomic profile of COVID-19-positive patients (n = 400 for proteomics, n = 483 for genomics), focusing on differential regulation between hospitalised and non-hospitalised COVID-19 patients. Signatures had their predictive capabilities tested using independent machine learning models such as Support Vector Machine (SVM), Random Forest (RF) and Logistic Regression (LR). Results: This study has identified 224 differentially expressed proteins involved in various inflammatory and immunological pathways in hospitalised COVID-19 patients compared to non-hospitalised COVID-19 patients. LGALS9 (p-value < 0.001), LAMP3 (p-value < 0.001), PRSS8 (p-value < 0.001) and AGRN (p-value < 0.001) were identified as the most statistically significant proteins. Several hundred rsIDs were queried across the top 10 significant signatures, identifying three significant SNPs on the FSTL3 gene showing a correlation with hospitalisation status. Conclusions: Our study has not only identified key signatures of COVID-19 patients with worsened health but has also demonstrated their predictive capabilities as potential biomarkers, which suggests a staple role in the worsened health effects caused by COVID-19.

Background: The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2 has posed unprecedented challenges to healthcare systems worldwide. Here, we have identified proteomic and genetic signatures for improved prognosis which is vital for COVID-19 research. Methods: We investigated the proteomic and genomic profile of COVID-19 positive patients (n=400 for proteomics, n=483 for genomics), focusing on differential regulation between hospitalised and non-hospitalised COVID-19 patients. Signatures had their predictive capabilities tested using independent machine learning models such as Support Vector Machine (SVM), Random Forest (RF) and Logistic Regression (LR). Results: This study has identified 224 differentially expressed proteins in hospitalised COVID-19 patients compared to non-hospitalised COVID-19 patients, involved in various inflammatory and immunological pathways. LGALS9 (p-value < 0.001), LAMP3 (p-value < 0.001), PRSS8 (p-value < 0.001) and AGRN (p-value < 0.001), were identified as the most statistically significant proteins. Several hundred rsIDs were queried across the top 10 significant signatures, identifying three significant SNPs on the FSTL3 gene showing correlation with hospitalisation status. Conclusion: Our study has not only identified key signatures of COVID-19 patients with worsened health but has also demonstrated their predictive capabilities as potential biomarkers, which suggests a staple role in the worsened health effects caused by COVID-19.

Background Health organizations and countries around the world have found it difficult to control the spread of COVID-19. To minimize the future impact on the UK National Health Service and improve patient care, there is a pressing need to identify individuals who are at a higher risk of being hospitalized because of severe COVID-19. Early targeted work was successful in identifying angiotensin-converting enzyme-2 receptors and type II transmembrane serine protease dependency as drivers of severe infection. Although a targeted approach highlights key pathways, a multiomics approach will provide a clearer and more comprehensive picture of severe COVID-19 etiology and progression. Objective The COVID-19 Response Study aims to carry out an integrated multiomics analysis to identify biomarkers in blood and saliva that could contribute to host susceptibility to SARS-CoV-2 and the development of severe COVID-19. Methods The COVID-19 Response Study aims to recruit 1000 people who recovered from SARS-CoV-2 infection in both community and hospital settings on the island of Ireland. This protocol describes the retrospective observational study component carried out in Northern Ireland (NI; Cohort A); the Republic of Ireland cohort will be described separately. For all NI participants (n=519), SARS-CoV-2 infection has been confirmed by reverse transcription-quantitative polymerase chain reaction. A prospective Cohort B of 40 patients is also being followed up at 1, 3, 6, and 12 months postinfection to assess longitudinal symptom frequency and immune response. Data will be sourced from whole blood, saliva samples, and clinical data from the electronic care records, the general health questionnaire, and a 12-item general health questionnaire mental health survey. Saliva and blood samples were processed to extract DNA and RNA before whole-genome sequencing, RNA sequencing, DNA methylation analysis, microbiome analysis, 16S ribosomal RNA gene sequencing, and proteomic analysis were performed on the plasma. Multiomics data will be combined with clinical data to produce sensitive and specific prognostic models for severity risk. Results An initial demographic and clinical profile of the NI Cohort A has been completed. A total of 249 hospitalized patients and 270 nonhospitalized patients were recruited, of whom 184 (64.3%) were female, and the mean age was 45.4 (SD 13) years. High levels of comorbidity were evident in the hospitalized cohort, with cardiovascular disease and metabolic and respiratory disorders being the most significant (P<.001), grouped according to the International Classification of Diseases 10 codes. Conclusions This study will provide a comprehensive opportunity to study the mechanisms of COVID-19 severity in recontactable participants. International Registered Report Identifier (IRRID) DERR1-10.2196/50733

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can misidentify Cutibacterium namnetense and Cutibacterium modestum as Cutibacterium acnes. We now describe how such MALDI-TOF MS misidentification explains previous reports of C. acnes isolates that could not be characterised using a multiplex PCR phylotyping assay.

Severe COVID-19 is characterised by an overactive pro-inflammatory response of the immune system which is associated with new persistent symptoms. Differences in the persistence of symptoms, plasma inflammatory proteins and antibodies between hospitalised and non-hospitalised cases were investigated in the current study. n=120 participants were recruited with informed consent, of whom n=60 were hospitalised with severe disease. Blood samples and detailed symptom data were collected 3 months after a positive PCR test for SARS-CoV-2. Plasma samples were analysed by OLINK Explore 384 inflammation panel and ACE2 neutralisation and IgG assays. R software was used to identify statistically significant differences in protein concentration in hospitalised cases, relative to non-hospitalised cases. Differences in anti-SARS-CoV-2 IgG antibody concentration, ACE2 neutralisation and symptoms were also analysed between hospitalisation subgroups. 14 proteins including angiopoietin-like protein 2 and C-X-C motif chemokine 17 were significantly elevated (by greater than +/− 0.20 log2 fold change; p < 0.05) at 3 months in the hospitalised study group, relative to non-hospitalised cases. The concentrations of BA.2 and BA.3 specific anti-SARS-CoV-2 IgG and spike receptor binding domain neutralising antibodies were more frequently detected in non-hospitalised cases. The hospitalised cohort more frequently reported persistence of muscle pain, headache, fatigue and shortness of breath. Characteristic changes in the plasma proteome and symptom clusters that persist 3 months after infection have been observed in hospitalised COVID-19 cases. This data could help inform clinical care requirements for longer term COVID-19 recovery. Supported by grants from UKRI, SFI and HSC R&D Office

Background With the impact of SARS-CoV-2 upon public health directly and socioeconomically, further information was required to inform policy decisions designed to limit virus spread. This study sought to contribute to serosurveillance work within Northern Ireland to track SARS-CoV-2 progression and guide health strategy. Methods Sera/plasma samples from clinical biochemistry laboratories were analysed for anti-SARS-CoV-2 immunoglobulins (Ig). Samples were assessed using an Elecsys anti-SARS-CoV-2 or anti-SARS-CoV-2 S ECLIA (Roche) on an automated Cobas-e-analyser. Samples were also assessed via ELISA (Euroimmun). A subset of samples assessed via Roche Elecsys anti-SARS-CoV-2 IgG assay were subsequently analysed in an ACE2 pseudoneutralisation assay using a V-PLEX SARS-CoV-2 Panel 7 for IgG and ACE2 by MesoScale Diagnostics Inc. Results Across three testing rounds (June-July 2020, November-December 2020 and June-July 2021 (rounds 1-3 respectively)), 4844 residual sera/plasma specimens were assayed for SARS-CoV-2 Ig. Seropositivity rates increased across the study, peaking at 11.6% during round 3. Varying trends in SARS-CoV-2 seropositivity were noted based on demographic factors. For instance, highest rates of seropositivity shifted from older to younger demographics across the study period. In round 3, alpha (B.1.1.7) variant neutralising antibodies were most frequently detected across age groups, with median concentration of anti-spike protein antibodies elevated in 50-69 year olds and anti-S1 RBD antibodies elevated in over 70s, relative to other age groups. Conclusions With seropositivity rates of <15% across the assessment period, it can be concluded that the significant proportion of the Northern Ireland population had not yet naturally contracted the virus by mid-2021.

OBJECTIVES:A 'high resolution' Single Locus Sequence Typing (SLST) scheme has been described for the anaerobic skin bacterium Cutibacterium acnes that seemingly discriminates sequence types (STs) to a level commensurate with previously described Multilocus Sequence Typing (MLST) methods (MLST4; MLST8; MLST9). However, no quantifiable evaluation of SLST versus MLST for differentiation of C. acnes strains, especially in relation to the subspecies of the bacterium, known as C. acnes subsp. acnes (type I), C. acnes subsp. defendens (type II) and C. acnes subsp. elongatum (type III), has been performed which is vital given its increasing use. To address this, we examined the discriminatory power of SLST versus MLST with a large group of isolates representative of all subspecies.METHODS:Simpson's index of diversity (D) was used for quantitative comparison of the resolving power of the SLST and MLST schemes for 186 isolates of C. acnes covering all three subspecies.RESULTS:When strains were considered collectively, SLST and all three MLST approaches had similar D values > 90%. However, at the subspecies level there were significant differences between the methods, most strikingly a reduced discrimination of type II and type III strains (D <80%) by SLST versus MLST8, and to a lesser extent MLST4. The MLST9 method also performed poorly for type II strains (D <70%), but did display the best results for type I (D = 90%). By combining the SLST locus with the camp2 gene sequence to create a novel and flexible high-resolution Bilocus Sequence Typing (BLST) scheme, known as CUTIS-SEQ typing (CUTIbacterium acneS BilocuS sEQuence Typing), we achieved improved resolution at both species and, critically, subspp. levels.CONCLUSIONS:CUTIS-SEQ provides an opportunity to improve differentiation of C. acnes isolates by SLST without significantly impacting laboratory workload, or compromising application to complex biological communities. A CUTIS-SEQ isolate database is now available as part of the C. acnes PubMLST database at https://pubmlst.org.

Objectives: Multiple myeloma (MM) is a malignant plasma cell neoplasm, requiring the integration of clinical examination, laboratory and radiological investigations for diagnosis. Detection and isotypic identification of the monoclonal protein(s) and measurement of other relevant biomarkers in serum and urine are pivotal analyses. However, occasionally this approach fails to characterize complex protein signatures. Here we describe the development and application of next generation mass spectrometry (MS) techniques, and a novel adaptation of immunofixation, to interrogate non-canonical monoclonal immunoproteins. Methods: Immunoprecipitation immunofixation (IP-IFE) was performed on a Sebia Hydrasys Scan2. Middle-down de novo sequencing and native MS were performed with multiple instruments (21T FT-ICR, Q Exactive HF, Orbitrap Fusion Lumos, and Orbitrap Eclipse). Post-acquisition data analysis was performed using Xcalibur Qual Browser, ProSight Lite, and TDValidator. Results: We adapted a novel variation of immunofixation electrophoresis (IFE) with an antibody-specific immunosubtraction step, providing insight into the clonal signature of gamma-zone monoclonal immunoglobulin (M-protein) species. We developed and applied advanced mass spectrometric techniques such as middle-down de novo sequencing to attain in-depth characterization of the primary sequence of an M-protein. Quaternary structures of M-proteins were elucidated by native MS, revealing a previously unprecedented non-covalently associated heterotetrameric immunoglobulin. Conclusions: Next generation proteomic solutions offer great potential for characterizing complex protein structures and may eventually replace current electrophoretic approaches for the identification and quantification of M-proteins. They can also contribute to greater understanding of MM pathogenesis, enabling classification of patients into new subtypes, improved risk stratification and the potential to inform decisions on future personalized treatment modalities.

Hand osteoarthritis (HOA) includes different subsets; a particular and uncommon form is erosive HOA (EHOA). Interleukin- (IL-) 1β plays a crucial role in the pathogenesis of osteoarthritis (OA); it is synthesized as an inactive precursor which requires the intervention of a cytosolic multiprotein complex, named inflammasome, for its activation. The aim of this study was to investigate the involvement of IL-1β and the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome in patients with EHOA and nonerosive HOA (NEHOA) compared to healthy controls. In particular, we evaluated the gene expression of IL-1β and NLRP3, the serum levels of IL-1β, IL-6, IL-17, and tumor necrosis factor- (TNF-) α, and the protein levels of IL-1β and NLRP3. We also assessed the relationships between IL-1β and NLRP3 and clinical, laboratory, and radiological findings. Fifty-four patients with HOA (25 EHOA and 29 NEHOA) and 20 healthy subjects were included in the study. Peripheral blood mononuclear cell (PBMC) gene and protein expressions of IL-1β and NLRP3 were quantified by quantitative real-time PCR and western blot. IL-1β, IL-6, IL-17, and TNF-α serum levels were determined by ELISA. IL-1β gene expression was significantly reduced (p = 0.0208) in EHOA compared to healthy controls. NLRP3 protein levels were significantly increased in the NEHOA group versus the control (p = 0.0063) and EHOA groups (p = 0.0038). IL-1β serum levels were not significantly different across the groups; IL-6, IL-17, and TNF-α were not detectable in any sample. IL-1β concentrations were negatively correlated with the Kellgren-Lawrence score in the whole population (r = −0.446; p = 0.0008) and in NEHOA (r = −0.608; p = 0.004), while IL-1β gene expression was positively correlated with the number of joint swellings in the EHOA group (r = 0.512; p = 0.011). Taken together, our results, showing poorly detectable IL-1β concentrations and minimal inflammasome activity in the PBMCs of HOA patients, suggest a low grade of systemic inflammation in HOA. This evidence does not preclude a possible involvement of these factors at the local level.

The anaerobic bacterium Propionibacterium acnes is believed to play an important role in the pathophysiology of the common skin disease acne vulgaris. Over the last 10 years our understanding of the taxonomic and intraspecies diversity of this bacterium has increased tremendously, and with it the realisation that particular strains are associated with skin health while others appear related to disease. This extensive review will cover our current knowledge regarding the association of P. acnes phylogroups, clonal complexes and sequence types with acne vulgaris based on multilocus sequence typing of isolates, and direct ribotyping of the P. acnes strain population in skin microbiome samples based on 16S rDNA metagenomic data. We will also consider how multi-omic and biochemical studies have facilitated our understanding of P. acnes pathogenicity and interactions with the host, thus providing insights into why certain lineages appear to have a heightened capacity to contribute to acne vulgaris development, while others are positively associated with skin health. We conclude with a discussion of new therapeutic strategies that are currently under investigation for acne vulgaris, including vaccination, and consider the potential of these treatments to also perturb beneficial lineages of P. acnes on the skin.