A high content microscopy-based platform for detecting antibodies to the nucleocapsid, spike and membrane proteins of SARS-CoV-2

The strong humoral immune response produced against the SARS-CoV-2 nucleocapsid (N) and spike (S) proteins has underpinned serological testing but the prevalence of antibody responses to other SARS-CoV-2 proteins, which may be of use as further serological markers, is still unclear. Cell-based serological screening platforms can fulfil a crucial niche in the identification of antibodies which recognise more complex folded epitopes or those incorporating post-translation modifications which may be undetectable by other methods used to investigate the antigenicity of the SARS-CoV-2 proteome. Here, we employed automated high content immunofluorescence microscopy (AHCIM) to assess the viability of such an approach as a method capable of assaying humoral immune responses against full length SARS-CoV-2 proteins in their native cellular state. We first demonstrate that AHCIM provides high sensitivity and specificity in the detection of SARS-CoV-2 N and S IgG. Assessing the prevalence of antibody responses to the SARS-CoV-2 structural membrane protein (M), we further find that 85% of COVID-19 patients within our sample set developed detectable M IgG responses (M sensitivity 85%, N sensitivity 93%, combined N + M sensitivity 95%). The identification of M as a serological marker of high prevalence may be of value in detecting additional COVID-19 cases during the era of mass SARS-CoV-2 vaccinations, where serological screening for SARS CoV-2 infections in vaccinated individuals is dependent on detection of antibodies against N. These findings highlight the advantages of using cell-based systems as serological screening platforms and raise the possibility of using M as a widespread serological marker alongside N and S.

Anti-Ganglioside Antibody Negative Miller Fisher Syndrome with Atypical Features: A Case Report

Miller Fisher Syndrome (MFS), a variant of Guillain Barre Syndrome (GBS), is an immune-mediated neuropathy presenting with the classical clinical triad of ophthalmoplegia, ataxia, and areflexia. Although the clinical triad is the cardinal diagnostic clue, it can also present with a variety of other atypical neurological symptoms and signs beyond the classical triad. IgG anti-GQ1b antibodies are a powerful serological marker for the diagnosis of MFS, however, they can be absent in 10-15% cases of MFS. Here, we are describing a case of a 55-year old lady with an anti-ganglioside negative case of MFS with ptosis and bulbar palsy, who improved with IVIg.

POS1425 ANTIBODIES TO PORPHYROMONAS GINGIVALIS ASSOCIATE WITH THE PRESENCE OF RHEUMATOID ARTHRITIS-RELATED AUTOANTIBODIES IN PATIENTS WITH PERIODONTITIS

Background:Epidemiologic studies have demonstrated a link between periodontitis (PD) and rheumatoid arthritis (RA), specifically RA characterized by anti-citrullinated protein antibodies (ACPA). The keystone pathogen driving PD, Porphyromonas gingivalis (Pg), is the only pathogen known to express peptidylarginine deiminase (PAD), a citrullinating enzyme. Hence, Pg has been proposed to be involved in triggering the ACPA response, by generating citrullinated antigens in an inflammatory milieu(1). Another major virulence factor of Pg is arginine gingipain B (RgpB), a proteinase which cleaves proteins so that P.PAD can access the site where citrullination takes place. We have previously shown elevated anti-RgpB IgG levels in ACPA+ RA patients, even before clinical onset(2, 3), and we hypothesize that anti-RgpB IgG could serve as a serological marker to identify PD patients with increased risk of developing ACPA+ RA.Objectives:Based on this hypothesis, we set out to investigate whether anti-RgpB IgG was associated with PD, PD severity, autoimmunity in general, and the ACPA response in particular.Methods:Anti-RgpB IgG, as well as RA- and systemic lupus erythematosus (SLE)-related autoantibodies targeting cyclic citrullinated peptide(s) (CCP2), rheumatoid factor (RF), dsDNA, cardiolipin, and β2 glycoprotein, were measured by ELISA in serum samples from the ParoKrank study, which is a well-characterized cohort of 805 patients with a first myocardial infarction and 805 matched controls, where periodontal status has been determined by dentists(4). In this study, individuals with PD (n=941) were compared to individuals without PD (n=557).Results:We detected significantly elevated (p<0,0001) anti-RgpB IgG levels in PD compared to non-PD individuals, with highest levels recorded in severe PD. Anti-RgpB IgG levels were significantly increased in PD patients positive for CCP2 and/or RF (n=50), when compared to PD patients negative for CCP2 and RF (n=507), p<0,05, and when compared to non-PD individuals positive for CCP2 and/or RF (n=62), p < 0,05. Notably, these differences were not seen for SLE-related autoantibodies. In addition, anti-RgpB IgG levels were significantly elevated amongst MI patients versus controls (p < 0,05), supporting the previous finding that PD is more common among MI patients(4).Conclusion:Our data demonstrates a specific association between severe PD, elevated anti-RgpB IgG levels and RA-related autoantibodies, supporting a role for Pg in linking PD to ACPA+ RA. Further investigation will be needed to confirm whether anti-RgpB IgG can be used as a serological marker to identify PD patients with increased risk of developing ACPA+ RA.References:[1]Rosenstein ED, Greenwald RA, Kushner LJ, Weissmann G. Hypothesis: the humoral immune response to oral bacteria provides a stimulus for the development of rheumatoid arthritis. Inflammation. 2004;28(6):311-8.[2]Kharlamova N, Jiang X, Sherina N, Potempa B, Israelsson L, Quirke AM, et al. Antibodies to Porphyromonas gingivalis Indicate Interaction Between Oral Infection, Smoking, and Risk Genes in Rheumatoid Arthritis Etiology. Arthritis Rheumatol. 2016;68(3):604-13.[3]Johansson L, Sherina N, Kharlamova N, Potempa B, Larsson B, Israelsson L, et al. Concentration of antibodies against Porphyromonas gingivalis is increased before the onset of symptoms of rheumatoid arthritis. Arthritis Res Ther. 2016;18(1):201.[4]Rydén L, Buhlin K, Ekstrand E, Faire Ud, Gustafsson A, Holmer J, et al. Periodontitis Increases the Risk of a First Myocardial Infarction. Circulation. 2016;133(6):576-83.Disclosure of Interests:None declared

Role of S100β Glial Protein as a Serological Marker for Analysis of Acute Ischemic Stroke

Background: Stroke is a major cause of neurological disability, which can be often predicted with serological markers. Glial-derived S100β protein is a potential biomarker for cerebral ischemia and may be helpful in predicting the severity, outcome, and recovery of stroke. Aim: This study aimed to study the role of S100β glial protein as a serological marker in predicting the severity of acute ischemic stroke (AIS), outcome, and functional recovery after 1 month. Methods: A hospital-based prospective case control study included 43 consecutive patients, >18 years old, who were admitted with acute middle cerebral artery (MCA) territory infarcts within 72 h of onset of neurological deficits. Control group comprised of 43 age-matched asymptomatic volunteers. Independent t-test and chi square test were used to compare the means and evaluate the association between protein level and various parameters. P ≤ .05 was statistically significant. Results: S100β protein level in AIS patients was significantly higher compared to controls ( P < .05). Elevated serum S100β protein level was found to be associated with larger infarct volumes, higher National Institute Health Stroke Scale scores, and higher modified Rankin Scale scores at admission ( P < .05). Patients with higher S100β protein levels at admission had poor recovery at 1 month compared to patients having normal S100β protein levels. Conclusion: S100β protein levels at admission after an acute MCA territory infarct may be used as a reliable serological tool in predicting the severity, outcome, and functional recovery in stroke.

P305 A location-based prediction model in Crohn’s disease regarding a novel serological marker, anti-Chitinase 3-like 1 autoantibodies

Background A defective neutrophil regulation in IBD is thought to play an important role in the onset or manifestation of IBD, since it could lead to damage of the intestinal mucosal barrier by infiltration of neutrophils in the inflamed mucosa and the accumulation of pathogens. Like neutrophils in the context of innate immune responses, immunoglobulin A (IgA) as an acquired immune response partakes in the defence of the intestinal epithelium. Under normal conditions, IgA would contribute to the elimination of microbes, but in connection with the loss of tolerance to CHI3L1 in IBD, IgA could participate in the CHI3L1-mediated improved adhesion and invasion of potentially pathogenic microorganisms. The tolerance brake to CHI3L1 and the occurrence of IgA auto-Abs to this particular target, the exact role and underlying mechanisms of CHI3L1 in the pathogenesis of IBD are still unclear. Methods We aim to determine the predictive potential of immunoglobulin subtypes of a novel serological marker, anti-chitinase 3-like 1 autoantibodies (aCHI3L1) regarding determination of disease phenotype, therapeutic strategy and long-term disease course in a prospective referral adult IBD patient cohort. Immunoblotting of antineutrophil-cytoplasmic antibody-positive sera of IBD patients on neutrophil proteins and MALDI-TOF mass spectrometry were used to identify autoantigenic targets in IBD. Sera of 257 CD and 180 UC patients were assayed for IgG, IgA, and secretory IgA (sIgA) type aCHI3L1 by enzyme-linked immunosorbent assay using recombinant CHI3L1 along with 86 healthy controls (HCONT). Results IgA type was more prevalent in CD than UC (29.2 vs. 11.1%) or HCONT (2.83%; p &lt; 0.0001 for both). However, sIgA subtype aCHI3L1 positivity was higher in both CD and UC compared with HCONT (39.3 and 32.8 vs. 4.65%, respectively; p &lt; 0.0001). The presence of both IgA and sIgA aCHI3L1 antibodies was associated with colonic involvement (p &lt; 0.0001 and p = 0.038, respectively) in CD. Complicated disease behaviour at sample procurement was associated with the presence of aCHI3L1 sIgA positivity (57.1% vs. 36.0%, p = 0.009). Whilst, IgA type aCH3L1 was more prevalent in patients with frequent relapse during the disease course in CD (46.9% vs. 25.7%, p = 0.005). In a group of patients with concomitant presence of pure inflammatory luminal disease and colon involvement at the time of the diagnosis, positivity for IgA or sIgA type aCH3L1 predicted a faster progression towards complicated disease course in time-dependent models. This association disappeared after merging subgroups of different disease locations. Conclusion CHI3L1 is a novel neutrophil autoantigenic target in IBD. Consideration of antibody classes along with location-based prediction can revolutionise the future of serology in IBD.