Clonally Expanded B Cells in Multiple Sclerosis Bind EBV EBNA1 and GlialCAM

Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system (CNS). B lymphocytes in the cerebrospinal fluid (CSF) of MS patients contribute to inflammation and secrete oligoclonal immunoglobulins. Epstein-Barr virus (EBV) infection has been linked to MS epidemiologically, but its pathological role remains unclear. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBNA1 and the CNS protein GlialCAM, and provide structural and in-vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements, and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment allowed for tracking the development of the naïve EBNA1-restricted antibody to a mature EBNA1/GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates the mouse model of MS and anti-EBNA1/GlialCAM antibodies are prevalent in MS patients. Our results provide a mechanistic link for the association between MS and EBV, and could guide the development of novel MS therapies.

Association of Serum Biomarkers with Post-Thrombolytic Early Neurological Improvement in Stroke: A Comprehensive Protein Microarray Analysis From INTRECIS Study

Objective Early neurological improvement (ENI) after intravenous thrombolysis is associated with favorable outcome, but associated serum biomarkers were not fully determined. We aimed to investigate the issue in a prospective cohort. Methods In INTRECIS study, five centers were designed to consecutively collect the blood sample from enrolled patients. Enrolled patients with ENI and without ENI were matched by propensity score matching with the ratio of 1:1. Preset 49 biomarkers were measured by protein microarray analysis. Enrichment of Gene Ontology and pathway, and protein-protein interaction network were analyzed in the identified biomarkers. Results Of 358 patients, 19 occurred ENI, who were assigned as ENI group, while 19 matched patients without ENI were assigned as Non ENI group. A total of 9 biomarkers were found different, among which levels of chemokine (C-C motif) ligand (CCL)-23, chemokine (C-X-C motif) ligand (CXCL)-12, insulin-like growth factor binding protein (IGFBP)-6, interleukin (IL)-5, lymphatic vessel endothelial hyaluronan receptor (LYVE)-1, plasminogen activator inhibitor (PAI)-1, platelet-derived growth factor (PDGF)-AA, suppression of tumorigenicity (ST)-2, and tumor necrosis factor (TNF)-α were higher in ENI group, compared with those in Non ENI group. Interpretation: Our finding indicated that pretreatment serum CCL-23, CXCL-12, IGFBP-6, IL-5, LYVE-1, PAI-1, PDGF-AA, ST-2, and TNF-α levels were associated with post-thrombolytic ENI in ischemic stroke. The role of these biomarkers warrant further investigation. Registration-URL : https://www.clinicaltrials.gov; Unique identifier: NCT02854592.

One year landscape of the inactivated virus vaccine triggered RBD-specific IgG, IgM and IgA responses against the Omicron variant

We reported the first map of vaccine stimulated RBD-specific antibody responses (IgG, IgM and IgA) against the Omicron variant. We generated the map using a protein microarray, by analyzing longitudinal sera collected spanning one year from individuals immunized with 3 doses of an inactivated virus vaccine. The IgG response to RBD-Omicron is: 1/3-1/5 that of RBD-wild type; ~6x higher for the booster dose vs. the 2nd dose; and reaches the plateau in about two weeks after the booster dose, then drops ~5x in another two weeks. Similar results were also obtained for IgM and IgA. Because of the high correlation between RBD-specific antibody response and the neutralization activity to authentic virus, we at least indirectly revealed the landscape of antibody protection against the Omicron variant throughout the vaccination stages. Our results strongly support the necessity of booster vaccination. However, post-booster vaccination may need to be considered.

Autoantibody Signatures Discovered by HuProt Protein Microarray to Enhance the Diagnosis of Lung Cancer

Background: This study aims to comprehensively discover novel autoantibodies (TAAbs) against tumor-associated antigens (TAAs) and establish diagnostic models for assisting in the diagnosis of lung cancer (LC) and discrimination of pulmonary nodules (PN).Methods: HuProt human microarray was used to discover the candidate TAAs and Enzyme-linked immunosorbent assay (ELISA) was performed to detect the level of TAAbs in 634 participants of two independent validation cohorts. Logistic regression analysis was used to construct models. Receiver operating characteristic curve (ROC) analysis was utilized to assess the diagnostic value of models.Results: Eleven TAAs were discovered by means of protein microarray and data analysis. The level of ten TAAbs (anti-SARS, anti-ZPR1, anti-FAM131A, anti-GGA3, anti-PRKCZ, anti-HDAC1, anti-GOLPH3, anti-NSG1, anti-CD84 and anti-EEA1) was higher in LC patients than that in NC of validation cohort 1 (P<0.05). The model 1 comprising 4 TAAbs (anti-ZPR1, anti-PRKCZ, anti-NSG1 and anti-CD84) and CEA reached an AUC of 0.813 (95%CI: 0.762-0.864) for diagnosing LC from normal individuals. 5 of 10 TAAbs (anti-SARS, anti-GOLPH3, anti-NSG1, anti-CD84 and anti-EEA1) existed a significant difference between malignant pulmonary nodules (MPN) and benign pulmonary nodules (BPN) patients in validation cohort 2 (P<0.05). Model 2 consisting of anti-EEA1, traditional biomarkers (CEA, CYFRA211 and CA125) and 3 CT characteristics (vascular notch sign, lobulation sign, mediastinal lymph node enlargement) could distinguish MPN from BPN patients with an AUC of 0.845 (sensitivity: 58.3%, specificity: 96.6%).Conclusions: High-throughput protein microarray is an efficient approach to discovering novel TAAbs which could increase the accuracy of lung cancer diagnosis in the clinic.

Magnitude and Breadth of Antibody Cross-reactivity Induced by Recombinant Influenza Hemagglutinin Trimer Vaccine Is Enhanced by Combination Adjuvants

The effects of adjuvants for increasing the immunogenicity of influenza vaccines are well known. However, the effect of adjuvants on increasing the breadth of cross-reactivity is less well understood. In this study we have performed a systematic screen of different toll-like receptor (TLR) agonists, with and without a squalene-in-water emulsion on the immunogenicity of a recombinant trimerized hemagglutinin (HA) vaccine in mice after single-dose administration. Antibody (Ab) cross-reactivity for other variants within and outside the immunizing subtype (homosubtypic and heterosubtypic cross-reactivity, respectively) was assessed using a protein microarray approach. Of all the formulations tested, a combination of CpG, MPLA and AddaVAX (termed “IVAX-1”) yielded the greatest breadth and magnitude of Ab responses, particularly against the HA1 region (which includes the variable head domain) of HA. Antigen-specific plasma cell labeling experiments show the components of IVAX-1 are synergistic. This adjuvant preferentially stimulates CD4 T cells to produce Th1>Th2 type (IgG2c>IgG1) antibodies and cytokine responses. Moreover, IVAX-1 induces identical homo- and heterosubtypic IgG and IgA cross-reactivity profiles when administered intranasally. Consistent with these observations, a single-cell transcriptomics analysis demonstrated significant increases in expression of IgG1, IgG2b and IgG2c genes of B cells in H5/IVAX-1 immunized mice relative to naïve mice, as well as significant increases in expression of the IFNg gene of both CD4 and CD8 T cells. These data support the use of adjuvants for enhancing the breath and durability of antibody responses of influenza virus vaccines.

Is the bioactivity of induced membranes time dependent?

Purpose The induced membrane technique (IMT) is a two-stage surgical procedure for reconstruction of bone defects. Bone grafting (second stage of IMT) is recommend after 4–8 weeks assuming the highest bioactivity of IMs. However, larger studies concerning the biology and maturation of IMs and a potential time dependency of the bioactivity are missing. Therefore, aim of this study was the time-dependent structural and cellular characterization of cement spacer IMs concomitantly to an analysis of membrane bioactivity. Methods IMs from 60 patients (35–82 years) were obtained at different maturation stages (1–16 weeks). IMs were studied by histology and co-culture with mesenchymal stem cells (MSC). IM lysates were analyzed by ELISA and protein microarray. Results Increasing vascularization and fibrosis were found in membranes older than 4 and 7 weeks, respectively. MSC grew out from all membranes and all membranes enhanced proliferation of cultured MSC. Osteocalcin and osteopontin (in membrane lysates or induced in MSC by membrane tissue) were found over all time points without significant differences. In contrast to alkaline phosphatase activity, increasing levels of osteoprotegerin were found in membranes. Conclusion The histological structure of IMs changes during growth and maturation, however, biologically active MSC and factors related to osteogenesis are found over all time points with minor changes. Thus, membranes older than 8 weeks exert regenerative capacities comparable to the younger ones. The postulated narrow time frame of 4–8 weeks until bone grafting can be questioned and surgeons may choose timing for the second operation more independently and based on other clinical factors.

Identification of the Recombinant Plasmodium vivax Surface-Related Antigen as a Possible Immune Evasion Factor Against Human Splenic Fibroblasts by Targeting ITGB1

Plasmodium vivax–infected erythrocytes can enter the spleen and evade spleen clearance to establish chronic infections. However, the mechanism underlying P. vivax immune evasion in the spleen is still unclear. Human splenic fibroblasts (HSF), also known as barrier cells, play an essential role in the immune function of spleen. A hypothesis holds that P. vivax—infected erythrocytes induce spleen structural remodeling to form barrier cells. Subsequently, these infected erythrocytes can selectively cytoadhere to these barrier cells to escape spleen clearance. In this work, we found that P. vivax surface-related antigen (PvSRA; PlasmoDB ID: PVX_084970), an exported protein on infected erythrocyte membrane, could bind with HSF. Considering the above hypothesis, we speculated that PvSRA might be involved in P. vivax immune evasion by changing HSF cell performance. To investigate this speculation, RNA sequencing, protein microarray, and bioinformatics analysis technologies were applied, and in vitro validations were further performed. The results showed that the recombinant PvSRA attracted HSF migration and interacted with HSF by targeting integrin β1 (ITGB1) along with changes in HSF cell performance, such as focal adhesion, extracellular matrix, actin cytoskeleton, and cell cycle. This study indicated that PvSRA might indeed participate in the immune evasion of P. vivax in the spleen by changing HSF function through PvSRA–ITGB1 axis.

Brain Injury in COVID-19 is Associated with Autoinflammation and Autoimmunity

COVID-19 has been associated with many neurological complications including stroke, delirium and encephalitis. Furthermore, many individuals experience a protracted post-viral syndrome which is dominated by neuropsychiatric symptoms, and is seemingly unrelated to COVID-19 severity. The true frequency and underlying mechanisms of neurological injury are unknown, but exaggerated host inflammatory responses appear to be a key driver of severe COVID-19 more broadly.We sought to investigate the dynamics of, and relationship between, serum markers of brain injury (neurofilament light [NfL], Glial Fibrillary Acidic Protein [GFAP] and total Tau) and markers of dysregulated host response including measures of autoinflammation (proinflammatory cytokines) and autoimmunity. Brain injury biomarkers were measured using the Quanterix Simoa HDx platform, cytokine profiling by Luminex (R&D) and autoantibodies by a custom protein microarray.During hospitalisation, patients with COVID-19 demonstrated elevations of NfL and GFAP in a severity-dependant manner, and there was evidence of ongoing active brain injury at follow-up 4 months later. Raised NfL and GFAP were associated with both elevations of pro-inflammatory cytokines and the presence of autoantibodies; autoantibodies were commonly seen against lung surfactant proteins as well as brain proteins such as myelin associated glycoprotein, but reactivity was seen to a large number of different antigens.Furthermore, a distinct process characterised by elevation of serum total Tau was seen in patients at follow-up, which appeared to be independent of initial disease severity and was not associated with dysregulated immune responses in the same manner as NfL and GFAP.