Differential patterns of cross-reactive antibody response against SARS-CoV-2 spike protein detected for chronically ill and healthy COVID-19 naïve individuals.

Immunity to previously encountered viruses can alter response to unrelated pathogens. We reasoned that similar mechanism may also involve SARS-CoV-2 and thereby affect the specificity and the quality of the immune response against the virus. Here, we employed high-throughput next generation phage display method to explore the link between antibody immune response to previously encountered antigens and spike (S) glycoprotein. By profiling the antibody response in COVID-19 naïve individuals with a diverse clinical history (including cardiovascular, neurological, or oncological diseases), we identified 15 highly antigenic epitopes on spike protein that showed cross-reactivity with antigens of seasonal, persistent, latent or chronic infections from common human viruses. We observed varying degrees of cross-reactivity of different viral antigens with S in an epitope-specific manner. The data show that pre-existing SARS-CoV-2 S1 and S2 cross-reactive serum antibody is readily detectable in pre-pandemic cohort. In the severe COVID-19 cases, we found differential antibody response to the 15 defined antigenic and cross-reactive epitopes on spike. We also noted that despite the high mutation rates of Omicron (B.1.1.529) variants of SARS-CoV-2, some of the epitopes overlapped with the described mutations. Finally, we propose that the resolved epitopes on spike if targeted by re-called antibody response from SARS-CoV-2 infections or vaccinations can function in chronically ill COVID-19 naïve/unvaccinated individuals as immunogenic targets to boost antibodies augmenting the chronic conditions. Understanding the relationships between prior antigen exposure at the antibody epitope level and the immune response to subsequent infections with viruses from a different strain is paramount to guiding strategies to exit the COVID-19 pandemic.

Melanoma-specific antigen-associated antitumor antibody reactivity as an immune-related biomarker for targeted immunotherapies.

Background: Immunotherapies, including cancer vaccines and immune checkpoint inhibitors have transformed the management of many cancers. However, a large number of patients show resistance to these immunotherapies and current research has provided limited findings for predicting response to precision immunotherapy treatments. Methods: Here, we applied the next generation phage display mimotope variation analysis (MVA) to profile antibody response and dissect the role of humoral immunity in targeted cancer therapies, namely anti-tumor dendritic cell vaccine (MelCancerVac®) and immunotherapy with anti-PD-1 monoclonal antibodies (pembrolizumab). Results: Analysis of the antibody immune response led to the characterization of epitopes that were linked to melanoma-associated and cancer-testis antigens (CTA) whose antibody response was induced upon MelCancerVac® treatments of lung cancer. Several of these epitopes aligned to antigens with strong immune response in patients with unresectable metastatic melanoma receiving anti-PD-1 therapy. Conclusions: This study provides insights into the differences and similarities in tumor-specific immunogenicity related to targeted immune treatments. The antibody epitopes as biomarkers reflect melanoma-associated features of immune response, and also provide insights into the molecular pathways contributing to the pathogenesis of cancer. Concluding, antibody epitope response can be useful in predicting anti-cancer immunity elicited by immunotherapy.

Antibody response to oral biofilm is a biomarker for acute coronary syndrome in periodontal disease.

Cumulative evidence over the last decades have supported the role of gum infections as a risk for future major cardiovascular events. The precise mechanism connecting coronary artery disease (CAD) with periodontal findings has remained elusive. Here, we employ next generation phage display mimotope-variation analysis (MVA) to identify the features of dysfunctional immune system that associate CAD with periodontitis. We identify a fine molecular description of the antigenic epitope repertoires of CAD and its most severe form - acute coronary syndrome (ACS) by profiling the antibody reactivity in a patient cohort with invasive heart examination and complete clinical oral assessment. Specifically, we identify a strong immune response to an EBV VP26 epitope mimicking multiple antigens of oral biofilm as a biomarker for the no-CAD group. With a 2-step biomarker test, we stratify subjects with periodontitis from healthy controls (balanced accuracy 84%), and then assess the risk for ACS with sensitivity 71-89% and specificity 67-100%, depending on the oral health status. Our findings highlight the importance of resolving the immune mechanisms related to severe heart conditions such as ACS in the background of oral health. Prospective validation of these findings will support incorporation of these non-invasive biomarkers into clinical practice.

Immune response to a conserved enteroviral epitope of the major capsid VP1 protein is associated with lower risk of cardiovascular disease.

BACKGROUND: Major cardiac events including myocardial infarction (MI) are associated with viral infections. However, how specific infections contribute to the cardiovascular insults has remained largely unclear. METHODS: We employed next generation phage display mimotope-variation analysis (MVA) to explore the link between antibody-based immune response and severe cardiovascular conditions. Here, we used a case-control design, including the first-stage discovery cohort (n = 100), along with cohorts for second-stage discovery (n = 329) and validation (n = 466). FINDINGS: We observed strong antibody response to the peptide antigens with Gly-Ile-X-Asp (G-I-X-D) core structure in healthy individuals but not in patients with MI. Analysis of the origin of this epitope linked it with the N-terminus of the VP1 protein of poliovirus 3 (PV3), but also other species of picornaviruses. Consistently, we found low levels of antibody response to the G-I-X-D epitope in individuals with severe cardiac disease complications. INTERPRETATION: Our findings imply that antibody response to the G-I-X-D epitope is associated with polio vaccinations and that high antibody levels to this epitope could discriminate healthy individuals from prospective MI patients as a blood-derived biomarker. Together, these findings highlight the importance of epitope-specific antibody response and suggest that protective immunity against the polio- and non-polio enteroviral infections support improved cardiovascular health. FUNDING: Estonian Ministry of Education (5.1-4/20/170), Estonian Research Council (PRG573, PRG805), H2020-MSCA-RISE-2016 (EU734791), H2020 PANBioRA (EU760921), European Union through the European Regional Development Fund (Project no. 2014-2020.4.01.15-0012), Helsinki University Hospital grants, Mary and Georg C. Ehrnrooth Foundation, Finnish Eye Foundation, Finska Läkaresällskapet, The Finnish Society of Sciences and Letters, Magnus Ehrnrooth Foundation and Sigrid Jusélius Foundation.

Identification of two highly antigenic epitope markers predicting multiple sclerosis in optic neuritis patients.

BACKGROUND: Optic neuritis (ON) can occur as an isolated episode or will develop to multiple sclerosis (MS) a chronic autoimmune disease. What predicts ON progression to MS remains poorly understood. METHODS: We characterised the antibody epitope repertoire in three independent clinical cohorts (discovery (n = 62), validation (n = 20) and external cohort (n = 421)) using mimotope variation analysis (MVA), a next generation phage display technology to identify epitopes that associate with prognosis of ON. FINDINGS: We observed distinct epitope profiles for ON, MS and the controls, whereas epitope repertoires of sera and CSF were highly similar. Two unique and highly immunogenic epitopes A and B were detected in subjects with ON progressing to MS. These epitopes A and B were strongly associated with herpesviral antigens (VCA p18 of  Epstein-Barr virus (EBV); gB of Cytomegalovirus (CMV)). ROC addressed 75% of MS subjects with ON onset correctly (at 75% sensitivity and 74.22% specificity) based on the two-epitope biomarker analysis. INTERPRETATION: This is the first report on epitope diagnostics for MS employing the unbiased strategy of MVA for identification of novel immunological features of disease. FUNDING: The Estonian Ministry of Education, The Estonian Research Council (PRG573, PRG805 and PSG691), H2020-MSCA-RISE-2016 (SZTEST), H2020-NMBP-2017 (PANBIORA), Helsinki University Hospital, Mary and Georg C. Ehrnrooth, Finnish Eye, Sigrid Jusélius and Magnus Ehrnrooth Foundations.

Prostaglandin D2 Receptor DP1 Antibodies Predict Vaccine-induced and Spontaneous Narcolepsy Type 1: Large-scale Study of Antibody Profiling.

BACKGROUND: Neuropathological findings support an autoimmune etiology as an underlying factor for loss of orexin-producing neurons in spontaneous narcolepsy type 1 (narcolepsy with cataplexy; sNT1) as well as in Pandemrix influenza vaccine-induced narcolepsy type 1 (Pdmx-NT1). The precise molecular target or antigens for the immune response have, however, remained elusive. METHODS: Here we have performed a comprehensive antigenic repertoire analysis of sera using the next-generation phage display method - mimotope variation analysis (MVA). Samples from 64 children and adolescents were analyzed: 10 with Pdmx-NT1, 6 with sNT1, 16 Pandemrix-vaccinated, 16 H1N1 infected, and 16 unvaccinated healthy individuals. The diagnosis of NT1 was defined by the American Academy of Sleep Medicine international criteria of sleep disorders v3. FINDINGS: Our data showed that although the immunoprofiles toward vaccination were generally similar in study groups, there were also striking differences in immunoprofiles between sNT1 and Pdmx-NT1 groups as compared with controls. Prominent immune response was observed to a peptide epitope derived from prostaglandin D2 receptor (DP1), as well as peptides homologous to B cell lymphoma 6 protein. Further validation confirmed that these can act as true antigenic targets in discriminating NT1 diseased along with a novel epitope of hemagglutinin of H1N1 to delineate exposure to H1N1. INTERPRETATION: We propose that DP1 is a novel molecular target of autoimmune response and presents a potential diagnostic biomarker for NT1. DP1 is involved in the regulation of non-rapid eye movement (NREM) sleep and thus alterations in its functions could contribute to the disturbed sleep regulation in NT1 that warrants further studies. Together our results also show that MVA is a helpful method for finding novel peptide antigens to classify human autoimmune diseases, possibly facilitating the design of better therapies.

Auxiliary Biomembranes as a Directional Delivery System To Control Biological Events in Cell-Laden Tissue-Engineering Scaffolds.

Delivery of growth factors is an indispensable part of tissue engineering. Here, we describe a detachable membrane-based release system composed of extracellular matrix components that can be attached to hydrogels to achieve directional release of bioactive molecules. This way, the release of cytokines/growth factors can be started at a desired point of tissue maturation or directly in vivo. As a model, we develop thin films of an interpenetrating network of double-cross-linked gelatin and hyaluronic acid derivatives. The use of the auxiliary release system with vascular endothelial growth factor results in extensive sprouting by encapsulated vascular endothelial cells. The presence of the release system with interleukin-4 results in clustering of encapsulated macrophages with a significant decrease in M1 macrophages (proinflammatory). This system can be used in conjunction with three-dimensional structures as an auxiliary system to control artificial tissue maturation and growth.

Targeted alternative splicing of TAF4: a new strategy for cell reprogramming.

Reprogramming of somatic cells has become a versatile tool for biomedical research and for regenerative medicine. In the current study, we show that manipulating alternative splicing (AS) is a highly potent strategy to produce cells for therapeutic applications. We demonstrate that silencing of hTAF4-TAFH activity of TAF4 converts human facial dermal fibroblasts to melanocyte-like (iMel) cells. iMel cells produce melanin and express microphthalmia-associated transcription factor (MITF) and its target genes at levels comparable to normal melanocytes. Reprogramming of melanoma cells by manipulation with hTAF4-TAFH activity upon TAFH RNAi enforces cell differentiation towards chondrogenic pathway, whereas ectoptic expression of TAF4 results in enhanced multipotency and neural crest-like features in melanoma cells. In both cell states, iMels and cancer cells, hTAF4-TAFH activity controls migration by supporting E- to N-cadherin switches. From our data, we conclude that targeted splicing of hTAF4-TAFH coordinates AS of other TFIID subunits, underscoring the role of TAF4 in synchronised changes of Pol II complex composition essential for efficient cellular reprogramming. Taken together, targeted AS of TAF4 provides a unique strategy for generation of iMels and recapitulating stages of melanoma progression.

GLI2 cell-specific activity is controlled at the level of transcription and RNA processing: Consequences to cancer metastasis.

High activity of GLI family zinc finger protein 2 (GLI2) promotes tumor progression. Removal of the repressor domain at the N terminus (GLI2∆N) by recombinant methods converts GLI2 into a powerful transcriptional activator. However, molecular mechanisms leading to the formation of GLI2∆N activator proteins have not been established. Herein we report for the first time that the functional activities of GLI2 are parted into different protein isoforms by alternative promoter usage, selection of alternative splicing, transcription initiation and termination sites. Functional studies using melanoma cells revealed that transcriptional regulation of GLI2 is TGFbeta-dependent and supports the predominant production of GLI2∆N and C-terminally truncated GLI2 (GLI2∆C) isoforms in cells with high migratory and invasive phenotype. Taken together, these results highlight the role of transcription and RNA processing as major processes in the regulation of GLI2 activity with severe impacts in cancer development.

N-terminally truncated BAF57 isoforms contribute to the diversity of SWI/SNF complexes in neurons.

The SWItch/Sucrose NonFermentable, a nucleosome remodeling complex (SWI/SNF) chromatin-remodelling complexes act upon the nucleosomal structure and regulate transcription, replication, repair of chromatin and splicing. In this study, we present evidence that human, mouse and rat genes encoding one of the SWI/SNF complex subunits, BAF57, undergo neuron-specific splicing of exons II, III and IV. Alternative splicing yields in at least three isoforms of BAF57 protein that have truncated N-termini (N-BAF57s). The transcripts encoding N-BAF57 isoforms are predominantly expressed in the nervous system. The biochemical fractionation data supported by the results of the co-immunoprecipitation analysis show that N-BAF57 isoforms associate into protein complexes together with Brg1, Brm, BAF155 and BAF170. Transient over-expression of N-BAF57 isoforms in non-neural cells affects the level of expression of certain neuron-restrictive silencer element-containing genes. Together these data suggest that neuronal isoforms of BAF57 contribute to functional SWI/SNF complexes regulating neurogenesis.