Association of apolipoprotein E4 to the number of cerebral microbleeds in patients recovered from COVID-19

Background: COVID-19 has been associated with cerebral microbleeds (CMB). Previously, an association of ApoE4 with COVID-19 severity and CMBs in autopsy was found. In this study, we investigated if carrying the Apoe4 allele relates to the number of CMBs in magnetic resonance imaging (MRI) in patients recovered from COVID-19. Material(s) and Method(s): Adult patients recovered from COVID-19 and a control group without a history of COVID-19 was recruited. Exclusion criteria were major neurologic disease, developmental disability or pregnancy. The participants underwent brain MRI 6 months after infection, and a blinded neuroradiologist analyzed the findings. ApoE was genotyped using a microarray. Statistical analysis was performed using the statistical software R. A negative binomial model was chosen based on the distribution of CMBs. Result(s): Of the 216 subjects that underwent MRI, 168 consented to genetic testing, additionally 2 patients were excluded due to extensive CMBs and 1 due to diffuse axonal injury. We included 113 COVID-19 patients (49 ICU-treated, 29 ward-treated and 35 home-isolated) and 52 controls. The most prevalent comorbidities were hypertension, asthma and diabetes. CMBs was found in 47 subjects, with the number of CMBs ranging from 0 to 26. The ApoeE4 allele was carried by 37%, equally distributed among the groups. After adjustment, age (aRR = 1.06, p = 0.007) and COVID-19 (aRR = 2.59, p = 0.038) were independently associated with CMBs. The ApoE4 allele (aRR = 2.16, p = 0.07, CI = 0.94-5.10) was not significant. Conclusion(s): Age and previous COVID-19, but not possession of the ApoeE4 allele, were independently associated with the number of CMBs.

Exploration of potential mechanism of SARS-CoV-2 induced immune inflammatory response leading to progression of atherosclerosis and prediction of traditional Chinese medicine for preventing and treating based on bioinformatics.

Objective To explore the core targets and important pathways of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) induced atherosclerosis (AS) progression from the perspective of immune inflammation, so as to predict the potential prevention and treatment of traditional Chinese medicine (TCM). Methods Microarray data were obtained from the Gene Expression Omnibus (GEO) database for coronavirus disease 2019 (COVID-19) patients and AS patients, and the "limmar" and "Venn" packages were used to screen out the common differentially expressed genes (DEGs) genes in both diseases. The gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses were performed on the common DEGs to annotate their functions and important pathways. The two gene sets were scored for immune cells and immune function to assess the level of immune cell infiltration. The protein-protein interaction (PPI) network was constructed by STRING database, and the CytoHubba plug-in of Cytoscape was used to identify the hub genes. Two external validation datasets were introduced to validate the hub genes and obtain the core genes. Immuno-infiltration analysis and gene set enrichment analysis (GSEA) were performed on the core genes respectively. Finally the potential TCM regulating the core genes were predicted by Coremine Medical database. Results A total of 7898 genes related to COVID-19, 471 genes related to AS progression;And 51 common DEGs, including 32 highly expressed genes and 19 low expressed genes were obtained. GO and KEGG analysis showed that common DEGs, which were mainly localized in cypermethrin-encapsulated vesicles, platelet alpha particles, phagocytic vesicle membranes and vesicles, were involved in many biological processes such as myeloid differentiation factor 88 (MyD88)-dependent Toll-like receptor signaling pathway transduction, interleukin-8 (IL-8) production and positive regulation, IL-6 production and positive regulation to play a role in regulating nicotinamide adenine dinucleotide phosphate oxidase activity, Toll-like receptor binding and lipopeptide and glycosaminoglycan binding through many biological pathways, including Toll-like receptor signaling pathways, neutrophil extracellular trap formation, complement and coagulation cascade reactions. The results of immune infiltration analysis demonstrated the state of immune microenvironment of COVID-19 and AS. A total of 5 hub genes were obtained after screening, among which Toll-like receptor 2 (TLR2), cluster of differentiation 163 (CD163) and complement C1q subcomponent subunit B (C1QB) genes passed external validation as core genes. The core genes showed strong correlation with immune process and inflammatory response in both immune infiltration analysis and GSEA enrichment analysis. A total of 35 TCMs, including Chuanxiong (Chuanxiong Rhizoma), Taoren (Persicae Semen), Danggui (Angelicae Sinensis Radix), Huangqin (Scutellariae Radix), Pugongying (Taraxaci Herba), Taizishen (Pseudostellariae Radix), Huangjing (Polygonati Rhizoma), could be used as potential therapeutic agents. Conclusion TLR2, CD163 and C1QB were the core molecules of SARS-CoV-2-mediated immune inflammatory response promoting AS progression, and targeting predicted herbs were potential drugs to slow down AS progression in COVID-19 patients.Copyright © 2023 Editorial Office of Chinese Traditional and Herbal Drugs. All rights reserved.

A linear B-cell epitope close to the furin cleavage site within the S1 domain of SARS-CoV-2 Spike protein discriminates the humoral immune response of nucleic acid- and protein-based vaccine cohorts.

Background: Understanding the humoral immune response towards viral infection and vaccination is instrumental in developing therapeutic tools to fight and restrict the viral spread of global pandemics. Of particular interest are the specificity and breadth of antibody reactivity in order to pinpoint immune dominant epitopes that remain immutable in viral variants. Methods: We used profiling with peptides derived from the Spike surface glycoprotein of SARS-CoV-2 to compare the antibody reactivity landscapes between patients and different vaccine cohorts. Initial screening was done with peptide microarrays while detailed results and validation data were obtained using peptide ELISA. Results: Overall, antibody patterns turned out to be individually distinct. However, plasma samples of patients conspicuously recognized epitopes covering the fusion peptide region and the connector domain of Spike S2. Both regions are evolutionarily conserved and are targets of antibodies that were shown to inhibit viral infection. Among vaccinees, we discovered an invariant Spike region (amino acids 657-671) N-terminal to the furin cleavage site that elicited a significantly stronger antibody response in AZD1222- and BNT162b2- compared to NVX-CoV2373-vaccinees. Conclusions: Understanding the exact function of antibodies recognizing amino acid region 657-671 of SARS-CoV-2 Spike glycoprotein and why nucleic acid-based vaccines elicit different responses from protein-based ones will be helpful for future vaccine design.

Microarray patches for managing infections at a global scale.

Since the first patent for micro array patches (MAPs) was filed in the 1970s, research on utilising MAPs as a drug delivery system has progressed significantly, evidenced by the transition from the simple 'poke and patch' of solid MAPs to the development of bio responsive systems such as hydrogel-forming and dissolving MAPs. In addition to the extensive research on MAPs for improving transdermal drug delivery, there is a growing interest in using these devices to manage infectious diseases. This is due to the minimally invasive nature of this drug delivery platform which enable patients to self-administer therapeutics without the aid of healthcare professionals. This review aims to provide a critical analysis on the potential utility of MAPs in managing infectious diseases which are still endemic at a global scale. The range of diseases covered in this review include tuberculosis, skin infections, malaria, methicillin-resistant Staphylococcus aureus infections and Covid-19. These diseases exert a considerable socioeconomic burden at a global scale with their impact magnified in low- and middle-income countries (LMICs). Due to the painless and minimally invasive nature of MAPs application, this technology also provides an efficient solution not only for the delivery of therapeutics but also for the administration of vaccine and prophylactic agents that could be used in preventing the spread and outbreak of emerging infections. Furthermore, the ability of MAPs to sample and collect dermal interstitial fluid that is rich in disease-related biomarkers could also open the avenue for MAPs to be utilised as a minimally invasive biosensor for the diagnosis of infectious diseases. The efficacy of MAPs along with the current limitations of such strategies to prevent and treat these infections will be discussed. Lastly, the clinical and translational hurdles associated with MAP technologies will also be critically discussed.

SARS-CoV-2 infection alters the breast milk glycomic profile

Breast milk is generally accepted as the perfect source of nutrition for the health and development of infants. It also assists in infant innate and adaptive immunity through many proteins that are decorated with glycans. Examples of these glycoproteins include IgA, IgG, and innate immune lectins. Maternal diet and environmental exposure such as pathogens and pollutants affect human milk composition including its glycoprofile. Despite altered glycosylation can have a consequence on the nursing infant's health and immunity, the current knowledge is still emerging in this area of study. COVID-19 has gained attention in recent years by causing severe morbidity and mortality. Similar to other infectious diseases such as influenza, our lab recently revealed alterations in glycome of plasma and different tissue samples of COVID-19 infected patients. Inspired by these findings, we are interested in disclosing the effect of SARS-CoV-2 on glycosylation of breast milk proteins. Toward this, we performed a large-scale systematic study using our high-throughput lectin microarray analysis technology. We analyzed 132 control samples (breast milk collected pre- COVID) and breast milk from 78 COVID-19 infected mothers. Our data showed there is a 4-fold increase in -2,3 sialic acid on glycoproteins that is associated with SARS-CoV-2 infection in lactating mothers. Lectin pulldown experiments further testified to these findings. Given the significance of -2,3 sialic acid glycan signature in infectious diseases, our finding could provide valuable insight into therapeutic development.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Recent Progress in Development and Application of DNA, Protein, Peptide, Glycan, Antibody, and Aptamer Microarrays.

Microarrays are one of the trailblazing technologies of the last two decades and have displayed their importance in all the associated fields of biology. They are widely explored to screen, identify, and gain insights on the characteristics traits of biomolecules (individually or in complex solutions). A wide variety of biomolecule-based microarrays (DNA microarrays, protein microarrays, glycan microarrays, antibody microarrays, peptide microarrays, and aptamer microarrays) are either commercially available or fabricated in-house by researchers to explore diverse substrates, surface coating, immobilization techniques, and detection strategies. The aim of this review is to explore the development of biomolecule-based microarray applications since 2018 onwards. Here, we have covered a different array of printing strategies, substrate surface modification, biomolecule immobilization strategies, detection techniques, and biomolecule-based microarray applications. The period of 2018-2022 focused on using biomolecule-based microarrays for the identification of biomarkers, detection of viruses, differentiation of multiple pathogens, etc. A few potential future applications of microarrays could be for personalized medicine, vaccine candidate screening, toxin screening, pathogen identification, and posttranslational modifications.

Comparison of disinfectants-induced gene expression profile: Potential adverse effects.

Despite their importance in combating the spread of the COVID-19 pandemic, adverse effects of disinfectants on human health, especially the respiratory system, have been of continuing concern to researchers. Considering that bronchi are the main target of sprayed disinfectants, we here treated the seven major active ingredients in disinfectant products accepted by the US EPA to human bronchial epithelial cells and determined the subtoxic levels. Then, we performed microarray analysis using total RNA obtained at the subtoxic level and designed a network representing disinfectant-induced cellular response using the KEGG pathway analysis technique. Polyhexamethylguanidine phosphate, a lung fibrosis inducer, was used as a reference material to verify the relationship between cell death and pathology. The derived results reveal potential adverse effects along with the need for an effective application strategy for each chemical.

Transcriptome Profiling in Autoimmune Diseases

Autoimmune diseases are a group of different inflammatory disorders characterized by systemic or localized inflammation, affecting approximately 0.1–1% of the general population. Several studies suggest that genetic risk loci are shared between different autoimmune diseases and pathogenic mechanisms may also be shared. The strategy of performing differential gene expression profiles in autoimmune disorders has unveiled new transcripts that may be shared among these disorders. Microarray technology and bioinformatics offer the most comprehensive molecular evaluations and it is widely used to understand the changes in gene expression in specific organs or in peripheral blood cells. The major goal of transcriptome studies is the identification of specific biomarkers for different diseases. It is believed that such knowledge will contribute to the development of new drugs, new strategies for early diagnosis, avoiding tissue autoimmune destruction, or even preventing the development of autoimmune disease. In this review, we primarily focused on the transcription profiles of three typical autoimmune disorders, including type 1 diabetes mellitus (destruction of pancreatic islet beta cells), systemic lupus erythematosus (immune complex systemic disorder affecting several organs and tissues), and multiple sclerosis (inflammatory and demyelinating disease of the nervous system). © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2014, 2022.

Development of a biomarker signature using grating-coupled fluorescence plasmonic microarray for diagnosis of MIS-C.

Multisystem inflammatory syndrome in children (MIS-C) is a rare but serious condition that can develop 4-6 weeks after a school age child becomes infected by SARS-CoV-2. To date, in the United States more than 8,862 cases of MIS-C have been identified and 72 deaths have occurred. This syndrome typically affects children between the ages of 5-13; 57% are Hispanic/Latino/Black/non-Hispanic, 61% of patients are males and 100% have either tested positive for SARS-CoV-2 or had direct contact with someone with COVID-19. Unfortunately, diagnosis of MIS-C is difficult, and delayed diagnosis can lead to cardiogenic shock, intensive care admission, and prolonged hospitalization. There is no validated biomarker for the rapid diagnosis of MIS-C. In this study, we used Grating-coupled Fluorescence Plasmonic (GCFP) microarray technology to develop biomarker signatures in pediatric salvia and serum samples from patients with MIS-C in the United States and Colombia. GCFP measures antibody-antigen interactions at individual regions of interest (ROIs) on a gold-coated diffraction grating sensor chip in a sandwich immunoassay to generate a fluorescent signal based on analyte presence within a sample. Using a microarray printer, we designed a first-generation biosensor chip with the capability of capturing 33 different analytes from 80  µ L of sample (saliva or serum). Here, we show potential biomarker signatures in both saliva and serum samples in six patient cohorts. In saliva samples, we noted occasional analyte outliers on the chip within individual samples and were able to compare those samples to 16S RNA microbiome data. These comparisons indicate differences in relative abundance of oral pathogens within those patients. Microsphere Immunoassay (MIA) of immunoglobulin isotypes was also performed on serum samples and revealed MIS-C patients had several COVID antigen-specific immunoglobulins that were significantly higher than other cohorts, thus identifying potential new targets for the second-generation biosensor chip. MIA also identified additional biomarkers for our second-generation chip, verified biomarker signatures generated on the first-generation chip, and aided in second-generation chip optimization. Interestingly, MIS-C samples from the United States had a more diverse and robust signature than the Colombian samples, which was also illustrated in the MIA cytokine data. These observations identify new MIS-C biomarkers and biomarker signatures for each of the cohorts. Ultimately, these tools may represent a potential diagnostic tool for use in the rapid identification of MIS-C.

Accelerating the development of vaccine microarray patches for epidemic response and equitable immunization coverage requires investment in microarray patch manufacturing facilities.

INTRODUCTION: There is a need for investment in manufacturing for vaccine microarray patches (vMAPs) to accelerate vMAP development and access. vMAPs could transform vaccines deployment and reach to everyone, everywhere. AREAS COVERED: We outline vMAPs' potential benefits for epidemic preparedness and for outreach in low- and lower-middle-income countries (LMICs), share lessons learned from pandemic response, and highlight that investment in manufacturing-at-risk could accelerate vMAP development. EXPERT OPINION: Pilot manufacturing capabilities are needed to produce clinical trial material and enable emergency response. Funding vMAP manufacturing scale-up in parallel to clinical proof-of-concept studies could accelerate vMAP approval and availability. Incentives could mitigate the risks of establishing multi-vMAP manufacturing facilities early.

Late acceptance hill climbing aided chaotic harmony search for feature selection: An empirical analysis on medical data

In today's era of data-driven digital society, there is a huge demand for optimized solutions that essentially reduce the cost of operation, thereby aiming to increase productivity. Processing a huge amount of data, like the Microarray based gene expression data, using machine learning and data mining algorithms has certain limitations in terms of memory and time requirements. This would be more concerning, when a dataset comes with redundant and non-important information. For example, many report-based medical datasets have several non-informative attributes which mislead the classification algorithms. To this end, researchers have been developing several feature selection algorithms that try to discard the redundant information from the raw datasets before feeding them to machine learning algorithms. Metaheuristic based optimization algorithms provide an excellent option to solve feature selection problems. In this paper, we propose a music-inspired harmony search (HS) algorithm based wrapper feature selection method. At the beginning, we use a chaotic mapping to initialize the population of the HS algorithm in order to better coverage of the search space. Further to complement the inferior exploitation of the HS algorithm, we integrate it with the Late Acceptance Hill Climbing (LAHC) method. Thus the combination of these two algorithms provides a good balance between the exploration and exploitation of the HS algorithm. We evaluate the proposed feature selection method on 15 UCI datasets and the obtained results are found to be better than many state-of-the-art methods both in terms of the classification accuracy and the number of features selected. To evaluate the effectiveness of our algorithm, we utilize a combination of precision, recall, F1 score, fitness value, and execution time as performance indicators. These metrics enable us to obtain a comprehensive assessment of the algorithm's abilities and limitations. We also apply our method on 3 microarray based gene expression datasets used for prediction of cancer to ensure the scalability and robustness as a feature selection method in real-life scenarios. In addition to this, we test our approach using the COVID-19 dataset, and it performs better than several metaheuristic based optimization techniques. © 2023

Novel functional monomer for the electrochemical synthesis of highly affine epitope-imprinted polymers

To address the lack of functional monomer diversity for the electrosynthesis of protein-selective molecularly imprinted polymers (MIPs), we introduce a new concept able to lead to a new class of functional monomers. This is based on conjugating an electropolymerizable monomer unit (umbelliferone) to an amino acid for closer mimicking of protein-based natural affinity ligands such as antibodies. As the first representative of this class of monomers an aspartate-umbelliferone (Asp-UMB) conjugate was synthesized and here we provide the proof for its suitability to generate highly affine MIPs for proteins by epitope imprinting. As model we used a heptapeptide (GFNCYFP) stemming from the receptor binding domain (RBD) of the SARS-CoV-2 spike protein to generate epitope-imprinted polymers able to recognize the parent RBD protein. For rapid optimization and assessment of the binding kinetics we prepared MIP microarrays on surface plasmon resonance imaging (SPRi) chips. First the peptides were microspotted on the bare gold surface of the chips followed by the electropolymerization of Asp-UMB. This resulted in ca. 2 nm thick, highly uniform, and electrically insulating polymer film, well suited both for hierarchical epitope imprinting and SPRi read-out. Taking advantage also of the on-chip optimization enabled by the microarray format the increased functional diversity of the new monomer resulted in highly affine MIPs with equilibrium dissociation constants in the lower picomolar range. © 2023 The Authors. Electroanalysis published by Wiley-VCH GmbH.

SARS-CoV-2 Host Receptors Expression in Human Pancreatic Islets Revealed Upregulation of ACE2 in Diabetic subjects

Background: It is well established that diabetic patients infected with COVID-19- are at higher risk of developing severe symptoms that may lead to death. Such observation argues for the possibility that SARS-CoV-2 may target and infect pancreatic islets. SARSCoV- 2 is thought to enter the cells through the binding of viral spike S1 protein to ACE2. The cellular entry process includes priming of the S protein by TMPRSS2 and ADAM17, which facilitate the binding and promote ACE2 shedding. To date, no conclusive evidence has emerged to address the expression of TMPRSS2 and ADMA17 or the interaction between SARS-CoV-2 and human pancreatic islets. Method(s): Microarray and RNA-sequencing (RNA-seq) expression data from human islets were used to profile the expression pattern of ACE2, ADAM17, and TMPRSS2 in diabetic and non-diabetic subjects. Result(s): Pancreatic islets express all three receptors regardless of diabetes status. ACE2 expression was significantly elevated in diabetic islets than non-diabetic. Female donors showed to have higher ACE2 expression compared to males, whereas ADAM17 and TMPRSS2 were not affected by gender. No difference in the expression of the three receptors in young (<=40 years old) compared to old (>=60 years old) islets. Obese donors (BMI>30) showed significantly higher expression levels of ADAM17 and TMPRSS2 as compared to non-obese (BMI<25). Expression of TMPRSS2 was associated positively with HbA1c and inversely with age, while ADAM17 and TMPRSS2 were associated positively with BMI. Muscle and subcutaneous adipose tissues showed similar expression of the three receptors in diabetic and nondiabetic donors. Conclusion(s): ACE2 expression is increased in diabetic human islets. More studies are warranted to understand the permissiveness of human pancreatic beta-cells to SARS-Cov-2 and whether variations of ACE2 expression could explain the severity of COVID-19 infection between diabetics and non-diabetic patients.

Exploring the Immunodominant Epitopes of SARS-CoV-2 Nucleocapsid Protein as Exposure Biomarker.

Background The nucleocapsid protein (N protein) of SARS-CoV-2 is undeniably a potent target for the development of diagnostic tools due to its abundant expression and lower immune evasion pressure compared to spike (S) protein. Methods Blood samples of active COVID-19 infections (n=71) and post-COVID-19 (n=11) were collected from a tertiary care hospital in India; pre-COVID-19 (n=12) sera samples served as controls. Real-time reverse transcriptase-PCR (rRT-PCR) confirmed pooled sera samples (n=5) were used with PEPperCHIP® SARS-CoV-2 Proteome Microarray (PEPperPRINT GmbH, Germany) to screen immunodominant epitopes of SARS-CoV-2. Highly immunodominant epitopes were then commercially synthesized and further validated for their immunoreactivity by dot-blot and ELISA. Results The lowest detectable concentration (LDC) of the N1 peptide in the dot-blot assay was 12.5 µg demonstrating it to be fairly immunoreactive compared to control sera. IgG titers against the contiguous peptide (N2: 156AIVLQLPQGTTLPKGFYAEGS176) was found to be significantly higher (p=0.018) in post-COVID-19 compared to pre-COVID-19 control sera. These results suggested that N2-specific IgG titers buildup over time as expected in post-COVID-19 sera samples, while a non-significant immunoreactivity of the N2 peptide was also observed in active-COVID-19 sera samples. However, there were no significant differences in the total IgG titers between active COVID-19 infections, post-COVID-19 and pre-COVID-19 controls. Conclusion The N2-specific IgG titers in post-COVID-19 samples demonstrated the potential of N protein as an exposure biomarker, particularly in sero-surveillance studies.

Lateral Flow Microarray-Based ELISA for Cytokines.

Cytokines are well known to be involved in numerous biological responses with diverse mechanisms of action, including the inflammatory process. The so-called "cytokine storm" has recently been associated with cases of severe COVID-19 infection.Lateral flow microarray (LFM) devices have been constructed for multiplex detection of cytokines. The LFM-cytokine rapid test involves the immobilization of an array of capture anti-cytokine antibodies. Here, we describe the methods to create and use multiplex lateral flow-based immunoassays based upon the enzyme-linked immunosorbent assay (ELISA).

Hybrid Peptide–Agarose Hydrogels for 3D Immunoassays

Recent advances in biosensing analytical platforms have brought relevant outcomes for novel diagnostic and therapy-oriented applications. In this context, 3D droplet microarrays, where hydrogels are used as matrices to stably entrap biomolecules onto analytical surfaces, potentially provide relevant advantages over conventional 2D assays, such as increased loading capacity, lower nonspecific binding, and enhanced signal-to-noise ratio. Here, we describe a hybrid hydrogel composed of a self-assembling peptide and commercial agarose (AG) as a suitable matrix for 3D microarray bioassays. The hybrid hydrogel is printable and self-adhesive and allows analyte diffusion. As a showcase example, we describe its application in a diagnostic immunoassay for the detection of SARS-CoV-2 infection. © 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Immune profiling of SARS-CoV-2 epitopes in asymptomatic and symptomatic pediatric and adult patients.

BACKGROUND: The infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has unpredictable manifestations of coronavirus disease (COVID-19) and variable clinical course with some patients being asymptomatic whereas others experiencing severe respiratory distress, or even death. We aimed to evaluate the immunoglobulin G (IgG) response towards linear peptides on a peptide array containing sequences from SARS-CoV-2, Middle East respiratory syndrome-related coronavirus (MERS) and common-cold coronaviruses 229E, OC43, NL63 and HKU1 antigens, in order to identify immunological indicators of disease outcome in SARS-CoV-2 infected patients. METHODS: We included in the study 79 subjects, comprising 19 pediatric and 30 adult SARS-CoV-2 infected patients with increasing disease severity, from mild to critical illness, and 30 uninfected subjects who were vaccinated with one dose of SARS-CoV-2 spike mRNA BNT162b2 vaccine. Serum samples were analyzed by a peptide microarray containing 5828 overlapping 15-mer synthetic peptides corresponding to the full SARS-CoV-2 proteome and selected linear epitopes of spike (S), envelope (E) and membrane (M) glycoproteins as well as nucleoprotein (N) of MERS, SARS and coronaviruses 229E, OC43, NL63 and HKU1 (isolates 1, 2 and 5). RESULTS: All patients exhibited high IgG reactivity against the central region and C-terminus peptides of both SARS-CoV-2 N and S proteins. Setting the threshold value for serum reactivity above 25,000 units, 100% and 81% of patients with severe disease, 36% and 29% of subjects with mild symptoms, and 8% and 17% of children younger than 8-years reacted against N and S proteins, respectively. Overall, the total number of peptides in the SARS-CoV-2 proteome targeted by serum samples was much higher in children compared to adults. Notably, we revealed a differential antibody response to SARS-CoV-2 peptides of M protein between adults, mainly reacting against the C-terminus epitopes, and children, who were highly responsive to the N-terminus of M protein. In addition, IgG signals against NS7B, NS8 and ORF10 peptides were found elevated mainly among adults with mild (63%) symptoms. Antibodies towards S and N proteins of other coronaviruses (MERS, 229E, OC43, NL63 and HKU1) were detected in all groups without a significant correlation with SARS-CoV-2 antibody levels. CONCLUSIONS: Overall, our results showed that antibodies elicited by specific linear epitopes of SARS-CoV-2 proteome are age dependent and related to COVID-19 clinical severity. Cross-reaction of antibodies to epitopes of other human coronaviruses was evident in all patients with distinct profiles between children and adult patients. Several SARS-CoV-2 peptides identified in this study are of particular interest for the development of vaccines and diagnostic tests to predict the clinical outcome of SARS-CoV-2 infection.

Epitranscriptomic N6-Methyladenosine Profile of SARS-CoV-2-Infected Human Lung Epithelial Cells.

N6-methyladenosine (m6A) is a dynamic posttranscriptional RNA modification that plays an important role in determining transcript fate. The functional consequence of m6A deposition is dictated by a group of host proteins that specifically recognize and bind the m6A modification, leading to changes in RNA stability, transport, splicing, or translation. The cellular m6A methylome undergoes changes during certain pathogenic conditions such as viral infections. However, how m6A modification of host cell transcripts and noncoding RNAs change during severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection has not been reported. Here, we define the epitranscriptomic m6A profile of SARS-CoV-2-infected human lung epithelial cells compared to uninfected controls. We identified mRNA and long and small noncoding RNA species that are differentially m6A modified in response to SARS-CoV-2 infection. The most significantly differentially methylated transcript was the precursor of microRNA-4486 (miRNA-4486), which showed significant increases in abundance and percentage of methylated transcripts in infected cells. Pathway analyses revealed that differentially methylated transcripts were significantly associated with several cancer-related pathways, protein processing in the endoplasmic reticulum, cell death, and proliferation. Upstream regulators predicted to be associated with the proteins encoded by differentially methylated mRNAs include several proteins involved in the type-I interferon response, inflammation, and cytokine signaling. IMPORTANCE Posttranscriptional modification of viral and cellular RNA by N6-methyladenosine (m6A) plays an important role in regulating the replication of many viruses and the cellular immune response to infection. We therefore sought to define the epitranscriptomic m6A profile of human lung epithelial cells infected with SARS-CoV-2. Our analyses demonstrate the differential methylation of both coding and noncoding cellular RNAs in SARS-CoV-2-infected cells compared to uninfected controls. Pathway analyses revealed that several of these RNAs may be involved in the cellular response to infection, such as type-I interferon. Our study implicates m6A modification of infected-cell RNA as a mechanism of posttranscriptional gene regulation during SARS-CoV-2 infection.

Epitope Coverage of Anti-SARS-CoV-2 Nucleocapsid IgA and IgG Antibodies Correlates with Protection against Re-Infection by New Variants in Subsequent Waves of the COVID-19 Pandemic.

The COVID-19 pandemic continues to affect individuals across the globe, with some individuals experiencing more severe disease than others. The relatively high frequency of re-infections and breakthrough infections observed with SARS-CoV-2 highlights the importance of extending our understanding of immunity to COVID-19. Here, we aim to shed light on the importance of antibody titres and epitope utilization in protection from re-infection. Health care workers are highly exposed to SARS-CoV-2 and are therefore also more likely to become re-infected. We utilized quantitative, multi-antigen, multi-epitope SARS-CoV-2 protein microarrays to measure IgG and IgA titres against various domains of the nucleocapsid and spike proteins. Potential re-infections in a large, diverse health care worker cohort (N = 300) during the second wave of the pandemic were identified by assessing the IgG anti-N titres before and after the second wave. We assessed epitope coverage and antibody titres between the 'single infection' and 're-infection' groups. Clear differences were observed in the breadth of the anti-N response before the second wave, with the epitope coverage for both IgG (p = 0.019) and IgA (p = 0.015) being significantly increased in those who did not become re-infected compared to those who did. Additionally, the IgG anti-N (p = 0.004) and anti-S titres (p = 0.018) were significantly higher in those not re-infected. These results highlight the importance of the breadth of elicited antibody epitope coverage following natural infection in protection from re-infection and disease in the COVID-19 pandemic.