Highly quantitative serological detection of anti-cytomegalovirus (CMV) antibodies

<p>Abstract</p> <p>Background</p> <p>Human cytomegalovirus infection is associated with a variety of pathological conditions including retinitis, pneumonia, hepatitis and encephalitis that may be transmitted congenitally, horizontally and parenterally and occurs both as a primary infection and as reactivation in immunocompromised individuals. Currently, there is a need for improved quantitative serological tests to document seropositivity with high sensitivity and specificity.</p> <p>Methods</p> <p>Here we investigated whether luciferase immunoprecipitation systems (LIPS) would provide a more quantitative and sensitive method for detecting anti-CMV antibodies. Four protein fragments of immunodominant regions of CMV antigens pp150 and pp65 were generated as <it>Renilla </it>luciferase (Ruc) fusion proteins and used in LIPS with two cohorts of CMV positive and negative sera samples previously tested by ELISA.</p> <p>Results</p> <p>Analysis of the antibody responses to two of these antigen fragments, pp150-d1 and pp150-d2, revealed geometric mean antibody titers in the first cohort that were 100–1000 fold higher in the CMV positive sera compared to the CMV negative samples (p < 0.0001) and infection status exactly matched the ELISA results for the 46 samples of the first cohort (100% sensitivity and 100% specificity). Two additional antigen fragments, pp65-d1 and pp65-d2 also showed robust antibody titers in some CMV-infected sera and yielded 50% and 96% sensitivity, respectively. Analysis of a second cohort of 70 samples using a mixture of the 4 antigens, which simplifies data collection and analysis, yielded values which correlated well with the sum of the values from the 4 separate tests (<it>r</it>_{<it>s </it>}= 0.93, p < 0.00001). While comparison of the LIPS results from this second cohort with ELISA showed 100% sensitivity, LIPS detected six additional CMV positive samples that were not detected by ELISA. Heat map analysis revealed that several of the LIPS positive/ELISA negative samples had positive LIPS immunoreactivity with 3–4 of the CMV antigens.</p> <p>Conclusion</p> <p>These results suggest that LIPS provides a highly robust and quantitative method for studying anti-CMV antibodies and has the potential to more accurately document CMV infection than standard ELISA.</p

Author Correction: Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases.

Emmanuelle Souzeau, who contributed to analysis of data, was inadvertently omitted from the author list in the originally published version of this Article. This has now been corrected in both the PDF and HTML versions of the Article

Rapid induction of autoantibodies during ARDS and septic shock

<p>Abstract</p> <p>Background</p> <p>Little is known about the induction of humoral responses directed against human autoantigens during acute inflammation. We utilized a highly sensitive antibody profiling technology to study autoantibodies in patients with acute respiratory distress syndrome (ARDS) and severe sepsis, conditions characterized by intensive immune activation leading to multiple organ dysfunction.</p> <p>Methods</p> <p>Using Luciferase Immunoprecipitation Systems (LIPS), a cohort of control, ARDS and sepsis patients were tested for antibodies to a panel of autoantigens. Autoantibody titers greater than the mean plus 3 SD of the 24 control samples were used to identify seropositive samples. Available longitudinal samples from different seropositive ARDS and sepsis patient samples, starting from within the first two days after admission to the intensive care, were then analyzed for changes in autoantibody over time.</p> <p>Results</p> <p>From screening patient plasma, 57% of ARDS and 46% of septic patients without ARDS demonstrated at least one statistically significant elevated autoantibody compared to the controls. Frequent high titer antibodies were detected against a spectrum of autoantigens including potassium channel regulator, gastric ATPase, glutamic decarboxylase-65 and several cytokines. Analysis of serial samples revealed that several seropositive patients had low autoantibodies at early time points that often rose precipitously and peaked between days 7-14. Further, the use of therapeutic doses of corticosteroids did not diminish the rise in autoantibody titers. In some cases, the patient autoantibody titers remained elevated through the last serum sample collected.</p> <p>Conclusion</p> <p>The rapid induction of autoantibodies in ARDS and severe sepsis suggests that ongoing systemic inflammation and associated tissue destruction mediate the break in tolerance against these self proteins.</p

Two Major Autoantibody Clusters in Systemic Lupus Erythematosus

Systemic lupus erythematosus is a chronic autoimmune disease of complex clinical presentation and etiology and is likely influenced by numerous genetic and environmental factors. While a large number of susceptibility genes have been identified, the production of antibodies against a distinct subset of nuclear proteins remains a primary distinguishing characteristic in disease diagnosis. However, the utility of autoantibody biomarkers for disease sub-classification and grouping remains elusive, in part, because of the difficulty in large scale profiling using a uniform, quantitative platform. In the present study serological profiles of several known SLE antigens, including Sm-D3, RNP-A, RNP-70k, Ro52, Ro60, and La, as well as other cytokine and neuronal antigens were obtained using the luciferase immunoprecipitation systems (LIPS) approach. The resulting autoantibody profiles revealed that 88% of a pilot cohort and 98% of a second independent cohort segregated into one of two distinct clusters defined by autoantibodies against Sm/anti-RNP or Ro/La autoantigens, proteins often involved in RNA binding activities. The Sm/RNP cluster was associated with a higher prevalence of serositis in comparison to the Ro/La cluster (P = 0.0022). However, from the available clinical information, no other clinical characteristics were associated with either cluster. In contrast, evaluation of autoantibodies on an individual basis revealed an association between anti-Sm (P = 0.006), RNP-A (P = 0.018) and RNP-70k (P = 0.010) autoantibodies and mucocutaneous symptoms and between anti-RNP-70k and musculoskeletal manifestations (P = 0.059). Serologically active, but clinically quiescent disease also had a higher prevalence of anti-IFN-α autoantibodies. Based on our findings that most SLE patients belong to either a Sm/RNP or Ro/La autoantigen cluster, these results suggest the possibility that alterations in RNA-RNA-binding protein interactions may play a critical role in triggering and/or the pathogenesis of SLE

Serological Studies Confirm the Novel Astrovirus HMOAstV-C as a Highly Prevalent Human Infectious Agent

Molecular identification of a microbe is the first step in determining its prevalence of infection and pathogenic potential. Detection of specific adaptive immune responses can provide insights into whether a microbe is a human infectious agent and its epidemiology. Here we characterized human anti-IgG antibody responses by luciferase immunoprecipitation systems (LIPS) against two protein fragments derived from the capsid protein of the novel HMOAstV-C astrovirus. While antibodies to the N-terminal fragment were not informative, the C-terminal capsid fragment of HMOAstV-C showed a high frequency of immunoreactivity with serum from healthy blood donors. In contrast, a similar C-terminal capsid fragment from the related HMOAstV-A astrovirus failed to show immunoreactivity. Detailed analysis of adult serum from the United Sates using a standardized threshold demonstrated HMOAstV-C seropositivity in approximately 65% of the samples. Evaluation of serum samples from different pediatric age groups revealed that the prevalence of antibodies in 6–12 month, 1–2 year, 2–5 year and 5–10 year olds was 20%, 23%, 32% and 36%, respectively, indicating rising seroprevalence with age. Additionally, 50% (11/22) of the 0–6 month old children showed anti-HMOAstV-C antibody responses, likely reflecting maternal antibodies. Together these results document human humoral responses to HMOAstV-C and validate LIPS as a facile and effective approach for identifying humoral responses to novel infectious agents

A common variant near TGFBR3 is associated with primary open angle glaucoma

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.Primary open angle glaucoma (POAG), a major cause of blindness worldwide, is a complex disease with a significant genetic contribution.We performed Exome Array (Illumina) analysis on 3504 POAG cases and 9746 controls with replication of the most significant findings in 9173 POAG cases and 26 780 controls across 18 collections of Asian, African and European descent. Apart from confirming strong evidence of association at CDKN2B-AS1 (rs2157719 [G], odds ratio [OR] = 0.71, P = 2.81 × 10−33), we observed one SNP showing significant association to POAG (CDC7–TGFBR3 rs1192415, ORG-allele = 1.13, Pmeta = 1.60 × 10−8). This particular SNP has previously been shown to be strongly associated with optic disc area and vertical cup-to-disc ratio, which are regarded as glaucoma-related quantitative traits. Our study now extends this by directly implicating it in POAG disease pathogenesis

Integrating genetic regulation and single-cell expression with GWAS prioritizes causal genes and cell types for glaucoma

Primary open-angle glaucoma (POAG), characterized by retinal ganglion cell death, is a leading cause of irreversible blindness worldwide. However, its molecular and cellular causes are not well understood. Elevated intraocular pressure (IOP) is a major risk factor, but many patients have normal IOP. Colocalization and Mendelian randomization analysis of &gt;240 POAG and IOP genome-wide association study (GWAS) loci and overlapping expression and splicing quantitative trait loci (e/sQTLs) in 49 GTEx tissues and retina prioritizes causal genes for 60% of loci. These genes are enriched in pathways implicated in extracellular matrix organization, cell adhesion, and vascular development. Analysis of single-nucleus RNA-seq of glaucoma-relevant eye tissues reveals that the POAG and IOP colocalizing genes and genome-wide associations are enriched in specific cell types in the aqueous outflow pathways, retina, optic nerve head, peripapillary sclera, and choroid. This study nominates IOP-dependent and independent regulatory mechanisms, genes, and cell types that may contribute to POAG pathogenesis.</p

Integrating genetic regulation and single-cell expression with GWAS prioritizes causal genes and cell types for glaucoma

Primary open-angle glaucoma (POAG), characterized by retinal ganglion cell death, is a leading cause of irreversible blindness worldwide. However, its molecular and cellular causes are not well understood. Elevated intraocular pressure (IOP) is a major risk factor, but many patients have normal IOP. Colocalization and Mendelian randomization analysis of &gt;240 POAG and IOP genome-wide association study (GWAS) loci and overlapping expression and splicing quantitative trait loci (e/sQTLs) in 49 GTEx tissues and retina prioritizes causal genes for 60% of loci. These genes are enriched in pathways implicated in extracellular matrix organization, cell adhesion, and vascular development. Analysis of single-nucleus RNA-seq of glaucoma-relevant eye tissues reveals that the POAG and IOP colocalizing genes and genome-wide associations are enriched in specific cell types in the aqueous outflow pathways, retina, optic nerve head, peripapillary sclera, and choroid. This study nominates IOP-dependent and independent regulatory mechanisms, genes, and cell types that may contribute to POAG pathogenesis.</p

Stabilized Coronavirus Spike Stem Elicits a Broadly Protective Antibody

Current coronavirus vaccines primarily target immunodominant epitopes in the S1 subunit, which are poorly conserved and susceptible to escape mutations, thus threatening vaccine efficacy. Here, we use structure-guided protein engineering to remove the S1 subunit from the MERS-CoV spike (S) glycoprotein and develop stabilized stem (SS) antigens. Vaccination with MERS SS elicits cross-reactive β-coronavirus antibody responses and protects mice against lethal MERS-CoV challenge. High-throughput screening of antibody secreting cells from MERS SS-immunized mice leads to discovery of a panel of cross-reactive monoclonal antibodies. Among them, antibody IgG22 binds with high affinity to both MERS-CoV and SARS-CoV-2 S proteins, and a combination of electron microscopy and crystal structures localizes the epitope to a conserved coiled-coil region in the S2 subunit. Passive transfer of IgG22 protects mice against both MERS-CoV and SARS-CoV-2 challenge. Collectively, these results provide proof-of-principle for cross-reactive coronavirus antibodies and inform the development of pan-coronavirus vaccines and therapeutic antibodies

DenseNet and Support Vector Machine classifications of major depressive disorder using vertex-wise cortical features

Major depressive disorder (MDD) is a complex psychiatric disorder that affects the lives of hundreds of millions of individuals around the globe. Even today, researchers debate if morphological alterations in the brain are linked to MDD, likely due to the heterogeneity of this disorder. The application of deep learning tools to neuroimaging data, capable of capturing complex non-linear patterns, has the potential to provide diagnostic and predictive biomarkers for MDD. However, previous attempts to demarcate MDD patients and healthy controls (HC) based on segmented cortical features via linear machine learning approaches have reported low accuracies. In this study, we used globally representative data from the ENIGMA-MDD working group containing an extensive sample of people with MDD (N=2,772) and HC (N=4,240), which allows a comprehensive analysis with generalizable results. Based on the hypothesis that integration of vertex-wise cortical features can improve classification performance, we evaluated the classification of a DenseNet and a Support Vector Machine (SVM), with the expectation that the former would outperform the latter. As we analyzed a multi-site sample, we additionally applied the ComBat harmonization tool to remove potential nuisance effects of site. We found that both classifiers exhibited close to chance performance (balanced accuracy DenseNet: 51%; SVM: 53%), when estimated on unseen sites. Slightly higher classification performance (balanced accuracy DenseNet: 58%; SVM: 55%) was found when the cross-validation folds contained subjects from all sites, indicating site effect. In conclusion, the integration of vertex-wise morphometric features and the use of the non-linear classifier did not lead to the differentiability between MDD and HC. Our results support the notion that MDD classification on this combination of features and classifiers is unfeasible