Predicting Variant Pathogenicity with Machine Learning

There are roughly 22,000 protein-coding genes in the human body, many of which play important roles in biological functions. The proteins fold in 3D space, and this is most often necessary for function. A genetic variant can disrupt the secondary structure of a protein (one aspect of structure) or eliminate a site important in protein-protein interaction or post-translational modification. The loss of function or deregulation can result in disease. Thus, there is great biomedical interest in identifying disease-causing single-nucleotide variants. We hypothesize that we can accurately predict variant pathogenicity. We used machine learning to predict the pathogenicity of a set of 28,369 single-nucleotide variants across 10 genes. The data are acquired from publicly available saturation mutagenesis data sets, which generate every possible amino acid substitution at every position in a protein. Our approach employs a support vector machine using linear, polynomial, and RBF kernel functions. The problem is implemented as a binary classification problem, where a label of 1 indicates a disease-causing variant and a label of 0 indicates a benign variant. The model predicts pathogenicity based on amino acid, post-translational modification, and secondary structure information. We cleaned and analyzed the data with custom Python scripts. Our results show average balanced accuracy scores for classifying pathogenicity of approximately 57.9%, 60.3%, and 60.3% for the linear, polynomial, and RBF kernels, respectively. Therefore, the model is an improvement over random guessing but has room for improvement.https://digitalscholarship.unlv.edu/durep_posters/1045/thumbnail.jp

Pre-exposure prophylaxis with OspA-specific human monoclonal antibodies protects mice against tick transmission of Lyme disease spirochetes

Background. Tick transmission of Borrelia spirochetes to humans results in significant morbidity from Lyme disease worldwide. Serum concentrations of antibodies against outer surface protein A (OspA) were shown to correlate with protection from infection with Borrelia burgdorferi, the primary cause of Lyme disease in the United States. Methods. Mice transgenic for human immunoglobulin genes were immunized with OspA protein of B. burgdorferi to generate human monoclonal antibodies (HuMabs) against OspA. HuMabs were generated and tested in in vitro borreliacidal assays and animal protection assays. Results. Nearly 100 unique OspA specific HuMabs were generated and four HuMabs (221-7, 857-2, 319-44, and 212-55) were selected as lead candidates based on borreliacidal activity. HuMab 319-44, 857-2 and 212-55 were borreliacidal against one or two Borrelia genospecies, whereas 221-7 was borreliacidal (IC50 \u3c 1nM) against B. burgdorferi, B. afzelii and B. garinii, the three main genospecies endemic in the US, Europe and Asia. All four HuMabs completely protected mice from infection at 10 mg/kg in a murine model of tick-mediated transmission of B. burgdorferi. Conclusions. Our study indicates that OspA-specific HuMabs can prevent the transmission of Borrelia and administration of these antibodies could be employed as pre-exposure prophylaxis for Lyme disease

Information mobility in complex networks

The concept of information mobility in complex networks is introduced on the basis of a stochastic process taking place in the network. The transition matrix for this process represents the probability that the information arising at a given node is transferred to a target one. We use the fractional powers of this transition matrix to investigate the stochastic process at fractional time intervals. The mobility coefficient is then introduced on the basis of the trace of these fractional powers of the stochastic matrix. The fractional time at which a network diffuses 50% of the information contained in its nodes (1/ k50 ) is also introduced. We then show that the scale-free random networks display better spread of information than the non scale-free ones. We study 38 real-world networks and analyze their performance in spreading information from their nodes. We find that some real-world networks perform even better than the scale-free networks with the same average degree and we point out some of the structural parameters that make this possible

Pre-exposure Immunoprophylaxis by Genetically Encoded DMAb anti-OspA Human Monoclonal Antibody to Prevent Lyme Disease

Tick transmission of Borrelia spirochetes to humans results in significant morbidity from Lyme disease. Animal studies have demonstrated that transmission of Borrelia from tick vector to the mammalian host can be blocked by antibodies against outer surface protein A (OspA). We have recently developed borreliacidal human IgG1 monoclonal antibodies (HuMabs) directed against OspA. HuMab 319-44 was borreliacidal against B. burgdorferi (IC50Borreliatransmission after a single dose of 2 mg/kg administered on the day of tick challenge. Since passively administered IgG1 antibodies do not have a sufficient half-life to provide protection for the 6-7 month peak risk period, we investigated a novel approach of vector-mediated gene transfer of HuMabs that could potentially provide protection against Lyme disease during the seasonal risk period. A modified HuMab, 319-44 mod, expressed by a synthetic DNA plasmid (DMAb) was optimized and characterized in in vitro OspA binding and bactericidal assays. To assess in vivo protection, mice were administered a single DMAb injection into the quadriceps followed by electroporation. The mice were then challenged by B. burgdorferi-infected nymphs. Tissue samples were monitored by dark-field microscopy for spirochete growth. Serum samples were analyzed by ELISA to determine antibody concentrations. The modified 319-44 DMAb maintained in vitro biological activity comparable to the un-modified wild type antibody, and formulation-based delivery of DMAb resulted in long-term expression. This led to effective pre-exposure prophylaxis preventing transmission of spirochetes in 80% of mice in the murine model of tick-transmitted Lyme disease. These studies represent the first demonstration of employing DNA transfer as a rapid, novel delivery system for biologically relevant functional full-length HuMAbs in an in vivo animal model and provide support for such an approach for pre-exposure immunoprophylaxis to prevent Lyme disease

Structural and Molecular Analysis of a Protective Epitope of Lyme Disease Antigen OspA and Antibody Interactions

The murine monoclonal antibody LA-2 recognizes a clinically protective epitope on outer surface protein (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease in North America. Human antibody equivalence to LA-2 is the best serologic correlate of protective antibody responses following OspA vaccination. Understanding the structural and functional basis of the LA-2 protective epitope is important for developing OspA-based vaccines and discovering prophylactic antibodies against Lyme disease. Here, we present a detailed structure-based analysis of the LA-2/OspA interaction interface and identification of residues mediating antibody recognition. Mutations were introduced into both OspA and LA-2 based on computational predictions on the crystal structure of the complex, and experimentally tested for in-vitro binding and borreliacidal activity. We find that Y32 and H49 on the LA-2 light chain, N52 on the LA-2 heavy chain and residues A208, N228 and N251 on OspA were the key constituents of OspA/LA-2 interface. These results reveal specific residues that may be exploited to modulate recognition of the protective epitope of OspA and have implications for design of vaccines against Lyme disease

Identification and Characterization of Human Monoclonal Antibodies for Immunoprophylaxis Against Enterotoxigenic Escherichia coli Infection

Background. Enterotoxigenic Escherichia coli (ETEC) cause diarrheal illness in infants in the developing world and travelers to endemic countries including military personnel. ETEC infection of the host involves colonization of the small intestinal epithelium and toxin secretion leading to watery diarrhea. There is currently no vaccine licensed to prevent ETEC. CFA/I is one of the most common colonization factor antigens (CFAs). The CFA/I adhesin subunit, CfaE, is required for ETEC adhesion to host intestinal cells. Human antibodies against CfaE have potential to block colonization of ETEC and serve as an immunoprophylactic against ETEC-related diarrhea. Methods. Mice transgenic for human immunoglobulin genes were immunized with CfaE to generate a panel of human monoclonal IgG1 antibodies (HuMAbs). The most potent IgG1 identified in the in vitro functional assays were selected and isotype switched to secretory IgA (sIgA) and tested in animal colonization assays via oral administration. Results. Over 300 unique anti-CfaE IgG1 HuMabs were identified. The lead IgG1 anti-CfaE HuMAbs completely inhibited hemagglutination and blocked adhesion of ETEC to Caco-2 cells. Epitope mapping studies revealed that HuMAbs recognized epitopes in the N-terminal domain of CfaE near the putative receptor binding site. Oral administration of anti-CfaE antibodies in either IgG or secretory IgA isotypes inhibited intestinal colonization in mice challenged with ETEC. A two to four log decrease of colony forming units was observed as compared to irrelevant isotype controls. Conclusions. We identified fully human monoclonal antibodies against CfaE adhesion domain that can be potentially employed as an immunoprophylaxis to prevent ETEC-related diarrhea

Anti-CfaE nanobodies provide broad cross-protection against major pathogenic enterotoxigenic Escherichia coli strains, with implications for vaccine design

Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. Development of vaccines against ETEC is very challenging due to the vast heterogeneity of the ETEC strains. An effective vaccines would have to be multicomponent to provide coverage of over ten ETEC strains with genetic variabilities. There is currently no vaccine licensed to prevent ETEC. Nanobodies are successful new biologics in treating mucosal infectious disease as they recognize conserved epitopes on hypervariable pathogens. Cocktails consisting of multiple nanobodies could provide even broader epitope coverage at a lower cost compared to monoclonal antibodies. Identification of conserved epitopes by nanobodies can also assist reverse engineering of an effective vaccine against ETEC. By screening nanobodies from immunized llamas and a naive yeast display library against adhesins of colonization factors, we identified single nanobodies that show cross-protective potency against eleven major pathogenic ETEC strains in vitro. Oral administration of nanobodies led to a significant reduction of bacterial colonization in animals. Moreover, nanobody-IgA fusion showed extended inhibitory activity in mouse colonization compared to commercial hyperimmune bovine colostrum product used for prevention of ETEC-induced diarrhea. Structural analysis revealed that nanobodies recognized a highly-conserved epitope within the putative receptor binding region of ETEC adhesins. Our findings support further rational design of a pan-ETEC vaccine to elicit robust immune responses targeting this conserved epitope

A cross-reactive human IgA monoclonal antibody blocks SARS-CoV-2 spike-ACE2 interaction

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity, or as a therapeutic, has yet been developed to SARS-CoV-2. In this study, we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks ACE2 receptor binding, by overlapping the ACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in 293 cells expressing ACE2. When converted to secretory IgA, MAb326 also neutralizes authentic SARS-CoV-2 virus while the IgG isotype shows no neutralization. Our results suggest that SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine

Consensus Definition of Misophonia: A Delphi Study

Misophonia is a disorder of decreased tolerance to specific sounds or their associated stimuli that has been characterized using different language and methodologies. The absence of a common understanding or foundational definition of misophonia hinders progress in research to understand the disorder and develop effective treatments for individuals suffering from misophonia. From June 2020 through January 2021, the authors conducted a study to determine whether a committee of experts with diverse expertise related to misophonia could develop a consensus definition of misophonia. An expert committee used a modified Delphi method to evaluate candidate definitional statements that were identified through a systematic review of the published literature. Over four rounds of iterative voting, revision, and exclusion, the committee made decisions to include, exclude, or revise these statements in the definition based on the currently available scientific and clinical evidence. A definitional statement was included in the final definition only after reaching consensus at 80% or more of the committee agreeing with its premise and phrasing. The results of this rigorous consensus-building process were compiled into a final definition of misophonia that is presented here. This definition will serve as an important step to bring cohesion to the growing field of researchers and clinicians who seek to better understand and support individuals experiencing misophonia

Anti-angiogenic tyrosine kinase inhibitors: what is their mechanism of action?

Tyrosine kinases are important cellular signaling proteins that have a variety of biological activities including cell proliferation and migration. Multiple kinases are involved in angiogenesis, including receptor tyrosine kinases such as the vascular endothelial growth factor receptor. Inhibition of angiogenic tyrosine kinases has been developed as a systemic treatment strategy for cancer. Three anti-angiogenic tyrosine kinase inhibitors (TKIs), sunitinib, sorafenib and pazopanib, with differential binding capacities to angiogenic kinases were recently approved for treatment of patients with advanced cancer (renal cell cancer, gastro-intestinal stromal tumors, and hepatocellular cancer). Many other anti-angiogenic TKIs are being studied in phase I-III clinical trials. In addition to their beneficial anti-tumor activity, clinical resistance and toxicities have also been observed with these agents. In this manuscript, we will give an overview of the design and development of anti-angiogenic TKIs. We describe their molecular structure and classification, their mechanism of action, and their inhibitory activity against specific kinase signaling pathways. In addition, we provide insight into what extent selective targeting of angiogenic kinases by TKIs may contribute to the clinically observed anti-tumor activity, resistance, and toxicity. We feel that it is of crucial importance to increase our understanding of the clinical mechanism of action of anti-angiogenic TKIs in order to further optimize their clinical efficacy