A cell-based Papain-like Protease (PLpro) activity assay for rapid detection of active SARS-CoV-2 infections and antivirals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are a continuous threat to human life. An urgent need remains for simple and fast tests that reliably detect active infections with SARS-CoV-2 and its variants in the early stage of infection. Here we introduce a simple and rapid activity-based diagnostic (ABDx) test that identifies SARS-CoV-2 infections by measuring the activity of a viral enzyme, Papain-Like protease (PLpro).

Characterization of two affinity matured Anti-Yersinia pestis F1 human antibodies with medical countermeasure potential.

Yersinia pestis, the causative agent of plague and a biological threat agent, presents an urgent need for novel medical countermeasures due to documented cases of naturally acquired antibiotic resistance and potential person-to-person spread during a pneumonic infection. Immunotherapy has been proposed as a way to circumvent current and future antibiotic resistance. Here, we describe the development and characterization of two affinity matured human antibodies (αF1Ig AM2 and αF1Ig AM8) that promote survival of mice after exposure to aerosolized Y.

Multidimensional mass profiles increase confidence in bacterial identification when using low-resolution mass spectrometers.

Field-forward analytical technologies, such as portable mass spectrometry (MS), enable essential capabilities for real-time monitoring and point-of-care diagnostic applications. Significant and recent investments improving the features of miniaturized mass spectrometers enable various new applications outside of small molecule detection. Most notably, the addition of tandem mass spectrometry scans (MS/MS) allows the instrument to isolate and fragment ions and increase the analytical specificity by measuring unique chemical signatures for ions of interest.

Direct selection of functional fluorescent-protein antibody fusions by yeast display.

Antibodies are important reagents for research, diagnostics, and therapeutics. Many examples of chimeric proteins combining the specific target recognition of antibodies with complementing functionalities such as fluorescence, toxicity or enzymatic activity have been described. However, antibodies selected solely on the basis of their binding specificities are not necessarily ideal candidates for the construction of chimeras.

Healthy humans can be a source of antibodies countering COVID-19.

Here, we describe the isolation of 18 unique anti SARS-CoV-2 human single-chain antibodies from an antibody library derived from healthy donors. The selection used a combination of phage and yeast display technologies and included counter-selection strategies meant to direct the selection of the receptor-binding motif (RBM) of SARS-CoV-2 spike protein's receptor binding domain (RBD2). Selected antibodies were characterized in various formats including IgG, using flow cytometry, ELISA, high throughput SPR, and fluorescence microscopy.

Warning Signs of Potential Black Swan Outbreaks in Infectious Disease.

Black swan events in infectious disease describe rare but devastatingly large outbreaks. While experts are skeptical that such events are predictable, it might be possible to identify the warning signs of a black swan event. Specifically, the initiation of an outbreak, key differentiating features could serve as alerts.

Nonclassical autophagy activation pathways are essential for production of infectious Influenza A virus in vitro.

Autophagy is a critical mechanism deployed by eukaryotic cells in response to stress, including viral infection, to boost the innate antimicrobial responses. However, an increasing number of pathogens hijack the autophagic machinery to facilitate their own replication. Influenza A virus (IAV), responsible for several global pandemics, has an intricate dependence on autophagy for successful replication in mammalian cells.

Engineered pH-Sensitive Protein G/IgG Interaction.

While natural protein-protein interactions have evolved to be induced by complex stimuli, rational design of interactions that can be switched-on-demand still remain challenging in the protein design world. Here, we demonstrate that a computationally redesigned natural interface for improved binding affinity could further be mutated to adopt a pH switchable interaction. The redesigned interface of Protein G/human IgG Fc domain (referred to as PrG/hIgG), when incorporated with histidine and glutamic acid on PrG (PrG-EHHE), showed a switch in binding affinity by 50-fold when the pH was altered from mild acidic to mild basic.

Construction, characterization and crystal structure of a fluorescent single-chain Fv chimera.

In vitro display technologies based on phage and yeast have a successful history of selecting single-chain variable fragment (scFv) antibodies against various targets. However, single-chain antibodies are often unstable and poorly expressed in Escherichia coli. Here, we explore the feasibility of converting scFv antibodies to an intrinsically fluorescent format by inserting the monomeric, stable fluorescent protein named thermal green, between the light- and heavy-chain variable regions.

Improving Detection of Disease Re-emergence Using a Web-Based Tool (RED Alert): Design and Case Analysis Study.

Background: Currently, the identification of infectious disease re-emergence is performed without describing specific quantitative criteria that can be used to identify re-emergence events consistently. This practice may lead to ineffective mitigation. In addition, identification of factors contributing to local disease re-emergence and assessment of global disease re-emergence require access to data about disease incidence and a large number of factors at the local level for the entire world.

Development of Anti- Human Antibodies with Features Required for Diagnostic and Therapeutic Applications.

Background: is a category A infective agent that causes bubonic, septicemic, and pneumonic plague. Notably, the acquisition of antimicrobial or multidrug resistance through natural or purposed means qualifies as a potential biothreat agent. Therefore, high-quality antibodies designed for accurate and sensitive diagnostics, and therapeutics potentiating or replacing traditional antibiotics are of utmost need for national security and public health preparedness.

Selection and verification of antibodies against the cytoplasmic domain of M2 of influenza, a transmembrane protein.

Interactions between the cytoplasmic domains of viral transmembrane proteins and host machinery often determine the outcome of viral infection. The M2 protein of influenza A has been identified as a key player in autophagy-mediated viral replication. Here, we describe the engineering and validation of an antibody specific for the cytoplasmic domain of the M2 protein.

A Gene Cluster That Encodes Histone Deacetylase Inhibitors Contributes to Bacterial Persistence and Antibiotic Tolerance in Burkholderia thailandensis.

Persister cells are genetically identical variants in a bacterial population that have phenotypically modified their physiology to survive environmental stress. In bacterial pathogens, persisters are able to survive antibiotic treatment and reinfect patients in a frustrating cycle of chronic infection. To better define core persistence mechanisms for therapeutics development, we performed transcriptomics analyses of populations enriched for persisters via three methods: flow sorting for low proton motive force, meropenem treatment, and culture aging.

Development of a Supervised Learning Algorithm for Detection of Potential Disease Reemergence: A Proof of Concept.

Infectious disease reemergence is an important yet ambiguous concept that lacks a quantitative definition. Currently, reemergence is identified without specific criteria describing what constitutes a reemergent event. This practice affects reproducible assessments of high-consequence public health events and disease response prioritization.

Selection and characterization of FcεRI phospho-ITAM specific antibodies.

Post-translational modifications, such as the phosphorylation of tyrosines, are often the initiation step for intracellular signaling cascades. Pan-reactive antibodies against modified amino acids (e.g.

Analytics for Investigation of Disease Outbreaks: Web-Based Analytics Facilitating Situational Awareness in Unfolding Disease Outbreaks.

Background: Information from historical infectious disease outbreaks provides real-world data about outbreaks and their impacts on affected populations. These data can be used to develop a picture of an unfolding outbreak in its early stages, when incoming information is sparse and isolated, to identify effective control measures and guide their implementation.

Objective: This study aimed to develop a publicly accessible Web-based visual analytic called Analytics for the Investigation of Disease Outbreaks (AIDO) that uses historical disease outbreak information for decision support and situational awareness of an unfolding outbreak.

Epidemiological Data Challenges: Planning for a More Robust Future Through Data Standards.

Accessible epidemiological data are of great value for emergency preparedness and response, understanding disease progression through a population, and building statistical and mechanistic disease models that enable forecasting. The status quo, however, renders acquiring and using such data difficult in practice. In many cases, a primary way of obtaining epidemiological data is through the internet, but the methods by which the data are presented to the public often differ drastically among institutions.

An extensible framework and database of infectious disease for biosurveillance.

Biosurveillance, a relatively young field, has recently increased in importance because of increasing emphasis on global health. Databases and tools describing particular subsets of disease are becoming increasingly common in the field. Here, we present an infectious disease database that includes diseases of biosurveillance relevance and an extensible framework for the easy expansion of the database.

Novel Use of Flu Surveillance Data: Evaluating Potential of Sentinel Populations for Early Detection of Influenza Outbreaks.

Influenza causes significant morbidity and mortality each year, with 2-8% of weekly outpatient visits around the United States for influenza-like-illness (ILI) during the peak of the season. Effective use of existing flu surveillance data allows officials to understand and predict current flu outbreaks and can contribute to reductions in influenza morbidity and mortality. Previous work used the 2009-2010 influenza season to investigate the possibility of using existing military and civilian surveillance systems to improve early detection of flu outbreaks.

Using phage display selected antibodies to dissect microbiomes for complete de novo genome sequencing of low abundance microbes.

Background: Single cell genomics has revolutionized microbial sequencing, but complete coverage of genomes in complex microbiomes is imperfect due to enormous variation in organismal abundance and amplification bias. Empirical methods that complement rapidly improving bioinformatic tools will improve characterization of microbiomes and facilitate better genome coverage for low abundance microbes.

Methods: We describe a new approach to sequencing individual species from microbiomes that combines antibody phage display against intact bacteria with fluorescence activated cell sorting (FACS).

Filtering "genic" open reading frames from genomic DNA samples for advanced annotation.

Background: In order to carry out experimental gene annotation, DNA encoding open reading frames (ORFs) derived from real genes (termed "genic") in the correct frame is required. When genes are correctly assigned, isolation of genic DNA for functional annotation can be carried out by PCR. However, not all genes are correctly assigned, and even when correctly assigned, gene products are often incorrectly folded when expressed in heterologous hosts.

A comprehensive analysis of filamentous phage display vectors for cytoplasmic proteins: an analysis with different fluorescent proteins.

Filamentous phage display has been extensively used to select proteins with binding properties of specific interest. Although many different display platforms using filamentous phage have been described, no comprehensive comparison of their abilities to display similar proteins has been conducted. This is particularly important for the display of cytoplasmic proteins, which are often poorly displayed with standard filamentous phage vectors.

A humanized anti-M2 scFv shows protective in vitro activity against influenza.

M2 is one of the most conserved influenza proteins, and has been widely prospected as a potential universal vaccine target, with protection predominantly mediated by antibodies. In this paper we describe the creation of a humanized single chain Fv from 14C2, a potent monoclonal antibody against M2. We show that the humanized scFv demonstrates similar activity to the parental mAb: it is able to recognize M2 in its native context on cell surfaces and is able to show protective in vitro activity against influenza, and so represents a potential lead antibody candidate for universal prophylactic or therapeutic intervention in influenza.

Fluorescence linked immunosorbant assays using microtiter plates.

Fluorescence methods are widely used in the detection of antibodies and other binding events. However, as a general screening and detection tool in microtiter plates, enzyme linked immunosorbant (ELISA) methods predominate. In this paper we explore all parameters for effective use of fluorescence as a plate based detection method, including which microtiter plates can be used, the most effective means of immobilization, and the use of different fluorescent dyes or fluorescent proteins.

Using T7 phage display to select GFP-based binders.

Filamentous phage do not display cytoplasmic proteins very effectively. As T7 is a cytoplasmic phage, released by cell lysis, it has been prospected as being more efficient for the display of such proteins. Here we investigate this proposition, using a family of GFP-based cytoplasmic proteins that are poorly expressed by traditional phage display.