Computationally restoring the potency of a clinical antibody against Omicron.

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3 and revealed how quickly viral escape can curtail effective options4,5. When the SARS-CoV-2 Omicron variant emerged in 2021, many antibody drug products lost potency, including Evusheld and its constituent, cilgavimab4-6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign and renew the efficacy of COV2-2130 against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and subsequent variants of concern, and provides protection in vivo against the strains tested: WA1/2020, BA.1.1 and BA.5. Deep mutational scanning of tens of thousands of pseudovirus variants reveals that 2130-1-0114-112 improves broad potency without increasing escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Our computational approach does not require experimental iterations or pre-existing binding data, thus enabling rapid response strategies to address escape variants or lessen escape vulnerabilities.

Computationally restoring the potency of a clinical antibody against SARS-CoV-2 Omicron subvariants.

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3, but also revealed how quickly viral escape can curtail effective options4,5. With the emergence of the SARS-CoV-2 Omicron variant in late 2021, many clinically used antibody drug products lost potency, including Evusheld™ and its constituent, cilgavimab4,6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies with a known clinical profile to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign COV2-2130 to rescue in vivo efficacy against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the contemporaneously dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and many variants of concern that subsequently emerged, and provides protection in vivo against the strains tested, WA1/2020, BA.1.1, and BA.5. Deep mutational scanning of tens of thousands pseudovirus variants reveals 2130-1-0114-112 improves broad potency without incurring additional escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Because our approach is computationally driven, not requiring experimental iterations or pre-existing binding data, it could enable rapid response strategies to address escape variants or pre-emptively mitigate escape vulnerabilities.

The CH···HC interaction in biphenyl is a delocalized, molecular-wide and entirely non-classical interaction: Results from FALDI analysis.

In this study we aim to determine the origin of the electron density describing a CH···HC interaction in planar and twisted conformers of biphenyl. In order to achieve this, the fragment, atomic, localized, delocalized, intra- and inter-atomic (FALDI) decomposition scheme was utilized to decompose the density in the inter-nuclear region between the ortho-hydrogens in both conformers. Importantly, the structural integrity, hence also topological properties, were fully preserved as no 'artificial' partitioning of molecules was implemented. FALDI-based qualitative and quantitative analysis revealed that the majority of electron density arises from two, non-classical and non-local effects: strong overlap of ortho CH σ-bonds, and long-range electron delocalization between the phenyl rings and ortho carbons and hydrogens. These effects resulted in a delocalized electron channel, that is, a density bridge or a bond path in a QTAIM terminology, linking the H-atoms in the planar conformer. The same effects and phenomena are present in both conformers of biphenyl. We show that the CH···HC interaction is a molecular-wide event due to large and long-range electron delocalization, and caution against approaches that investigate CH···HC interactions without fully taking into account the remainder of the molecule.

The Wilms Tumor Gene wt1a Contributes to Blood-Cerebrospinal Fluid Barrier Function in Zebrafish.

The Wilms tumor suppressor gene Wt1 encodes a zinc finger transcription factor, which is highly conserved among vertebrates. It is a key regulator of urogenital development and homeostasis but also plays a role in other organs including the spleen and the heart. More recently additional functions for Wt1 in the mammalian central nervous system have been described. In contrast to mammals, bony fish possess two paralogous Wt1 genes, namely wt1a and wt1b. By performing detailed in situ hybridization analyses during zebrafish development, we discovered new expression domains for wt1a in the dorsal hindbrain, the caudal medulla and the spinal cord. Marker analysis identified wt1a expressing cells of the dorsal hindbrain as ependymal cells of the choroid plexus in the myelencephalic ventricle. The choroid plexus acts as a blood-cerebrospinal fluid barrier and thus is crucial for brain homeostasis. By employing wt1a mutant larvae and a dye accumulation assay with fluorescent tracers we demonstrate that Wt1a is required for proper choroid plexus formation and function. Thus, Wt1a contributes to the barrier properties of the choroid plexus in zebrafish, revealing an unexpected role for Wt1 in the zebrafish brain.

Molecular Orbitals Support Energy-Stabilizing "Bonding" Nature of Bader's Bond Paths.

Our MO-based findings proved a bonding nature of each density bridge (DB, or a bond path with an associated critical point, CP) on a Bader molecular graph. A DB pinpoints universal physical and net energy-lowering processes that might, but do not have to, lead to a chemical bond formation. Physical processes leading to electron density (ED) concentration in internuclear regions of three distinctively different homopolar H,H atom-pairs as well as classical C-C and C-H covalent bonds were found to be exactly the same. Notably, properties of individual MOs are internuclear-region specific as they (i) concentrate, deplete, or do not contribute to ED at a CP and (ii) delocalize electron-pairs through either in- (positive) or out-of-phase (negative) interference. Importantly, dominance of a net ED concentration and positive e--pairs delocalization made by a number of σ-bonding MOs is a common feature at a CP. This feature was found for the covalently bonded atoms as well as homopolar H,H atom-pairs investigated. The latter refer to a DB-free H,H atom-pair of the bay in the twisted biphenyl (Bph) and DB-linked H,H atom-pairs (i) in cubic Li4H4, where each H atom is involved in three highly repulsive interactions (over +80 kcal/mol), and (ii) in a weak attractive interaction when sterically clashing in the planar Bph.

Wilms Tumor 1b Expression Defines a Pro-regenerative Macrophage Subtype and Is Required for Organ Regeneration in the Zebrafish.

Organ regeneration is preceded by the recruitment of innate immune cells, which play an active role during repair and regrowth. Here, we studied macrophage subtypes during organ regeneration in the zebrafish, an animal model with a high regenerative capacity. We identified a macrophage subpopulation expressing Wilms tumor 1b (wt1b), which accumulates within regenerating tissues. This wt1b+ macrophage population exhibited an overall pro-regenerative gene expression profile and different migratory behavior compared to the remainder of the macrophages. Functional studies showed that wt1b regulates macrophage migration and retention at the injury area. Furthermore, wt1b-null mutant zebrafish presented signs of impaired macrophage differentiation, delayed fin growth upon caudal fin amputation, and reduced cardiomyocyte proliferation following cardiac injury that correlated with altered macrophage recruitment to the regenerating areas. We describe a pro-regenerative macrophage subtype in the zebrafish and a role for wt1b in organ regeneration.

Kidney regeneration in fish.

Age-related diseases, such as kidney diseases, are becoming more prevalent in aging societies. Currently, patients with reduced kidney function require dialysis or organ transplants. Those who suffer from kidney disease would benefit from regenerative therapies. Thus, one of the ultimate goals of regeneration research is to enhance an individual's capacity of self-repairing damaged tissue; something that fish models can contribute towards. Kidney structures are conserved among vertebrates highlighting the opportunities for fish to act as human disease models. Here, different species can offer respective advantages. An understanding of the different modes of regeneration can help to visualize the differences in mammalian and fish regenerative capacity. The remarkable regenerative capacity of fish is well known, but kidney regeneration is an understudied area. The kinetics of kidney regeneration allows one to investigate early damage responses, as well as the initiation and completion of repair. Age-related reductions in regeneration are an additional societal problem; again an area where fish models can be of help. Age-matched experiments between varied vertebrate species will help us to learn from those that do or do not exhibit age-related phenotypes. The goal of such experiments is not only to outline important age-related factors and pathways, but, in addition, to see if age-related decreases in regenerative capacity can be reduced. Widening our knowledge of this very complex process will help to address many of the unanswered questions in the field.

Function and Regulation of the Wilms' Tumor Suppressor 1 (WT1) Gene in Fish.

The Wilms' tumor suppressor gene Wt1 is highly conserved among vertebrates. In contrast to mammals, most fish species possess two wt1 paralogs that have been named wt1a and wt1b. Concerning wt1 in fish, most work so far has been done using zebrafish, focusing on the embryonic kidney, the pronephros. In this chapter we will describe the structure and development of the pronephros as well as the role that the wt1 genes play in the embryonic zebrafish kidney. We also discuss Wt1 target genes and describe the potential function of the Wt1 proteins in the adult kidney. Finally we will summarize data on the role of Wt1 outside of the kidney.

Fluorescence-Activated Cell Sorting (FACS) Protocol for Podocyte Isolation in Adult Zebrafish.

Zebrafish is becoming a very important model for studying human diseases. The conserved structure of the nephrons in the kidney allows the user to answer questions relating to study human kidney disorders. Wt1a-expressing podocytes are the most important cells within the glomeruli of adult zebrafish. In order to understand the molecular characteristics of these cells, within damage models, we have established a method for isolating them.

A Conceptual Architecture for National Biosurveillance: Moving Beyond Situational Awareness to Enable Digital Detection of Emerging Threats.

Despite numerous calls for improvement, the US biosurveillance enterprise remains a patchwork of uncoordinated systems that fail to take advantage of the rapid progress in information processing, communication, and analytics made in the past decade. By synthesizing components from the extensive biosurveillance literature, we propose a conceptual framework for a national biosurveillance architecture and provide suggestions for implementation. The framework differs from the current federal biosurveillance development pathway in that it is not focused on systems useful for "situational awareness" but is instead focused on the long-term goal of having true warning capabilities. Therefore, a guiding design objective is the ability to digitally detect emerging threats that span jurisdictional boundaries, because attempting to solve the most challenging biosurveillance problem first provides the strongest foundation to meet simpler surveillance objectives. Core components of the vision are: (1) a whole-of-government approach to support currently disparate federal surveillance efforts that have a common data need, including those for food safety, vaccine and medical product safety, and infectious disease surveillance; (2) an information architecture that enables secure national access to electronic health records, yet does not require that data be sent to a centralized location for surveillance analysis; (3) an inference architecture that leverages advances in "big data" analytics and learning inference engines-a significant departure from the statistical process control paradigm that underpins nearly all current syndromic surveillance systems; and (4) an organizational architecture with a governance model aimed at establishing national biosurveillance as a critical part of the US national infrastructure. Although it will take many years to implement, and a national campaign of education and debate to acquire public buy-in for such a comprehensive system, the potential benefits warrant increased consideration by the US government.

Molecular specification of germ layers in vertebrate embryos.

In order to generate the tissues and organs of a multicellular organism, different cell types have to be generated during embryonic development. The first step in this process of cellular diversification is the formation of the three germ layers: ectoderm, endoderm and mesoderm. The ectoderm gives rise to the nervous system, epidermis and various neural crest-derived tissues, the endoderm goes on to form the gastrointestinal, respiratory and urinary systems as well as many endocrine glands, and the mesoderm will form the notochord, axial skeleton, cartilage, connective tissue, trunk muscles, kidneys and blood. Classic experiments in amphibian embryos revealed the tissue interactions involved in germ layer formation and provided the groundwork for the identification of secreted and intracellular factors involved in this process. We will begin this review by summarising the key findings of those studies. We will then evaluate them in the light of more recent genetic studies that helped clarify which of the previously identified factors are required for germ layer formation in vivo, and to what extent the mechanisms identified in amphibians are conserved across other vertebrate species. Collectively, these studies have started to reveal the gene regulatory network (GRN) underlying vertebrate germ layer specification and we will conclude our review by providing examples how our understanding of this GRN can be employed to differentiate stem cells in a targeted fashion for therapeutic purposes.

Coco regulates dorsoventral specification of germ layers via inhibition of TGFß signalling.

One of the earliest steps in embryonic development is the specification of the germ layers, the subdivision of the blastula embryo into endoderm, mesoderm and ectoderm. Maternally expressed members of the Transforming Growth Factor ß (TGFß) family influence all three germ layers; the ligands are required to induce endoderm and mesoderm, whereas inhibitors are required for formation of the ectoderm. Here, we demonstrate a vital role for maternal Coco, a secreted antagonist of TGFß signalling, in this process. We show that Coco is required to prevent Activin and Nodal signals in the dorsal marginal side of the embryo from invading the prospective ectoderm, thereby restricting endoderm- and mesoderm-inducing signals to the vegetal and marginal zones of the pre-gastrula Xenopus laevis embryo.

Neuronatin promotes neural lineage in ESCs via Ca(2+) signaling.

Neural induction is the first step in the formation of the vertebrate central nervous system. The emerging consensus of the mechanisms underlying neural induction is the combined influences from inhibiting bone morphogenetic protein (BMP) signaling and activating fibroblast growth factor (FGF)/Erk signaling, which act extrinsically via either autocrine or paracrine fashions. However, do intrinsic forces (cues) exist and do they play decisive roles in neural induction? These questions remain to be answered. Here, we have identified a novel neural initiator, neuronatin (Nnat), which acts as an intrinsic factor to promote neural fate in mammals and Xenopus. ESCs lacking this intrinsic factor fail to undergo neural induction despite the inhibition of the BMP pathway. We show that Nnat initiates neural induction in ESCs through increasing intracellular Ca(2+) ([Ca(2+) ](i)) by antagonizing Ca(2+) -ATPase isoform 2 (sarco/endoplasmic reticulum Ca(2+) -ATPase isoform 2) in the endoplasmic reticulum, which in turn increases the phosphorylation of Erk1/2 and inhibits the BMP4 pathway and leads to neural induction in conjunction with FGF/Erk pathway.

A simulation model of intraherd transmission of foot and mouth disease with reference to disease spread before and after clinical diagnosis.

Intraherd transmission of foot and mouth disease virus (FMDV) was examined using a simulation model for a hypothetical 1,000-cow dairy, assuming clinical diagnosis was made when at least 1% (10 cows) or 5% (50 cows) had clinical signs of FMD, I index case cow, and transition state distributions for the latent, subclinically infectious, and clinically infectious periods of FMD calculated from published data. Estimates assumed for the number of animal-to-animal contacts (k) adequate for transmission ranged from 0.6 to 9.0 per hour (13.7-216.0 per day). A total of 40,000 iterations (5,000 for each scenario, assessing 4 adequate contact rates and 2 detection criteria) were run. The model predicted that FMD would not be diagnosed in the herd until 10.0-13.5 days after the index case cow had become infected, at which time between 65% and 97% of the cows (646-967 cows) to nearly 100% (978-996 cows) would already have become infected with the virus, if the number of cows showing clinical signs of FMD at the time of diagnosis were 10 or 50, respectively. At the time of diagnosis, the simulated number of infectious cattle varied substantially from 82-472 to 476-537 cows, depending on adequate contact rate and whether the diagnosis was made when 10 or 50 animals were showing clinical signs, respectively. The simulated number of infectious cows increased rapidly during the first few days after diagnosis. In the scenario where at least 10 cows showing clinical signs was necessary before a clinical diagnosis was made, each day after diagnosis, the number of infectious animals increased by nearly 100 to more than 200 cases per day up to day 5, assuming 0.57-9.0 animal-to-animal contacts per hour, respectively. Results obtained when it was assumed that at least 50 clinical cases were present at the time of diagnosis showed smaller relative increases because nearly one-half of the herd was projected to be infected at the time of diagnosis. From these results, it is clear that once an individual in a herd becomes infected with FMDV, herd infectivity is not static, rather it accelerates as would be expected as long as there are sufficient susceptible animals to sustain the increasing transmission rate, after which time the rate at which new infections occurs will diminish. Results indicate that biosecurity strategies aimed at minimizing both intraherd and interherd contact will be critical in minimizing the spread of FMD before the initial diagnosis is made. In addition, simulations suggest that very early clinical diagnosis of FMD and effective isolation or depopulation and disposal will be critical in limiting the number of infectious animals capable of transmitting the virus to other herds and thus in timely control of an epidemic. Early diagnosis will rely on early virus detection from animals in the preclinical phase of infection, rather than waiting for clinical signs to manifest in sufficient numbers to be noticed and to warrant investigation.

Benefit-cost analysis of vaccination and preemptive slaughter as a means of eradicating foot-and-mouth disease.

OBJECTIVE: To assess relative costs and benefits of vaccination and preemptive herd slaughter to control transmission of foot-and-mouth disease (FMD) virus (FMDV). SAMPLE POPULATION: 2,238 herds and 5 sale yards located in Fresno, Kings, and Tulare counties of California. PROCEDURE: Direct costs associated with indemnity, slaughter, cleaning and disinfecting livestock premises, and vaccination were compared for various eradication strategies. Additional cost, total program cost, net benefit, and benefit-cost value (B/C) for each supplemental strategy were estimated, based in part on results of published model simulations for FMD. Sensitivity analyses were conducted. RESULTS: Mean herd indemnity payments were estimated to be dollars 2.6 million and dollars 110,359 for dairy and nondairy herds, respectively. Cost to clean and disinfect livestock premises ranged from dollars 18,062 to dollars 60,205. Mean vaccination cost was dollars 2,960/herd. Total eradication cost ranged from dollars 61 million to dollars 551 million. All supplemental strategies involving use of vaccination were economically efficient (B/C range, 5.0 to 10.1) and feasible, whereas supplemental strategies involving use of slaughter programs were not economically efficient (B-C, 0.05 to 0.8) or feasible. CONCLUSIONS AND CLINICAL RELEVANCE: Vaccination with a highly efficacious vaccine may be a cost-effective strategy for control of FMD if vaccinated animals are not subsequently slaughtered and there is no future adverse economic impact, such as trade restrictions. Although less preferable than the baseline eradication program, selective slaughter of highest-risk herds was preferable to other preemptive slaughter strategies. However, indirect costs can be expected to contribute substantially more than direct costs to the total cost of eradication programs.

Description of an epidemic simulation model for use in evaluating strategies to control an outbreak of foot-and-mouth disease.

OBJECTIVE: To develop a spatial epidemic model to simulate intraherd and interherd transmission of foot-and-mouth disease (FMD) virus. SAMPLE POPULATION: 2,238 herds, representing beef, dairy, swine, goats, and sheep, and 5 sale yards located in Fresno, Kings, and Tulare counties of California. PROCEDURE: Using Monte-Carlo simulations, a spatial stochastic epidemic simulation model was developed to identify new herds that would acquire FMD following random selection of an index herd and to assess progression of an epidemic after implementation of mandatory control strategies. RESULTS: The model included species-specific transition periods for FMD infection, locations of herds, rates of direct and indirect contacts among herds, and probability distributions derived from expert opinions on probabilities of transmission by direct and indirect contact, as well as reduction in contact following implementation of restrictions on movements in designated infected areas and surveillance zones. Models of supplemental control programs included slaughter of all animals within a specified distance of infected herds, slaughter of only high-risk animals identified by use of a model simulation, and vaccination of all animals within a 5- to 50-km radius of infected herds. CONCLUSIONS AND CLINICAL RELEVANCE: The FMD model represents a tool for use in planning biosecurity and emergency-response programs and in comparing potential benefits of various strategies for control and eradication of FMD appropriate for specific populations.

Results of epidemic simulation modeling to evaluate strategies to control an outbreak of foot-and-mouth disease.

OBJECTIVE: To assess estimated effectiveness of control and eradication procedures for foot-and-mouth disease (FMD) in a region of California. SAMPLE POPULATION: 2,238 herds and 5 sale yards in Fresno, Kings, andTulare counties of California. PROCEDURE: A spatial stochastic model was used to simulate hypothetical epidemics of FMD for specified control scenarios that included a baseline eradication strategy mandated by USDA and supplemental control strategies of slaughter or vaccination of all animals within a specified distance of infected herds, slaughter of only high-risk animals identified by use of a model simulation, and expansion of infected and surveillance zones. RESULTS: Median number of herds affected varied from 1 to 385 (17% of all herds), depending on type of index herd and delay in diagnosis of FMD. Percentage of herds infected decreased from that of the baseline eradication strategy by expanding the designated infected area from 10 to 20 km (48%), vaccinating within a 50-km radius of an infected herd (41%), slaughtering the 10 highest-risk herds for each infected herd (39%), and slaughtering all animals within 5 km of an infected herd (24%). CONCLUSIONS AND CLINICAL RELEVANCE: Results for the model provided a means of assessing the relative merits of potential strategies for control and eradication of FMD should it enter the US livestock population. For the study region, preemptive slaughter of highest-risk herds and vaccination of all animals within a specified distance of an infected herd consistently decreased size and duration of an epidemic, compared with the baseline eradication strategy.