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At least 1,369 records · Page 76
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Magnetic field sensor based on cascaded microfiber coupler with magnetic fluid

A kind of magnetic field sensor based on cascaded microfiber coupler with magnetic fluid is proposed and experimentally demonstrated. The magnetic fluid is utilized as the cladding of the fused regions of the cascaded microfiber coupler. As the interference valley wavelength of the sensing structure is sensitive to the ambient variation, considering the magnetic-field-dependent refractive index of magnetic fluid, the proposed structure is employed for magnetic field sensing. The effective coupling length for each coupling region of the as-fabricated cascaded microfiber coupler is 6031 μm. The achieved sensitivity is 125 pm/Oe, which is about three times larger than that of the previously similar structure based on the single microfiber coupler. Experimental results indicate that the sensing sensitivity can be easily improved by increasing the effective coupling length or cascading more microfiber couplers. The proposed magnetic field sensor is attractive due to its low cost, immunity to electromagnetic interference, as well as high sensitivity, which also has the potentials in other tunable all-fiber photonic devices, such as filter.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2016

Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies

Significance Conjugate vaccines have proven to be an effective and safe strategy for reducing the incidence of disease caused by bacterial pathogens. However, the manufacture of these vaccines is technically demanding, inefficient, and expensive, thereby limiting their widespread use. Here, we describe an alternative methodology for generating glycoconjugate vaccines whereby recombinant polysaccharide biosynthesis is coordinated with vesicle formation in nonpathogenic Escherichia coli , resulting in glycosylated outer membrane vesicles (glycOMVs) that can effectively deliver pathogen-mimetic glycotopes to the immune system. An attractive feature of our approach is the fact that different plasmid-encoded polysaccharide biosynthetic pathways can be readily transformed into E. coli , enabling a “plug-and-play” platform for the on-demand creation of glycOMV vaccine candidates that carry heterologous glycotopes from numerous pathogenic bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Molecular characterization of a fungal gasdermin-like protein

Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified regulator of cell death-1 ( rcd-1 ), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassa . rcd-1 alleles are highly polymorphic and fall into two haplogroups in N. crassa populations. Coexpression of alleles from the two haplogroups, rcd-1–1 and rcd-1–2 , is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of rcd-1 -dependent cell death. Based on in silico analyses, we found that RCD-1 is a remote homolog of the N-terminal pore-forming domain of gasdermin, the executioner protein of a highly inflammatory cell death reaction termed pyroptosis, which plays a key role in mammalian innate immunity. We show that RCD-1 localizes to the cell periphery and that cellular localization of RCD-1 was correlated with conserved positively charged residues on predicted amphipathic α-helices, as shown for murine gasdermin-D. Similar to gasdermin, RCD-1 binds acidic phospholipids in vitro, notably, cardiolipin and phosphatidylserine, and interacts with liposomes containing such lipids. The RCD-1 incompatibility system was reconstituted in human 293T cells, where coexpression of incompatible rcd-1–1/rcd-1–2 alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin and rcd-1 . This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Regulatory T Cell Modulation by CBP/EP300 Bromodomain Inhibition

Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors. Here we describe the discovery and characterization of potent and selective small molecule inhibitors for the bromodomains of CREBBP/EP300 that engage their target in cellular assays. We use these tools to demonstrate a critical role for CREBBP/EP300 bromodomains in regulatory T cell biology. Because regulatory T cell recruitment to tumors is a major mechanism of immune evasion by cancer cells, our data highlight the importance of CREBBP/EP300 bromodomain inhibition as a novel, small molecule-based approach for cancer immunotherapy.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2019

Conformational flexibility and inhibitor binding to unphosphorylated interleukin-1 receptor–associated kinase 4 (IRAK4)

Interleukin-1 receptor–associated kinase 4 (IRAK4) is a key factor in innate immune and inflammatory responses, performing a critical role in signal transduction downstream of Toll-like receptors and interleukin-1 (IL-1) receptors. Upon ligand binding and via its N-terminal death domain, IRAK4 is recruited to an oligomeric receptor that is proximal to the Myddosome signaling complex, inducing IRAK4 kinase domain dimerization, autophosphorylation, and activation. To date, all known IRAK4 structures are in the active conformation, precluding a good understanding of IRAK4's conformational dynamics. To address this issue, here we first solved three crystal structures of the IRAK4 kinase domain (at ≤2.6 Å resolution), in its unphosphorylated, inactive state bound to either the ATP analog AMP-PNP or to one of the two small-molecule inhibitors JH-I-25 and JH-I-17. The structures disclosed that although the structure in complex with AMP-PNP is in an “αC-out” inactive conformation, those in complex with type I inhibitors assume an active “Asp–Phe–Gly (DFG)-in” and “αC-in” conformation. The ability of unphosphorylated IRAK4 to take on variable conformations prompted us to screen for small-molecule inhibitors that bind preferentially to unphosphorylated IRAK4, leading to the identification of ponatinib and HG-12-6. Solving the structures of unphosphorylated IRAK4 in complex with these two inhibitors, we found that they both bind as type II inhibitors with IRAK4 in a “DFG-out” conformation. Collectively, these structures reveal conformational flexibility of unphosphorylated IRAK4 and provide unexpected insights into the potential use of small molecules to modulate IRAK4 activity in cancer, autoimmunity, and inflammation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2017

Structure of a novel antibacterial toxin that exploits elongation factor Tu to cleave specific transfer RNAs

Contact-dependent growth inhibition (CDI) is a mechanism of inter-cellular competition in which Gram-negative bacteria exchange polymorphic toxins using type V secretion systems. Here, we present structures of the CDI toxin from Escherichia coli NC101 in ternary complex with its cognate immunity protein and elongation factor Tu (EF-Tu). The toxin binds exclusively to domain 2 of EF-Tu, partially overlapping the site that interacts with the 3'-end of aminoacyl-tRNA (aa-tRNA). The toxin exerts a unique ribonuclease activity that cleaves the single-stranded 3'-end from tRNAs that contain guanine discriminator nucleotides. EF-Tu is required to support this tRNase activity in vitro , suggesting the toxin specifically cleaves substrate in the context of GTP·EF-Tu·aa-tRNA complexes. However, superimposition of the toxin domain onto previously solved GTP·EF-Tu·aa-tRNA structures reveals potential steric clashes with both aa-tRNA and the switch I region of EF-Tu. Further, the toxin induces conformational changes in EF-Tu, displacing a β-hairpin loop that forms a critical salt-bridge contact with the 3'-terminal adenylate of aa-tRNA. Altogether, these observations suggest that the toxin remodels GTP·EF-Tu·aa-tRNA complexes to free the 3'-end of aa-tRNA for entry into the nuclease active site.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Autocrine and paracrine interferon signalling as ‘ring vaccination’ and ‘contact tracing’ strategies to suppress virus infection in a host

The innate immune response, particularly the interferon response, represents a first line of defence against viral infections. The interferon molecules produced from infected cells act through autocrine and paracrine signalling to turn host cells into an antiviral state. Although the molecular mechanisms of IFN signalling have been well characterized, how the interferon response collectively contribute to the regulation of host cells to stop or suppress viral infection during early infection remain unclear. Here, we use mathematical models to delineate the roles of the autocrine and the paracrine signalling, and show that their impacts on viral spread are dependent on how infection proceeds. In particular, we found that when infection is well-mixed, the paracrine signalling is not as effective; by contrast, when infection spreads in a spatial manner, a likely scenario during initial infection in tissue, the paracrine signalling can impede the spread of infection by decreasing the number of susceptible cells close to the site of infection. Furthermore, we argue that the interferon response can be seen as a parallel to population-level epidemic prevention strategies such as ‘contact tracing’ or ‘ring vaccination’. Thus, our results here may have implications for the outbreak control at the population scale more broadly.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Constitutive and inducible oleoresin defenses share genetic architectures and mechanisms in Pinus taeda

The oleoresin defense system of loblolly pine (Pinus taeda) protects trees from insects and pathogens and is an important source of renewable biofuels and chemicals, but the genetic basis of oleoresin production is poorly understood. We characterized the genetic architecture of oleoresin flow, resin canal number, stem wood terpene content, and monoterpene composition in two clonal populations of P. taeda. We used quantitative genetic analyses, genome-wide association studies (GWASs), multiplex network learning, and gene expression profiling to elucidate shared gene networks underlying defense traits and to identify high-quality candidates for breeding and engineering loblolly pine. Genetic analyses revealed polygenic inheritance and trait-to-trait correlations provide strong evidence for shared genes regulating constitutive and induced oleoresin flow. We identified 236 single nucleotide polymorphisms associated with oleoresin flow, resin canal number, and terpene composition and highlight candidate genes likely involved in terpene biosynthesis, cambial meristem reprogramming, and pathogen perception and immune signaling. Fourteen GWAS candidates were methyl jasmonate-responsive in tissues where resin canals initiate and terpene production occurs. Integrating quantitative genetics, GWAS, gene expression, and multiplex network analyses enabled the prioritization of high-quality candidate genes. This work advances the development of more resilient loblolly pine optimized for ecological performance, renewable chemical, and biofuel production.

genome-wide association study↗
DOE OSTI2020

Molecular Dialogues between Early Divergent Fungi and Bacteria in an Antagonism versus a Mutualism

Animals and plants interact with microbes by engaging specific surveillance systems, regulatory networks, and response modules that allow for accommodation of mutualists and defense against antagonists. Antimicrobial defense responses are mediated in both animals and plants by innate immunity systems that owe their functional similarities to convergent evolution. Like animals and plants, fungi interact with bacteria. However, the principles governing these relations are only now being discovered. In a study system of host and nonhost fungi interacting with a bacterium isolated from the host, we found that bacteria used a common gene repertoire to engage both partners. In contrast, fungal responses to bacteria differed dramatically between the host and nonhost. These findings suggest that as in animals and plants, the genetic makeup of the fungus determines whether bacterial partners are perceived as mutualists or antagonists and what specific regulatory networks and response modules are initiated during each encounter.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

The Single Cells and Cell Populations Viability Estimation in vitro by the Time-Domain Impedance Spectroscopy

In the present study, we investigate the sensitivity and applicability of the time-domain electrical impedance spectroscopy (EIS) techniques, namely Fourier-EIS and adaptive filtering based EIS (AF-EIS), for studying cells' populations and single living cells in natural physiological environment in vitro. Using ultra-violet radiation for decreasing cell life-span we demonstrate the possibility to distinguish the living cells' population from the dead cells by both Fourier-EIS and AF-EIS. However, determining the viability of the single cell requires significant sensitivity, which is inaccessible with the conventional Fourier-EIS contrary to AF-EIS. The latter result stems from the high noise immunity of the AF-EIS, which also makes it possible to provide the measurements using the safe for cells 15-mV excitation voltage and 10–100 nA current response. The developed single-cell AF-EIS approach opens a direct roadmap for creating accurate, robust, and easy to implement toxin and radiation hazard sensors with the living cell as an acting element and proposes a solution for actual ecological and healthcare problems.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2019

Genome sequence of the model rice variety KitaakeX

Abstract Background The availability of thousands of complete rice genome sequences from diverse varieties and accessions has laid the foundation for in-depth exploration of the rice genome. One drawback to these collections is that most of these rice varieties have long life cycles, and/or low transformation efficiencies, which limits their usefulness as model organisms for functional genomics studies. In contrast, the rice variety Kitaake has a rapid life cycle (9 weeks seed to seed) and is easy to transform and propagate. For these reasons, Kitaake has emerged as a model for studies of diverse monocotyledonous species. Results Here, we report the de novo genome sequencing and analysis of Oryza sativa ssp. japonica variety KitaakeX, a Kitaake plant carrying the rice XA21 immune receptor. Our KitaakeX sequence assembly contains 377.6 Mb, consisting of 33 scaffolds (476 contigs) with a contig N50 of 1.4 Mb. Complementing the assembly are detailed gene annotations of 35,594 protein coding genes. We identified 331,335 genomic variations between KitaakeX and Nipponbare (ssp. japonica ), and 2,785,991 variations between KitaakeX and Zhenshan97 (ssp. indica ). We also compared Kitaake resequencing reads to the KitaakeX assembly and identified 219 small variations. The high-quality genome of the model rice plant KitaakeX will accelerate rice functional genomics. Conclusions The high quality, de novo assembly of the KitaakeX genome will serve as a useful reference genome for rice and will accelerate functional genomics studies of rice and other species.

Nipponbare↗
DOE OSTI2016

Elevated blood lead and cadmium levels associated with chronic infections among non-smokers in a cross-sectional analysis of NHANES data

Background: Experimental animal studies, in vitro experiments, and clinical assessments have shown that metal toxicity can impair immune responses. We analyzed data from a United States representative National Health and Nutrition Examination Survey (NHANES) to explore associations between chronic infections and elevated blood concentrations of lead and cadmium among non-smoking NHANES participants. Methods: NHANES data from 1999 to 2012 were examined and weighted to represent the United States population. Multivariable logistic regression was used to estimate adjusted odds ratios (AOR) and 95 % confidence intervals (CI) for heavy metal associations with seropositivity for Helicobacter pylori, Toxoplasma gondii, and Hepatitis B virus (HBV) infections. Results: Available 2-year survey cycles for infection seroprevalence varied by pathogen, from 1 to 7 cycles. Available sample size, disease seroprevalence, and participant age range also varied by pathogen of interest. After controlling for demographic characteristics and general health condition, an elevated blood lead level above the survey population median was significantly associated with seropositivity for all three pathogens (AORs = 1.2–1.5). In addition, an elevated blood cadmium level above the median was significantly associated with HBV (AOR = 1.5; 95 % CI = 1.2–2.0) and H. pylori (AOR = 1.5; 95 % CI = 1.2–1.7) seropositivity. Age-specific analyses for H. pylori and T. gondii indicated stronger associations among children under 13 years of age, particularly for lead exposure and H. pylori seropositivity, and weaker associations among those over 35 years of age. Conclusions: The results of this cross-sectional human health survey suggest that the immunological effects of lead and cadmium toxicity may be associated with an increased susceptibility to chronic infections.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Differential methods for assessing sensitivity in biological models

Differential sensitivity analysis is indispensable in fitting parameters, understanding uncertainty, and forecasting the results of both thought and lab experiments. Although there are many methods currently available for performing differential sensitivity analysis of biological models, it can be difficult to determine which method is best suited for a particular model. In this paper, we explain a variety of differential sensitivity methods and assess their value in some typical biological models. First, we explain the mathematical basis for three numerical methods: adjoint sensitivity analysis, complex perturbation sensitivity analysis, and forward mode sensitivity analysis. We then carry out four instructive case studies. (a) The CARRGO model for tumor-immune interaction highlights the additional information that differential sensitivity analysis provides beyond traditional naive sensitivity methods, (b) the deterministic SIR model demonstrates the value of using second-order sensitivity in refining model predictions, (c) the stochastic SIR model shows how differential sensitivity can be attacked in stochastic modeling, and (d) a discrete birth-death-migration model illustrates how the complex perturbation method of differential sensitivity can be generalized to a broader range of biological models. Finally, we compare the speed, accuracy, and ease of use of these methods. We find that forward mode automatic differentiation has the quickest computational time, while the complex perturbation method is the simplest to implement and the most generalizable.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Modeling dynamics of acute HIV infection incorporating density-dependent cell death and multiplicity of infection

Understanding the dynamics of acute HIV infection can offer valuable insights into the early stages of viral behavior, potentially helping uncover various aspects of HIV pathogenesis. The standard viral dynamics model explains HIV viral dynamics during acute infection reasonably well. However, the model makes simplifying assumptions, neglecting some aspects of HIV infection. For instance, in the standard model, target cells are infected by a single HIV virion. Yet, cellular multiplicity of infection (MOI) may have considerable effects in pathogenesis and viral evolution. Further, when using the standard model, we take constant infected cell death rates, simplifying the dynamic immune responses. Here, we use four models—1) the standard viral dynamics model, 2) an alternate model incorporating cellular MOI, 3) a model assuming density-dependent death rate of infected cells and 4) a model combining (2) and (3)—to investigate acute infection dynamics in 43 people living with HIV very early after HIV exposure. We find that all models qualitatively describe the data, but none of the tested models is by itself the best to capture different kinds of heterogeneity. Instead, different models describe differing features of the dynamics more accurately. For example, while the standard viral dynamics model may be the most parsimonious across study participants by the corrected Akaike Information Criterion (AICc), we find that viral peaks are better explained by a model allowing for cellular MOI, using a linear regression analysis as analyzed by R 2 . These results suggest that heterogeneity in within-host viral dynamics cannot be captured by a single model. Depending on the specific aspect of interest, a corresponding model should be employed.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Characterization and vaccine potential of outer membrane vesicles produced by Haemophilus parasuis

Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swine and is capable of causing a systemic infection, resulting in high morbidity and mortality. H. parasuis isolates display a wide range of virulence and virulence factors are largely unknown. Commercial bacterins are often used to vaccinate swine against H. parasuis , though strain variability and lack of cross-reactivity can make this an ineffective means of protection. Outer membrane vesicles (OMV) are spherical structures naturally released from the membrane of bacteria and OMV are often enriched in toxins, signaling molecules and other bacterial components. Examination of OMV structures has led to identification of virulence factors in a number of bacteria and they have been successfully used as subunit vaccines. We have isolated OMV from both virulent and avirulent strains of H. parasuis , have examined their protein content and assessed their ability to induce an immune response in the host. Lastly, vaccination with purified OMV derived from the virulent H. parasuis Nagasaki strain provided protection against challenge with a lethal dose of the bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 10 5 –10 6 and neutralizing antibody titers of approximately 10 3 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deletedEBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-lengthGP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc,and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. In conclusion, our data further support the development of Fc fusions of GP as a candidate vaccine for human use.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Mouse IgG2c Fc loop residues promote greater receptor-binding affinity than mouse IgG2b or human IgG1

The structures of non-human antibodies are largely unstudied despite the potential for the identification of alternative structural motifs and physical properties that will benefit a basic understanding of protein and immune system evolution as well as highlight unexplored motifs to improve therapeutic monoclonal antibody. Here we probe the structure and receptor-binding properties of the mouse IgG2c crystallizable fragment (Fc) to compare to mouse IgG2b and human IgG1 Fcs. Models of mIgG2c Fc determined by x-ray crystallography with a complex-type biantennary (to 2.05 Å) or a truncated (1)GlcNAc asparagine-linked (N)-glycan attached (to 2.04 Å) show differences in key regions related to mouse Fc γ receptor IV (mFcγRIV) binding. Mouse IgG2c forms different non-bonded interactions between the BC, DE and FG loops than the highly-conserved mIgG2b and binds to FcγRIV with 4.7-fold greater affinity in the complex-type glycoform. Secondary structural elements surrounding the Asn297 site of glycosylation form longer beta strands in the truncated mIgG2c Fc glycoform when compared to mIgG2c with the complex-type N-glycan. Solution NMR spectroscopy of the N-linked (1)GlcNAc residues show differences between mIgG2b, 2c and hIgG1 Fc that correlate to receptor binding affinity. Mutations targeting differences in mIgG2 DE and FG loops decreased affinity of mIgG2c for FcγRIV and increased affinity of mIgG2b. Changes in NMR spectra of the mutant Fc proteins mirrored these changes in affinity. Our studies identified structural and functional differences in highly conserved molecules that were not predicted from primary sequence comparison.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Differential laboratory passaging of SARS-CoV-2 viral stocks impacts the in vitro assessment of neutralizing antibodies

Viral populations in natural infections can have a high degree of sequence diversity, which can directly impact immune escape. However, antibody potency is often tested in vitro with a relatively clonal viral populations, such as laboratory virus or pseudotyped virus stocks, which may not accurately represent the genetic diversity of circulating viral genotypes. This can affect the validity of viral phenotype assays, such as antibody neutralization assays. To address this issue, we tested whether recombinant virus carrying SARS-CoV-2 spike (VSV-SARS-CoV-2-S) stocks could be made more genetically diverse by passage, and if a stock passaged under selective pressure was more capable of escaping monoclonal antibody (mAb) neutralization than unpassaged stock or than viral stock passaged without selective pressures. We passaged VSV-SARS-CoV-2-S four times concurrently in three cell lines and then six times with or without polyclonal antiserum selection pressure. All three of the monoclonal antibodies tested neutralized the viral population present in the unpassaged stock. The viral inoculum derived from serial passage without antiserum selection pressure was neutralized by two of the three mAbs. However, the viral inoculum derived from serial passage under antiserum selection pressure escaped neutralization by all three mAbs. Deep sequencing revealed the rapid acquisition of multiple mutations associated with antibody escape in the VSV-SARS-CoV-2-S that had been passaged in the presence of antiserum, including key mutations present in currently circulating Omicron subvariants. These data indicate that viral stock that was generated under polyclonal antiserum selection pressure better reflects the natural environment of the circulating virus and may yield more biologically relevant outcomes in phenotypic assays. Thus, mAb assessment assays that utilize a more genetically diverse, biologically relevant, virus stock may yield data that are relevant for prediction of mAb efficacy and for enhancing biosurveillance.

60 APPLIED LIFE SCIENCES↗