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At least 1,711 records · Page 95
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Disrupting autorepression circuitry generates “open-loop lethality” to yield escape-resistant antiviral agents

Across biological scales, gene-regulatory networks employ autorepression (negative feedback) to maintain homeostasis and minimize failure from aberrant expression. In this study, we present a proof of concept that disrupting transcriptional negative feedback dysregulates viral gene expression to therapeutically inhibit replication and confers a high evolutionary barrier to resistance. We find that nucleic-acid decoys mimicking cis-regulatory sites act as “feedback disruptors,” break homeostasis, and increase viral transcription factors to cytotoxic levels (termed “open-loop lethality”). Feedback disruptors against herpesviruses reduced viral replication >2-logs without activating innate immunity, showed sub-nM IC 50 , synergized with standard-of-care antivirals, and inhibited virus replication in mice. In contrast to approved antivirals where resistance rapidly emerged, no feedback-disruptor escape mutants evolved in long-term cultures. For SARS-CoV-2, disruption of a putative feedback circuit also generated open-loop lethality, reducing viral titers by >1-log. These results demonstrate that generating open-loop lethality, via negative-feedback disruption, may yield a class of antimicrobials with a high genetic barrier to resistance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

helixCAM: A platform for programmable cellular assembly in bacteria and human cells

Interactions between cells are indispensable for signaling and creating structure. The ability to direct precise cell-cell interactions would be powerful for engineering tissues, understanding signaling pathways, and directing immune cell targeting. In humans, intercellular interactions are mediated by cell adhesion molecules (CAMs). However, endogenous CAMs are natively expressed by many cells and tend to have cross-reactivity, making them unsuitable for programming specific interactions. Here, we showcase “helixCAM,” a platform for engineering synthetic CAMs by presenting coiled-coil peptides on the cell surface. helixCAMs were able to create specific cell-cell interactions and direct patterned aggregate formation in bacteria and human cells. Based on coiled-coil interaction principles, we built a set of rationally designed helixCAM libraries, which led to the discovery of additional high-performance helixCAM pairs. Finally, we applied this helixCAM toolkit for various multicellular engineering applications, such as spherical layering, adherent cell targeting, and surface patterning.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Structural Basis for Molecular Discrimination by a 3',3'-cGAMP Sensing Riboswitch

Cyclic dinucleotides are second messengers that target the adaptor STING and stimulate the innate immune response in mammals. Besides protein receptors, there are bacterial riboswitches that selectively recognize cyclic dinucleotides. We recently discovered a natural riboswitch that targets 3',3'-cGAMP, which is distinguished from the endogenous mammalian signal 2',3'-cGAMP by its backbone connectivity. Here, we report on structures of the aptamer domain of the 3',3'-cGAMP riboswitch from Geobacter in the 3',3'-cGAMP and c-di-GMP bound states. The riboswitch adopts a tuning forklike architecture with a junctional ligand-binding pocket and different orientations of the arms are correlated with the identity of the bound cyclic dinucleotide. Subsequent biochemical experiments revealed that specificity of ligand recognition can be affected by point mutations outside of the binding pocket, which has implications for both the assignment and reengineering of riboswitches in this structural class.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Structural Constraints of Vaccine-Induced Tier-2 Autologous HIV Neutralizing Antibodies Targeting the Receptor-Binding Site

Antibodies that neutralize autologous transmitted/founder (TF) HIV occur in most HIV-infected individuals and can evolve to neutralization breadth. Autologous neutralizing antibodies (nAbs) against neutralization-resistant (Tier-2) viruses are rarely induced by vaccination. Whereas broadly neutralizing antibody (bnAb)-HIV-Envelope structures have been defined, the structures of autologous nAbs have not. Here, we show that immunization with TF mutant Envs gp140 oligomers induced high-titer, V5-dependent plasma neutralization for a Tier-2 autologous TF evolved mutant virus. Structural analysis of autologous nAb DH427 revealed binding to V5, demonstrating the source of narrow nAb specificity and explaining the failure to acquire breadth. Thus, oligomeric TF Envs can elicit autologous nAbs to Tier-2 HIVs, but induction of bnAbs will require targeting of precursors of B cell lineages that can mature to heterologous neutralization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Systematic Analysis of Splice-Site-Creating Mutations in Cancer

For the past decade, cancer genomic studies have focused on mutations leading to splice-site disruption, overlooking those having splice-creating potential. Here, we applied a bioinformatic tool, MiSplice, for the large-scale discovery of splice-site-creating mutations (SCMs) across 8,656 TCGA tumors. We report 1,964 originally mis-annotated mutations having clear evidence of creating alternative splice junctions. TP53 and GATA3 have 26 and 18 SCMs, respectively, and ATRX has 5 from lower-grade gliomas. Mutations in 11 genes, including PARP1, BRCA1, and BAP1, were experimentally validated for splice-site-creating function. Notably, we found that neoantigens induced by SCMs are likely several folds more immunogenic compared to missense mutations, exemplified by the recurrent GATA3 SCM. Further, high expression of PD-1 and PD-L1 was observed in tumors with SCMs, suggesting candidates for immune blockade therapy. Finally, our work highlights the importance of integrating DNA and RNA data for understanding the functional and the clinical implications of mutations in human diseases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Intricate Genetic Programs Controlling Dormancy in Mycobacterium tuberculosis

Mycobacterium tuberculosis (MTB) displays the remarkable ability to transition in and out of dormancy, a hallmark of the pathogen’s capacity to evade the immune system and exploit susceptible individuals. Uncovering the gene regulatory programs that underlie the phenotypic shifts in MTB during disease latency and reactivation has posed a challenge. We develop an experimental system to precisely control dissolved oxygen levels in MTB cultures in order to capture the transcriptional events that unfold as MTB transitions into and out of hypoxia-induced dormancy. Using a comprehensive genome-wide transcription factor binding map and insights from network topology analysis, we identify regulatory circuits that deterministically drive sequential transitions across six transcriptionally and functionally distinct states encompassing more than three-fifths of the MTB genome. The architecture of the genetic programs explains the transcriptional dynamics underlying synchronous entry of cells into a dormant state that is primed to infect the host upon encountering favorable conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Antibody:CD47 ratio regulates macrophage phagocytosis through competitive receptor phosphorylation

Cancer immunotherapies often modulate macrophage effector function by introducing either targeting antibodies that activate Fcγ receptors (FcγRs) or blocking antibodies that disrupt inhibitory SIRPα-CD47 engagement. However, how these competing signals are integrated is poorly understood, raising questions about how to effectively titrate immune responses. Here, we find that macrophage phagocytic decisions are regulated by the ratio of activating ligand to inhibitory ligand over a broad range of absolute molecular densities. Using both endogenous and chimeric receptors, we show that activating:inhibitory ligand ratios of at least 10:1 are required to promote phagocytosis of model antibody-opsonized CD47-inhibited targets and that lowering that ratio reduces FcγR phosphorylation because of inhibitory phosphatases recruited to CD47-bound SIRPα. We demonstrate that ratiometric signaling is critical for phagocytosis of tumor cells and can be modified by blocking SIRPα, indicating that balancing targeting and blocking antibodies may be important for controlling macrophage phagocytosis in cancer immunotherapy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Rapid identification of neutralizing antibodies against SARS-CoV-2 variants by mRNA display

The increasing prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with the ability to escape existing humoral protection conferred by previous infection and/or immunization necessitates the discovery of broadly reactive neutralizing antibodies (nAbs). Utilizing mRNA display, we identify a set of antibodies against SARS-CoV-2 spike (S) proteins and characterize the structures of nAbs that recognize epitopes in the S1 subunit of the S glycoprotein. These structural studies reveal distinct binding modes for several antibodies, including the targeting of rare cryptic epitopes in the receptor-binding domain (RBD) of S that interact with angiotensin-converting enzyme 2 (ACE2) to initiate infection, as well as the S1 subdomain 1. Further, we engineer a potent ACE2-blocking nAb to sustain binding to S RBD with the E484K and L452R substitutions found in multiple SARS-CoV-2 variants. We demonstrate that mRNA display is an approach for the rapid identification of nAbs that can be used in combination to combat emerging SARS-CoV-2 variants.

SARS-CoV-2↗
DOE OSTI2023

Molecular determinants of cross-reactivity and potency by VH3-33 antibodies against the Plasmodium falciparum circumsporozoite protein

IGHV3-33 -encoded antibodies are prevalent in the human humoral response against the Plasmodium falciparum circumsporozoite protein (PfCSP). Among VH3-33 antibodies, cross-reactivity between PfCSP major repeat (NANP), minor (NVDP), and junctional (NPDP) motifs is associated with high affinity and potent parasite inhibition. However, the molecular basis of antibody cross-reactivity and the relationship with efficacy remain unresolved. Here, we perform an extensive structure-function characterization of 12 VH3-33 anti-PfCSP monoclonal antibodies (mAbs) with varying degrees of cross-reactivity induced by immunization of mice expressing a human immunoglobulin gene repertoire. We identify residues in the antibody paratope that mediate cross-reactive binding and delineate four distinct epitope conformations induced by antibody binding, with one consistently associated with high protective efficacy and another that confers comparably potent inhibition of parasite liver invasion. Our data show a link between molecular features of cross-reactive VH3-33 mAb binding to PfCSP and mAb potency, relevant for the development of antibody-based interventions against malaria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Multi-platform ’Omics Analysis of Human Ebola Virus Disease Pathogenesis

The pathogenesis of human Ebola virus disease (EVD) is complex. EVD is characterized by high levels of virus replication and dissemination, dysregulated immune responses, extensive virus- and host-mediated tissue damage, and disordered coagulation. To clarify how host responses contribute to EVD pathophysiology, we performed multi-platform ’omics analysis of peripheral blood mononuclear cells and plasma from EVD patients. Our results indicate that EVD molecular signatures overlap with those of sepsis, imply that pancreatic enzymes contribute to tissue damage in fatal EVD, and suggest that Ebola virus infection may induce aberrant neutrophils whose activity could explain hallmarks of fatal EVD. Moreover, integrated biomarker prediction identified putative biomarkers from different data platforms that differentiated survivors and fatalities early after infection. We report this work reveals insight into EVD pathogenesis, suggests an effective approach for biomarker identification, and provides an important community resource for further analysis of human EVD severity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

HIV-1 Neutralizing Antibody Signatures and Application to Epitope-Targeted Vaccine Design

Eliciting HIV-1-specific broadly neutralizing antibodies (bNAbs) remains a challenge for vaccine development, and the potential of passively delivered bNAbs for prophylaxis and therapeutics is being explored. We used neutralization data from four large virus panels to comprehensively map viral signatures associated with bNAb sensitivity, including amino acids, hypervariable region characteristics, and clade effects across four different classes of bNAbs. The bNAb signatures defined for the variable loop 2 (V2) epitope region of HIV-1 Env were then employed to inform immunogen design in a proof-of-concept exploration of signature-based epitope targeted (SET) vaccines. V2 bNAb signature-guided mutations were introduced into Env 459C to create a trivalent vaccine, and immunization of guinea pigs with V2- SET vaccines resulted in increased breadth of NAb responses compared with Env 459C alone. These data demonstrate that bNAb signatures can be utilized to engineer HIV-1 Env vaccine immunogens capable of eliciting antibody responses with greater neutralization breadth.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

A Combination of Human Broadly Neutralizing Antibodies against Hepatitis B Virus HBsAg with Distinct Epitopes Suppresses Escape Mutations

Although there is no effective cure for chronic hepatitis B virus (HBV) infection, antibodies are protective and correlate with recovery from infection. To examine the human antibody response to HBV, we screened 124 vaccinated and 20 infected, spontaneously recovered individuals. The selected individuals produced shared clones of broadly neutralizing antibodies (bNAbs) that targeted 3 non-overlapping epitopes on the HBV S antigen (HBsAg). Single bNAbs protected humanized mice against infection but selected for resistance mutations in mice with prior established infection. In contrast, infection was controlled by a combination of bNAbs targeting non-overlapping epitopes with complementary sensitivity to mutations that commonly emerge during human infection. The co-crystal structure of one of the bNAbs with an HBsAg peptide epitope revealed a stabilized hairpin loop. This structure, which contains residues frequently mutated in clinical immune escape variants, provides a molecular explanation for why immunotherapy for HBV infection may require combinations of complementary bNAbs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

D614G Spike Mutation Increases SARS CoV-2 Susceptibility to Neutralization

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein acquired a D614G mutation early in the pandemic that confers greater infectivity and is now the globally dominant form. To determine whether D614G might also mediate neutralization escape that could compromise vaccine efficacy, sera from spike-immunized mice, nonhuman primates, and humans were evaluated for neutralization of pseudoviruses bearing either D614 or G614 spike. In all cases, the G614 pseudovirus was moderately more susceptible to neutralization. The G614 pseudovirus also was more susceptible to neutralization by receptor-binding domain (RBD) monoclonal antibodies and convalescent sera from people infected with either form of the virus. Here, negative stain electron microscopy revealed a higher percentage of the 1-RBD “up” conformation in the G614 spike, suggesting increased epitope exposure as a mechanism of enhanced vulnerability to neutralization. Based on these findings, the D614G mutation is not expected to be an obstacle for current vaccine development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

SARS-CoV-2 variant B.1.1.7 is susceptible to neutralizing antibodies elicited by ancestral spike vaccines

All current vaccines for COVID-19 utilize ancestral SARS-CoV-2 spike with the goal of generating protective neutralizing antibodies. The recent emergence and rapid spread of several SARS-CoV-2 variants carrying multiple spike mutations raise concerns about possible immune escape. One variant, first identified in the United Kingdom (B.1.1.7, also called 20I/501Y.V1), contains eight spike mutations with potential to impact antibody therapy, vaccine efficacy, and risk of reinfection. Here in this paper, we show that B.1.1.7 remains sensitive to neutralization, albeit at moderately reduced levels (~2-fold), by serum samples from convalescent individuals and recipients of an mRNA vaccine (mRNA-1273, Moderna) and a protein nanoparticle vaccine (NVX-CoV2373, Novavax). A subset of monoclonal antibodies to the receptor binding domain (RBD) of spike are less effective against the variant, while others are largely unaffected. Lastly, these findings indicate that variant B.1.1.7 is unlikely to be a major concern for current vaccines or for an increased risk of reinfection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Structure of the Inmazeb cocktail and resistance to Ebola virus escape

Monoclonal antibodies can provide important pre- or post-exposure protection against infectious disease for those not yet vaccinated or in individuals that fail to mount a protective immune response after vaccination. Inmazeb (REGN-EB3), a three-antibody cocktail against Ebola virus, lessened disease and improved survival in a controlled trial. Here, we present the cryo-EM structure at 3.1 Å of the Ebola virus glycoprotein, determined without symmetry averaging, in a simultaneous complex with the antibodies in the Inmazeb cocktail. This structure allows the modeling of previously disordered portions of the glycoprotein glycan cap, maps the non-overlapping epitopes of Inmazeb, and illuminates the basis for complementary activities and residues critical for resistance to escape by these and other clinically relevant antibodies. We further provide direct evidence that Inmazeb protects against the rapid emergence of escape mutants, whereas monotherapies even against conserved epitopes do not, supporting the benefit of a cocktail versus a monotherapy approach.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Protecting honey bees through microbiome engineering

Honey bees are indispensable insects. Their pollination services support modern agriculture and natural ecosystems. Managed honey bee colonies face increasing threats to their survival, ranging from environmental stressors that include agrochemicals to infestations of arthropod pests and infections with microbial pathogens. Like humans, honey bees have a native gut microbiome that supports their health. However, the bee gut microbiome has a simpler composition than the gut microbiome of mammals, and its main constituent bacterial species can be easily cultured outside of the host. Furthermore, this experimental tractability and the need for new methods for protecting hive health have made honey bees a testbed for synthetic microbiomes augmented with probiotic bacteria and engineered DNA. Here, we discuss the natural benefits of bee gut bacteria, recent progress in genetically modifying these bacteria, and how symbiont-mediated RNA interference and other microbiome engineering approaches can boost bee immunity and suppress bee pathogens and parasites. Finally, we discuss how emerging methods for microbiome engineering and biocontainment could be applied to honey bees and used to address challenges in translating these proof-of-principle achievements into safe and effective technologies for field applications at scale.

Biological and medical sciences↗
DOE OSTI2019

Within-host mathematical models of hepatitis B virus infection: Past, present, and future

Mathematical modeling has been instrumental in enhancing our understanding of the viral dynamics of hepatitis B virus (HBV) infection. We give a primer on HBV infection in humans and a brief overview of the development of within-host mathematical models of HBV infection. In the last decade, models have advanced from considering chronic HBV infections under therapy to the pathogenesis of infection. We also summarize estimates of key viral dynamic parameters that have varied greatly among studies, and show that they impact model predictions. Here, future directions for mathematical modeling of HBV infection are proposed to better understand emerging therapies, the HBV life cycle, predicting cure, and the mechanisms involved in the immune response to HBV.

59 BASIC BIOLOGICAL SCIENCES↗