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Smith, D. M.

Publications and source records attributed to Smith, D. M..

At least 55 records · Page 3Linked to original sources
bioRxiv2022

Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause severe disease

Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against coronaviruses that cause severe disease, for anticipating novel pandemic-causing viruses, and to respond more effectively to SARS-CoV-2 variants. The emergence of the Omicron variant of SARS-CoV-2 has illustrated the limitations of solely targeting the receptor binding domain (RBD) of the envelope Spike (S)-protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors that target a conserved S2 region in the fusion machinery on betacoronavirus spikes. Select bnAbs show broad in vivo protection against all three pathogenic betacoronaviruses, SARS-CoV-1, SARS-CoV-2 and MERS-CoV, that have spilled over into humans in the past 20 years to cause severe disease. The bnAbs provide new opportunities for antibody-based interventions and key insights for developing pan-betacoronavirus vaccines.

immunology↗
bioRxiv2022

BOLD cofluctuation 'events' are predicted from static functional connectivity

Recent work identified single time points ("events") of high regional cofluctuation in functional Magnetic Resonance Imaging (fMRI) which contain more large-scale brain network information than other, low cofluctuation time points. This suggested that events might be a discrete, temporally sparse signal which drives functional connectivity (FC) over the timeseries. However, a different, not yet explored possibility is that network information differences between time points are driven by sampling variability on a constant, static, noisy signal. Using a combination of real and simulated data, we examined the relationship between cofluctuation and network structure and asked if this relationship was unique, or if it could arise from sampling variability alone. First, we show that events are not discrete - there is a gradually increasing relationship between network structure and cofluctuation; [~]50% of samples show very strong network structure. Second, using simulations we show that this relationship is predicted from sampling variability on static FC. Finally, we show that randomly selected points can capture network structure about as well as events, largely because of their temporal spacing. Together, these results suggest that, while events exhibit particularly strong representations of static FC, there is little evidence that events are unique timepoints that drive FC structure. Instead, a parsimonious explanation for the data is that events arise from a single static, but noisy, FC structure. HIGHLIGHTSO_LIPast results suggested high cofluctuation BOLD "events" drive fMRI functional connectivity, FC C_LIO_LIHere, events were examined in both real fMRI data and a stationary null model to test this model C_LIO_LIIn real data, >50% of BOLD timepoints show high modularity and similarity to time- averaged FC C_LIO_LIStationary null models identified events with similar behavior to real data C_LIO_LIEvents may not be a transient driver of static FC, but rather an expected outcome of it. C_LI

neuroscience↗
bioRxiv2022

Antisense oligonucleotide-based therapeutic strategy for progranulin-deficient frontotemporal dementia

Heterozygous GRN (progranulin) mutations cause frontotemporal dementia (FTD) due to haploinsufficiency, and increasing progranulin levels is a major therapeutic goal. Several microRNAs, including miR-29b, negatively regulate progranulin protein levels. Antisense oligonucleotides (ASOs) are emerging as a promising therapeutic modality for neurological diseases, but strategies for increasing target protein levels are limited. Here, we tested the efficacy of ASOs as enhancers of progranulin expression by sterically blocking the miR-29b binding site in the 3 UTR of the human GRN mRNA. We found 16 ASOs that increase progranulin protein in a dose-dependent manner in neuroglioma cells. A subset of these ASOs also increased progranulin protein in iPSC-derived neurons and in a humanized GRN mouse model. In FRET-based assays, the ASOs effectively competed miR-29b from binding to the GRN 3 UTR RNA. The ASOs increased levels of newly synthesized progranulin protein by increasing its translation, as revealed by ribosomal profiling. Together, our results demonstrate that ASOs can be used to effectively increase target protein levels by partially blocking miR binding sites. This ASO strategy may be therapeutically feasible for progranulin-deficient FTD as well as other conditions of haploinsufficiency.

neuroscience↗
bioRxiv2021

Distinguishing COVID-19 infection and vaccination history by T cell reactivity

SARS-CoV-2 infection and COVID-19 vaccines elicit memory T cell responses. Here, we report the development of two new pools of Experimentally-defined T cell epitopes derived from the non-spike Remainder of the SARS-CoV-2 proteome (CD4RE and CD8RE). The combination of T cell responses to these new pools and Spike (S) were used to discriminate four groups of subjects with different SARS-CoV-2 infection and COVID-19 vaccine status: non-infected, non-vaccinated (I-V-); infected and non-vaccinated (I+V-); infected and then vaccinated (I+V+); and non-infected and vaccinated (I-V+). The overall classification accuracy based on 30 subjects/group was 89.2% in the original cohort and 88.5% in a validation cohort of 96 subjects. The T cell classification scheme was applicable to different mRNA vaccines, and different lengths of time post-infection/post-vaccination. T cell responses from breakthrough infections (infected vaccinees, V+I+) were also effectively segregated from the responses of vaccinated subjects using the same classification tool system. When all five groups where combined, for a total of 239 different subjects, the classification scheme performance was 86.6%. We anticipate that a T cell-based immunodiagnostic scheme able to classify subjects based on their vaccination and natural infection history will be an important tool for longitudinal monitoring of vaccination and aid in establishing SARS-CoV-2 correlates of protection.

immunology↗
bioRxiv2021

Allosteric regulation of the proteasomes catalytic sites by the proteasome activator PA28gamma/REGgamma

Proteasome Activator 28{gamma} (PA28{gamma}) is a member of the 11S family of proteasomal regulators that is constitutively expressed in the nucleus and is implicated in certain cancers, lupus, rheumatoid arthritis, and Poly-glutamine neurodegenerative diseases. However, how PA28{gamma} functions in protein degradation remains unclear. Though PA28{gamma}s mechanism has been investigated for some time, many alternative hypotheses have not been tested: e.g. 1) substrate selection, 2) allosteric upregulation of the Trypsin-like catalytic site, 3) allosteric inhibition of the Chymotrypsin- and Caspase-like catalytic sites, 4) conversion of the Chymotrypsin- or Caspase-like sites to new Trypsin-like catalytic sites, and 5) gate-opening in combination with these. The purpose of this study was to conclusively determine how PA28{gamma} regulates proteasome function. Here, we rigorously and definitively show that PA28{gamma} uses an allosteric mechanism to upregulate the proteolytic activity of the 20S proteasomes Trypsin-like catalytic site. Using a constitutively open channel proteasome, we were able to dissociate gating affects from catalytic affects demonstrating that the PA28{gamma}-increases the affinity (Km) and Vmax for Trypsin-like peptide substrates. Mutagenesis of PA28{gamma} also reveals that it does not select for (i.e. filter) peptide substrates, and does not change the specificity of the other active sites to trypsin-like. Further, using Cryo-EM we were able to visualize the C7 symmetric PA28{gamma}-20S proteasome complex at 4.4[A] validating its expected 11S-like quaternary structure and proteasome binding mode. The results of this study provide unambiguous evidence that PA28{gamma} functions by allosterically upregulating the T-L like site in the 20S proteasome. Significance StatementThis study rigorously demonstrates that PA28g allosterically activates the b-2 proteolytic site of the 20S proteasome directly without affecting 20S gating. Further, we generated the first human 11S-human 20S proteasome cryo-EM structure of the PA28g-20S complex showing that, despite its different affects on 20S activity, it has a similar quaternary structure as the other 11S family members. The significance of these findings is paramount as the b-2 site is responsible for post-basic cleavage and suggests that PA28g is specialized to degrade positively charged DNA binding proteins. Further, b-2 upregulation via PA28g could provide a protective effect against poly-glutamine expanded proteins, like Huntingtin. This work provides a framework for PA28g drug development to treat PA28g addicted cancers and Huntingtons Disease.

biochemistry↗
bioRxiv2021

Brain hubs defined in the group do not overlap with regions of high inter-individual variability

Connector hubs are brain regions with links to multiple networks. These regions are hypothesized to play a critical role in brain function. While hubs are often identified based on group-average functional magnetic resonance imaging (fMRI) data, there is considerable inter-subject variation in the functional connectivity profiles of the brain, especially in association regions where hubs tend to be located. Here we investigated how group hubs are related to locations of inter-individual variability, to better understand if hubs are (a) relatively conserved across people, (b) locations with malleable connectivity, leading individuals to show variable hub profiles, or (c) artifacts arising from cross-person variation. To answer this question, we compared the locations of hubs and regions of strong idiosyncratic functional connectivity ("variants") in both the Midnight Scan Club and Human Connectome Project datasets. Group hubs defined based on the participation coefficient did not overlap strongly with variants. These hubs have relatively strong similarity across participants and consistent cross-network profiles. Consistency across participants was further improved when participation coefficient hubs were allowed to shift slightly in local position. Thus, our results demonstrate that group hubs defined with the participation coefficient are generally consistent across people, suggesting they may represent conserved cross-network bridges. More caution is warranted with alternative hub measures, such as community density, which are based on spatial proximity and show higher correspondence to locations of individual variability.

neuroscience↗
bioRxiv2021

A science friction story. Molecular interactions in semiflexible polymer networks

Established model theories, developed to capture the mechanical behavior of soft complex materials composed of semiflexible polymers assume entropic interactions between filaments to determine the mechanical response. In recent studies, the general accepted tube model has been challenged in terms of its basic assumption about filament-filament interactions, but also because of its predictions regarding the frequency dependence of the elastic modulus in the intermediate frequency regime. A central question is how molecular interactions and friction between network constituents influence the rheological response of isotropic entangled networks of semiflexible polymers. It was shown that friction forces between aligned pairs of actin filaments are not negligible. Here, we systematically investigate the influence of friction forces and attractive interactions on network rheology by means of a targeted surface modification. We show that these forces have a qualitative and quantitative influence on the viscoelastic properties of semiflexible polymer networks and contribute to the response to nonlinear deformations. By comparing two polymer model systems with respect to their surface compositions we give a possible explanation about the origin of acting forces on a molecular level.

biophysics↗
bioRxiv2021

Two common and distinct forms of variation in human functional brain networks

The cortex has a characteristic layout with specialized functional areas forming distributed large-scale networks. However, substantial work shows striking variation in this organization across people, which relates to differences in behavior. While most prior work treats all individual differences as equivalent and primarily linked to boundary shifts between the borders of regions, here we show that cortical variants actually occur in two different forms. In addition to border shifts, variants also occur at a distance from their typical position, forming ectopic intrusions. Both forms of variants are common across individuals, but the forms differ in their location, network associations, and activations during tasks, patterns that replicate across datasets and methods of definition. Border shift variants also track significantly more with shared genetics than ectopic variants, suggesting a closer link between ectopic variants and environmental influences. Further, variant properties are categorically different between subgroups of individuals. Exploratory evidence suggests that variants can predict individual differences in behavior, but the two forms differ in which behavioral phenotypes they predict. This work argues that individual differences in brain organization commonly occur in two dissociable forms - border shifts and ectopic intrusions - suggesting that these types of variation are indexing distinct forms of cortical variation that must be separately accounted for in the analysis of cortical systems across people. This work expands our knowledge of cortical variation in humans and helps reconceptualize the discussion of how cortical systems variability arises and links to individual differences in cognition and behavior.

neuroscience↗
bioRxiv2021

Targeted isolation of panels of diverse human broadly neutralizing antibodies against SARS-like viruses

The emergence of current SARS-CoV-2 variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy 1-7. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed 8, 9. Notably, several recent studies have revealed that vaccination of recovered COVID-19 donors results in enhanced nAb responses compared to SARS-CoV-2 infection or vaccination alone 10-13. Here, we utilized a targeted donor selection strategy to isolate a large panel of broadly neutralizing antibodies (bnAbs) to sarbecoviruses from two such donors. Many of the bnAbs are remarkably effective in neutralization against sarbecoviruses that use ACE2 for viral entry and a substantial fraction also show notable binding to non-ACE2-using sarbecoviruses. The bnAbs are equally effective against most SARS-CoV-2 VOCs and many neutralize the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor binding domain (RBD) as opposed to strain-specific nAbs to the receptor binding site that are commonly elicited in SARS-CoV-2 infection and vaccination 14-18. Consistent with targeting of conserved sites, select RBD bnAbs exhibited in vivo protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model. The generation of a large panel of potent bnAbs provides new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and, importantly, provides a molecular basis for effective design of pan-sarbecovirus vaccines.

immunology↗
bioRxiv2021

Broadly neutralizing antibodies to SARS-related viruses can be readily induced in rhesus macaques

To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting neutralizing antibody responses against multiple CoVs. Because of the phylogenetic similarity to humans, rhesus macaques are an animal model of choice for many virus-challenge and vaccine-evaluation studies, including SARS-CoV-2. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein generates potent receptor binding domain cross- neutralizing antibody (nAb) responses to both SARS-CoV-2 and SARS-CoV-1, in contrast to human infection or vaccination where responses are typically SARS-CoV-2-specific. Furthermore, the macaque nAbs are equally effective against SARS-CoV-2 variants of concern. Structural studies show that different immunodominant sites are targeted by the two primate species. Human antibodies generally target epitopes strongly overlapping the ACE2 receptor binding site (RBS), whereas the macaque antibodies recognize a relatively conserved region proximal to the RBS that represents another potential pan-SARS-related virus site rarely targeted by human antibodies. B cell repertoire differences between the two primates appear to significantly influence the vaccine response and suggest care in the use of rhesus macaques in evaluation of vaccines to SARS-related viruses intended for human use. ONE SENTENCE SUMMARYBroadly neutralizing antibodies to an unappreciated site of conservation in the RBD in SARS- related viruses can be readily induced in rhesus macaques because of distinct properties of the naive macaque B cell repertoire that suggest prudence in the use of the macaque model in SARS vaccine evaluation and design.

immunology↗
bioRxiv2021

Glucose-dependent miR-125b is a negative regulator of β-cell function

Impaired pancreatic {beta}-cell function and insulin secretion are hallmarks of type 2 diabetes. MicroRNAs are short non-coding RNAs that silence gene expression, vital for the development and function of {beta}-cells. We have previously shown that {beta}-cell specific deletion of the important energy sensor AMP-activated protein kinase (AMPK) results in increased miR-125b-5p levels. Nevertheless, the function of this miRNA in {beta}-cells is unclear. We hypothesized that miR-125b-5p expression is regulated by glucose and that this miRNA mediates some of the deleterious effects of hyperglycaemia in {beta}-cells. Here we show that islet miR-125b-5p expression is up-regulated by glucose in an AMPK-dependent manner and that short-term miR-125b-5p overexpression impairs glucose stimulated insulin secretion (GSIS) in the mouse insulinoma MIN6 cells and in human islets. An unbiased high-throughput screen in MIN6 cells identified multiple miR-125b-5p targets, including the transporter of lysosomal hydrolases M6pr and the mitochondrial fission regulator Mtfp1. Inactivation of miR-125b-5p in the human {beta}-cell line EndoC{beta}-H1 shortened mitochondria and enhanced GSIS, whilst mice overexpressing miR-125b-5p selectively in {beta}-cells (MIR125B-Tg) were hyperglycaemic and glucose intolerant. MIR125B-Tg {beta}-cells contained enlarged lysosomal structures and showed reduced insulin content and secretion. Collectively, we identify miR-125b as a glucose-controlled regulator of organelle dynamics that modulates insulin secretion. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗
bioRxiv2021

A protective broadly cross-reactive human antibody defines a conserved site of vulnerability on beta-coronavirus spikes

Broadly neutralizing antibodies (bnAbs) to coronaviruses (CoVs) are valuable in their own right as prophylactic and therapeutic reagents to treat diverse CoVs and, importantly, as templates for rational pan-CoV vaccine design. We recently described a bnAb, CC40.8, from a coronavirus disease 2019 (COVID-19)-convalescent donor that exhibits broad reactivity with human beta-coronaviruses ({beta}-CoVs). Here, we showed that CC40.8 targets the conserved S2 stem-helix region of the coronavirus spike fusion machinery. We determined a crystal structure of CC40.8 Fab with a SARS-CoV-2 S2 stem-peptide at 1.6 [A] resolution and found that the peptide adopted a mainly helical structure. Conserved residues in {beta}-CoVs interacted with CC40.8 antibody, thereby providing a molecular basis for its broad reactivity. CC40.8 exhibited in vivo protective efficacy against SARS-CoV-2 challenge in two animal models. In both models, CC40.8-treated animals exhibited less weight loss and reduced lung viral titers compared to controls. Furthermore, we noted CC40.8-like bnAbs are relatively rare in human COVID-19 infection and therefore their elicitation may require rational structure-based vaccine design strategies. Overall, our study describes a target on {beta}-CoV spike proteins for protective antibodies that may facilitate the development of pan-{beta}-CoV vaccines. SUMMARYA human mAb isolated from a COVID-19 donor defines a protective cross-neutralizing epitope for pan-{beta}-CoV vaccine design strategies

immunology↗
bioRxiv2021

Negligible impact of SARS-CoV-2 variants on CD4+ and CD8+ T cell reactivity in COVID-19 exposed donors and vaccinees.

The emergence of SARS-CoV-2 variants highlighted the need to better understand adaptive immune responses to this virus. It is important to address whether also CD4+ and CD8+ T cell responses are affected, because of the role they play in disease resolution and modulation of COVID-19 disease severity. Here we performed a comprehensive analysis of SARS-CoV-2-specific CD4+ and CD8+ T cell responses from COVID-19 convalescent subjects recognizing the ancestral strain, compared to variant lineages B.1.1.7, B.1.351, P.1, and CAL.20C as well as recipients of the Moderna (mRNA-1273) or Pfizer/BioNTech (BNT162b2) COVID-19 vaccines. Similarly, we demonstrate that the sequences of the vast majority of SARS-CoV-2 T cell epitopes are not affected by the mutations found in the variants analyzed. Overall, the results demonstrate that CD4+ and CD8+ T cell responses in convalescent COVID-19 subjects or COVID-19 mRNA vaccinees are not substantially affected by mutations found in the SARS-CoV-2 variants.

immunology↗
bioRxiv2021

MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution

Technical challenges remain in the sequencing of RNA viruses due to their high intra-host diversity. This bottleneck is particularly pronounced when interrogating long-range co-evolution given the read-length limitations of next-generation sequencing platforms. This has hampered the direct observation of long-range genetic interactions that code for protein-protein interfaces with relevance in both drug and vaccine development. Here we overcome these technical limitations by developing a nanopore-based long-range viral sequencing pipeline that yields accurate single molecule sequences of circulating virions from clinical samples. We demonstrate its utility in observing the evolution of individual HIV Gag-Pol genomes in response to antiviral pressure. Our pipeline, called Multi-read Hairpin Mediated Error-correction Reaction (MrHAMER), yields >1000s viral genomes per sample at 99.9% accuracy, maintains the original proportion of sequenced virions present in a complex mixture, and allows the detection of rare viral genomes with their associated mutations present at <1% frequency. This method facilitates scalable investigation of genetic correlates of resistance to both antiviral therapy and immune pressure, and enable the identification of novel host-viral and viral-viral interfaces that can be modulated for therapeutic benefit.

genomics↗
bioRxiv2020

Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases

T cells are involved in control of SARS-CoV-2 infection. To establish the patterns of immunodominance of different SARS-CoV-2 antigens, and precisely measure virus-specific CD4+ and CD8+ T cells, we studied epitope-specific T cell responses of approximately 100 convalescent COVID-19 cases. The SARS-CoV-2 proteome was probed using 1,925 peptides spanning the entire genome, ensuring an unbiased coverage of HLA alleles for class II responses. For HLA class I, we studied an additional 5,600 predicted binding epitopes for 28 prominent HLA class I alleles, accounting for wide global coverage. We identified several hundred HLA-restricted SARS-CoV-2-derived epitopes. Distinct patterns of immunodominance were observed, which differed for CD4+ T cells, CD8+ T cells, and antibodies. The class I and class II epitopes were combined into new epitope megapools to facilitate identification and quantification of SARS-CoV-2-specific CD4+ and CD8+ T cells.

immunology↗
bioRxiv2020

Cross-reactive serum and memory B cell responses to spike protein in SARS-CoV-2 and endemic coronavirus infection

Pre-existing immune responses to seasonal endemic coronaviruses could have profound consequences for antibody responses to SARS-CoV-2, either induced in natural infection or through vaccination. Such consequences are well established in the influenza and flavivirus fields. A first step to establish whether pre-existing responses can impact SARS-CoV-2 infection is to understand the nature and extent of cross-reactivity in humans to coronaviruses. We compared serum antibody and memory B cell responses to coronavirus spike (S) proteins from pre-pandemic and SARS-CoV-2 convalescent donors using a series of binding and functional assays. We found weak evidence of pre-existing SARS-CoV-2 cross-reactive serum antibodies in pre-pandemic donors. However, we found stronger evidence of pre-existing cross-reactive memory B cells that were activated on SARS-CoV-2 infection. Monoclonal antibodies (mAbs) isolated from the donors showed varying degrees of cross-reactivity with betacoronaviruses, including SARS and endemic coronaviruses. None of the cross-reactive mAbs were neutralizing except for one that targeted the S2 subunit of the S protein. The results suggest that pre-existing immunity to endemic coronaviruses should be considered in evaluating antibody responses to SARS-CoV-2.

immunology↗
bioRxiv2020

Rapid isolation of potent SARS-CoV-2 neutralizing antibodies and protection in a small animal model

The development of countermeasures to prevent and treat COVID-19 is a global health priority. In under 7 weeks, we enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate serum and monoclonal antibody responses, adapted our high throughput antibody isolation, production and characterization pipeline to rapidly screen over 1000 antigen-specific antibodies, and established an animal model to test protection. We report multiple highly potent neutralizing antibodies (nAbs) and show that passive transfer of a nAb provides protection against high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.

immunology↗