Biology subjects

Newman, A.

Publications and source records attributed to Newman, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Integrated Characterization of Proximal and Distal Nerve Stumps in a Model of Chronic Motor Nerve Transection

Prolonged peripheral nerve discontinuity subjects the proximal stump to chronic axotomy and the distal stump to chronic denervation, resulting in pathological changes that may compromise regeneration following delayed repair. Although these changes have been investigated in both nerve stumps, much of the existing evidence derives from mixed sensorimotor nerve models, leaving the histopathological changes of chronic motor nerve injury incompletely understood. Addressing this gap requires a well-defined, reproducible model that maintains prolonged motor nerve discontinuity and enables concurrent characterization of both nerve stumps. This study aimed to establish a chronic motor nerve transection model and characterize the distinct structural and microenvironmental changes occurring in both nerve stumps. The buccal branch of the facial nerve was transected in Lewis rats, and the nerve ends were secured to adjacent muscle to prevent spontaneous reconnection. At 10 weeks post-injury (p.i.), proximal and distal stumps were compared with uninjured nerves using histological, immunofluorescence, and morphometric analyses. Persistent functional impairment was observed throughout the study period. The proximal stump exhibited traumatic neuroma formation, altered axonal morphology, and loss of myelin integrity, but largely preserved its underlying nerve architecture and microenvironment. In contrast, the distal stump exhibited reduced neurofilament expression, clusters of small-caliber myelinated axons, and increased myelinated axon density compared to both uninjured nerves and the proximal chronically injured segment. These changes were accompanied by endoneurial tube atrophy, increased collagen I deposition, vascular remodeling, and immune-cell accumulation, indicating extensive structural and microenvironmental remodeling. Collectively, these findings demonstrate that chronic motor nerve transection produces two distinct pathological compartments: a proximal stump characterized by persistent but disorganized regeneration-associated features and a distal stump characterized by pronounced degenerative and microenvironmental changes. This study establishes a reproducible model of chronic peripheral nerve injury and provides a foundation for investigating how these compartment-specific pathologies contribute to regenerative failure and for developing targeted therapeutic strategies to improve regeneration following delayed nerve repair.

neuroscience↗

bioRxiv2026

Sexual Dimorphism and Geographic Variation in Washington Bobcat (Lynx rufus) Skulls

Sexual dimorphism is a well-studied phenomenon in which males and females of a species exhibit different phenotypic characteristics. However, the degree of sexual dimorphism within a species may vary among populations and may be influenced by geographic factors such as seasonality, prey availability, and population density. In this study, we examined the effects of sexual dimorphism and geographic region on the craniomandibular morphology of bobcats (Lynx rufus) in Washington State. Using digital calipers, we measured 13 craniomandibular traits from 35 males and 42 females. We found that the degree of skull dimorphism does not differ between the eastern and western side of the Cascade Mountains. However, we found significant effects of sex and geographic region on craniomandibular morphology. Across the geographic regions, males were significantly larger than females in most craniomandibular traits, excluding postorbital constriction breadth (POC). After correcting for expected size differences, only condylobasal length (CBL) was disproportionately longer in males, indicating subtle shape dimorphism within this trait, that is largely unexplained by allometry. Mechanical advantage (MA) of the temporalis and masseter muscles did not differ between the sexes at the canine or the molar bite points. Comparisons between populations east and west of the Cascade Range revealed that while sex remains the primary indicator of overall size, there is a small regional effect on size as western bobcats are slightly larger than eastern bobcats. These regional differences highlight how environmental pressures including local prey availability and severe winter conditions may influence functional skull design. Future ecological and behavioral studies are needed to further clarify how these localized environmental drivers shape phenotypic variation across populations.

evolutionary biology↗

bioRxiv2026

B Cell Receptor Signaling Sets Boundaries for Selection of Broadly Neutralizing Antibody Functional Mutations

B cell signaling is required for germinal center (GC) selection and synergizes with T cell-help leading to differentiation into effector cells and a protective antibody response. Here, we studied the relationship between B cell signaling and the selection of functional mutations in a humanized mouse model of a CD4 binding-site specific HIV-1 broadly neutralizing antibody precursor. While BCR signaling increased with antigen affinity, immunization-induced frequency of a functional mutation was inversely related to affinity and favored a gain in association rate. Antigen-specific GC B cells and key mutation frequency were higher in the mid-affinity (0.5 - 5M) than in either the higher or lower affinity group, and were consistent with the significantly higher serum neutralization titers in the mid-range group. Our studies show that BCR signaling imposes boundaries (upper/lower) for selection of antibody functional mutations and support a "Goldilocks Zone" model that defines the favored BCR signaling strength for GC selection.

immunology↗

bioRxiv2026

BCL11B predetermines a persister state in breast cancer that is reversed by TNFα

The frequent development of chemoresistance in cancer presents a significant clinical challenge, yet the underlying causes for heterogeneous drug responses remain largely elusive. Here, we systematically assessed the cellular differentiation status of human breast cancer cells utilizing single-cell atlases and identified a distinct population of immature basal-like cancer cells marked by BCL11B. Notably, higher levels of BCL11B+ cancer cells are significantly associated with early relapse in breast cancer patients who received chemotherapy. Functioning as a central regulator, BCL11B delineates an immature cell state that preferentially transitions to a drug-resistant persister state during treatment through multiple pre-existing and adaptive drug-resistance programs. Importantly, the cytokine tumor necrosis factor alpha (TNF) is revealed as a natural inhibitor of BCL11B and can directly reverse the emergence of chemoresistant persister cells. Therefore, we identify BCL11B as an unappreciated pre-determinant of drug response and a therapeutic target for a subset of breast cancer patients at high risk of developing chemoresistance.

Cancer Biology↗

bioRxiv2025

Tau interactions with inner nuclear envelope proteins modulates chromatin

Abstract/SummaryThe intracellular re-distribution of the neuronal microtubule-associated protein Tau, from the axon into the somatodendritic compartment, is a physiological stress-related event and occurs early in Alzheimers disease (AD). Nuclear envelope distortions have been linked to the presence and aggregation of pathological Tau near the nucleus in these diseases. How physiologically increased soma Tau levels, enabling Tau interactions with the nucleus, impact nuclear integrity and neuronal physiology is unclear. Combining proximity biotinylation interactomics with chromatin imaging and molecular assays, we show that soluble Tau interacts with proteins coordinating chromatin at the inner nuclear membrane, including lamin B receptor and SUN1. This interaction promotes nuclear envelope invaginations and damage and changes the coordination of DNA at the nuclear lamina. Increasing somatodendritic Tau is sufficient to upregulate the expression of multiple transcription factors implicated in AD pathogenesis and to reduce expression of genes involved in cholesterol biosynthesis, which seem coordinated at lamin associated domains. These nuclear envelope-related mechanisms suggest that physiological, neuronal stress-related somatodendritic Tau missorting can initiate chromatin-related cascades important for early changes in AD and tauopathies.

neuroscience↗

bioRxiv2025

Nuc domain electrostatics drive the trans cleavage activity of CRISPR-Cas12a

The trans cleavage activity of type V CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas12a system has been widely used for the detection of biomolecules. Different Cas12a orthologues exhibit faster or slower trans cleavage kinetics, making some orthologues more suited for sensitive molecular detection. Ionic strength of reaction buffers, and mutations that change the electrostatic environment near the RuvC active site have also been reported to strongly influence trans cleavage kinetics. Studying three commonly used Cas12a orthologues (FnCas12a, AsCas12a, and LbCas12a), we report that electrostatic interactions near the RuvC active site are critical for their trans cleavage activity. Alanine substitution of arginine and lysine residues in the Nuc domain can abolish trans cleavage while modestly reducing cis cleavage. Substitutions of the RuvC lid and substitutions to introduce positively charged residues in the Nuc could enhance both cis and trans cleavage. These Cas12a variants improved DNA detection and genome editing efficiency. Overall this study provides a blueprint for future rational engineering of Cas12a nucleases for their trans cleavage activities. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/684486v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@6de4aeorg.highwire.dtl.DTLVardef@c06f37org.highwire.dtl.DTLVardef@1c5c1d0org.highwire.dtl.DTLVardef@375002_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Integrating 12 Spatial and Single Cell Technologies to Characterise Tumour Neighbourhoods and Cellular Interactions in three Skin Cancer Types

Cutaneous squamous cell carcinoma (cSCC), basal cell carcinoma (BCC), and melanoma - the three major skin cancers - collectively comprise over 70% of all cancer cases. Despite their prevalence, much understanding of cellular interactions in the skin cancer microenvironment is needed, both in the outer skin layer where the cancer originates and at the deeper junctional and dermal layers into which it progresses. To address this gap, we integrated 12 complementary spatial and single-cell technologies to generate orthogonally-validated cell signatures, spatial maps, and interactomes for cSCC, BCC, and melanoma. Through comprehensive comparisons and integrating these spatial methods, we provided practical benchmarking guidelines for experimental design and analysis. By identifying keratinocyte cancer cells and melanomas, we found distinct signatures of these cells compared to non-cancer keratinocytes and melanocytes. Spatial integration of transcriptomics, proteomics and glycomics uncovered cancer niches enriched for cancer initiating cells (melanocytes or keratinocytes) and fibroblast and T-cell (MKFT) clusters, with altered tyrosine and pyrimidine metabolism. Ligand-receptor analysis across >700 cell-type combinations and >1.5 million interactions highlighted key roles for CD44, integrins, and collagens, with CD44-FGF2 emerging as a potential therapeutic target. Consistently, melanoma showed strong MFT interactions, validated by Opal Polaris, RNAScope, Proximal Ligation Assay and two additional single-cell spatial platforms (making a total of 14 technologies). For population-scale generalisation, genetic associations from >500,000 individuals were mapped onto spatial skin tissues, identifying SNPs enriched in domains containing melanocytes and T cells and their ligand-receptor pairs, shedding light on functional mechanisms linking genetic heritability to cells within cancer tissue. We built an interactive multiomics resource for exploring spatially-resolved molecular signatures and cellular crosstalk in skin cancer, available at https://skincanceratlas.com.

cancer biology↗

bioRxiv2025

CRISPR-Cas12a REC2 - NUC interactions drive target-strand cleavage and constrain trans cleavage.

CRISPR-Cas12a effects RNA-guided cleavage of dsDNA in cis, after which it remains catalytically active and non-specifically cleaves ssDNA in trans. Native host-defence by Cas12a employs cis cleavage, which can be repurposed for the genome editing of other organisms, and trans cleavage can be used for in vitro DNA detection. Cas12a orthologues have high structural similarity and a conserved mechanism of DNA cleavage, yet highly different efficacies when applied for genome editing or DNA detection. By comparing three well characterised Cas12a orthologues (FnCas12a, LbCas12a, and AsCas12a), we sought to determine what drives their different cis and trans cleavage, and how this relates to their applied function. We integrated in vitro DNA cleavage kinetics with molecular dynamics simulations, plasmid interference in E. coli, and genome editing in human cell lines. We report large differences in cis cleavage kinetics between orthologues, which may be driven by dynamic REC2-NUC interactions. We generated and tested REC2 and NUC mutants, including a hitherto unstudied NUC loop, integrity of which is critical for the function of Cas12 orthologues. In total, our in vitro, in vivo, and in silico survey of Cas12a orthologues highlights key properties that drive their function in biotechnology applications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/644851v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@18c702aorg.highwire.dtl.DTLVardef@11816d1org.highwire.dtl.DTLVardef@822316org.highwire.dtl.DTLVardef@1cda6bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Parkinson's Paradox: Alpha-synuclein's Selective Strike on SNc Dopamine Neurons over VTA

In synucleinopathies, including Parkinsons disease (PD), dopamine neurons in the substantia nigra pars compacta (SNc) exhibit greater vulnerability to degeneration than those in the ventral tegmental area (VTA). While -synuclein (Syn) pathology is implicated in nigral dopamine neuron loss, the mechanisms by which Syn affects neuronal activity and midbrain dopamine network connectivity prior to cell death remain unclear. This study tested the hypothesis that elevated Syn expression induces pathophysiological changes in firing activity and disrupts network connectivity dynamics of dopamine neurons before neuronal loss. We employed two mouse models of synucleinopathy: preformed Syn fibril (PFF) injection and AAV-mediated expression of human Syn (hSyn) under the control of the tyrosine hydroxylase (TH) promoter, both targeting the VTA and SNc. Four weeks post-injection, brain sections underwent histological, electrophysiological, and network analyses. Immunohistochemistry for TH, hSyn, and phospho-Ser129 Syn assessed Syn expression and dopaminergic neuron alterations. Neuronal viability was evaluated using two complementary approaches: quantification of TH+ or FOX3+ and TUNEL labeling. Importantly, these analyses revealed no significant changes in neuronal counts or TUNEL+ cells at this time point, confirming that subsequent functional assessments captured pre-neurodegenerative, Syn-induced alterations rather than late-stage neurodegeneration. Electrophysiological recordings revealed a differential effect of hSyn expression. SNc dopamine neurons exhibited significantly increased baseline firing rates, whereas VTA dopamine neurons remained unchanged. These findings indicate a region-specific vulnerability to Syn-induced hyperactivity of dopamine neurons. Further analysis revealed impaired homeostatic firing rate regulation in SNc, but not VTA, dopamine neurons, demonstrated by a reduced capacity to recover baseline firing following hyperpolarization. Collectively, our results demonstrate that, prior to neurodegeneration, elevated Syn expression differentially disrupts both basal firing activity and network stability of SNc dopamine neurons, while sparing VTA dopamine neurons. By identifying neurophysiological changes preceding dopaminergic neuron loss, these findings provide critical insights into the pathophysiological mechanisms predisposing SNc neurons to degeneration in Parkinsons disease. Significance StatementA central question in Parkinsons disease research is why dopamine neurons in the substantia nigra pars compacta (SNc) are more vulnerable than those in the ventral tegmental area (VTA). This study reveals that alpha-synuclein (Syn) pathology differentially impacts dopamine neuronal activity and network connectivity, causing changes in the SNc before neuronal loss occurs, but not in the VTA. These findings provide a mechanism to explain the differential resilience of these neighboring dopamine neuron populations and provide insights into Parkinsons disease progression. The methodologies developed in this study establish a foundation for investigating network topology in deep brain structures and its role in neurodegenerative disorders.

neuroscience↗

bioRxiv2025

Design of SARS-CoV-2 RBD Immunogens to Focus Immune Responses Towards Conserved Coronavirus Epitopes

SARS-CoV-2 continues to evolve, with new variants emerging that evade pre-existing immunity and limit the efficacy of existing vaccines. One approach towards developing superior, variant-proof vaccines is to engineer immunogens that preferentially elicit antibodies with broad cross-reactivity against SARS-CoV-2 and its variants by targeting conserved epitopes on spike. The inner and outer faces of the Receptor Binding Domain (RBD) are two such conserved regions targeted by antibodies that recognize diverse human and animal coronaviruses. To promote the elicitation of such antibodies by vaccination, we engineered "resurfaced" RBD immunogens that contained mutations at exposed RBD residues outside the target epitopes. In the context of pre-existing immunity, these vaccine candidates aim to disfavor the elicitation of strain-specific antibodies against the immunodominant Receptor Binding Motif (RBM) while boosting the induction of inner and outer face antibodies. The engineered resurfaced RBD immunogens were stable, lacked binding to monoclonal antibodies with limited breadth, and maintained strong interactions with target broadly neutralizing antibodies. When used as vaccines, they limited humoral responses against the RBM as intended. Multimerization on nanoparticles further increased the immunogenicity of the resurfaced RBDs immunogens, thus supporting resurfacing as a promising immunogen design approach to rationally shift natural immune responses to develop more protective vaccines.

immunology↗

bioRxiv2025

MicroRNA signatures of VO2peak in older adult participants of the Study of Muscle, Mobility and Aging

BackgroundPeak oxygen consumption during exercise (VO2peak), is a direct measure of cardiorespiratory fitness (CF), a key indicator of physical function and overall health. However, the molecular changes that underpin VO2peak variation are not clear. Our objective is to understand the miRNA signatures that relate to VO2peak variation, which could provide insights to novel mechanisms that contribute to low VO2peak. MethodsWe used small RNA sequencing to analyze serum samples from 72 participants (70-79 yrs old, 53% female) of the Study of Muscle, Mobility and Aging (SOMMA). We analyzed samples from individuals with low or high VO2peak (N=18/group) as well as samples from 36 randomly selected participants spanning the entire spectrum of VO2peak. We used LIMMA analysis package for regression analysis and to identify differentially expressed miRNAs. We used receiver operating characteristic curve analysis to evaluate the Area Under the Curve (AUC) and sensitivity and specificity rates. ResultsWe identified 1,055 miRNAs expressed in all serum samples. Expression of 65 miRNAs differed between participants with low and high VO2peak (p < 0.05). After p-value adjustment, expression of 5 miRNAs (miR-1301-3p, -431-5p, -501-5p, -519a-3p, and -18a-3p) remained significantly different (FDR = 0.05). The five miRNAs had AUC ranging from 0.77 to 0.84. The optimal sensitivity and specificity ranged from 70 to 80% and 80 to 90%, respectively. After adjustment for age and sex covariates, 46 miRNAs significantly correlated with VO2peak (p < 0.05) and miR-519a-3p remained significant based on adjusted of p-values. ConclusionsWe identified a miRNA signature of VO2peak in older individuals that might provide insights to novel mechanisms that drive low VO2peak. Future studies will validate the findings in a larger, longitudinal study cohort.

molecular biology↗

bioRxiv2024

Structural and antigenic characterization of novel and diverse Henipavirus glycoproteins

Henipaviruses, a genus within the Paramyxoviridae family, include the highly virulent Nipah and Hendra viruses that cause reoccurring outbreaks of deadly disease. Recent discoveries of several new Paramyxoviridae species, including the zoonotic Langya virus, have revealed much higher antigenic diversity than currently characterized and prompted the reorganization of these viruses into the Henipavirus and Parahenipavirus genera. Here, to explore the limits of structural and antigenic variation in both genera, collectively referred to here as HNVs, we constructed an expanded, antigenically diverse panel of HNV fusion and attachment glycoproteins from 56 unique HNV strains that better reflects global HNV diversity. We expressed and purified the fusion protein ectodomains and the attachment protein head domains and studied their biochemical, biophysical and structural properties. We performed immunization experiments in mice leading to the elicitation of antibodies reactive to multiple HNV fusion proteins. Cryo-electron microscopy structures of diverse fusion proteins elucidated molecular determinants of differential pre-fusion state metastability and higher order contacts. A crystal structure of the Gamak virus attachment head domain revealed an additional domain appended to the conserved 6-bladed, {beta}-propeller fold. Taken together, these studies expand the known structural and antigenic limits of the HNVs, reveal new cross-reactive epitopes within both genera and provide foundational data for the development of broadly reactive countermeasures.

biochemistry↗

bioRxiv2024

SpaMTP: Integrative Statistical Analysis and Visualisation of Spatial Metabolomics and Transcriptomics data.

The ability to spatially measure multi-modal data provides an unprecedented opportunity to comprehensively explore molecular regulation at transcriptional, translational and metabolic levels to acquire insights on cellular activities underpinning health and disease. However, there is currently a lack of analytical tools to integrate complementary information across different spatial-omics data modalities, particularly with respect to spatial metabolomics data, which is becoming increasingly invaluable. We introduce SpaMTP, a versatile software that implements an end-to-end integrative analysis of spatial metabolomics and transcriptomics data. Based in R, SpaMTP bridges processing functionalities for metabolomics data from Cardinal with user-friendly cell-centric analyses implemented in Seurat. Furthermore, SpaMTPs comprehensive analysis pipeline covers (1) automated mass-to-charge ratio (m/z) metabolite annotation; (2) a wide range of metabolite-gene based downstream statistical analyses including differential expression, pathway analysis, and correlation analysis; (3) integrative spatial-omics analysis; and (4) a suite of visualisation functions. For flexibility and interoperability, SpaMTP includes various functions for data import/export and object conversion, enabling seamless integration with other R and Python packages. We demonstrated the utility of SpaMTP to draw new biological understandings through analysing two biological system. We believe this software and implemented methods will be broadly utilised in spatial multi-omics and spatial metabolomics analyses.

bioinformatics↗

bioRxiv2024

Unraveling lncRNA Diversity at a Single Cell Resolution and in a Spatial Context across Different Cancer Types

Long non-coding RNAs (lncRNAs) play pivotal roles in gene regulation and disease, including cancer. Overcoming the limitations of lncRNA analysis with bulk data, we analyzed single-cell and spatial transcriptomics data to uncover 354937 novel lncRNAs and their functions across 13 cancer types. LncRNA functions were assessed by identifying their cell-type specificity and distinct spatial distributions across different tissue regions. First, lncRNAs were computationally validated by comparing to existing databases, and experimentally validated using spatial long read sequencing methods. Further, genome-wide computation of spatial-autocorrelation identified coexpression of lncRNAs with cancer-associated protein coding genes across the tissue. Additionally, genomic co-localization of lncRNAs with regulatory features and disease-associated genetic variants suggest possible functional association. The identified lncRNAs were analyzed for responses to immunotherapy and prognostic value, revealing cancer-outcome associated lncRNAs. We have made this novel resource available as an open website SPanC-Lnc hosted on AWS cloud to serve as a pan-cancer atlas of single cell- and spatially-resolved lncRNAs. These can complement established biomarkers because they reflect the unique characteristics of specific cell populations within tumors, offering new insights into disease progression and treatment response.

cancer biology↗

bioRxiv2024

Sub-clinical glutamate receptor antagonist combinations prevent progressive demyelination.

Multiple sclerosis (MS) affects almost 3 million people globally who suffer demyelination as a series of relapses and remissions that tend towards progressive deterioration over time. The proximate cause is auto-immune attack by the adaptive immune system; therapies directed against this are effective during the relapsing-remitting phase but are less effective or ineffective during progression where other injury mechanisms may be significant. LPS- and cuprizone-induced experimental demyelination share features of progressive demyelination in MS but the underlying mechanisms are not well understood. We show here that these demyelination models can be reproduced ex vivo using short protocols, revealing that combined antagonism of two types of glutamate receptor, NMDA and AMPA, using clinically approved antagonists at sub-clinical doses, can protect against these forms of demyelination. Combined low dose therapy was subsequently shown to be effective in vivo against dietary cuprizone and experimental autoimmune-encephalomyelitis (EAE) models of demyelination and, in particular, protected the smaller myelinated axons that are the main substrate of the function loss in progressive MS.

neuroscience↗

bioRxiv2024

Autoimmune antibody-induced neuronal hyperactivity triggers pathological Tau in IgLON5 disease

Anti-IgLON5 disease is an autoimmune disease, in which autoantibodies (AABs) against the neuronal cell surface protein IgLON5 lead to profound brain dysfunction and Tau pathology. How -IgLON5 AABs cause neuronal Tau protein pathology and neurodegeneration remains unclear. We find that patient-derived -IgLON5 AABs cluster IgLON5 proteins with other cell surface proteins, leading to neuronal hyperactivity that triggers pathological Tau missorting and phosphorylation, typically observed early in Tau-related neurodegenerative diseases. In wildtype mice, -IgLON5 AABs induce hippocampal Tau phosphorylation and neuroinflammatory responses. Our findings establish a causal link between the -IgLON5 AABs and Tau pathology in anti-IgLON5 disease patients, and highlight the role of neuronal hyperactivity as a disease-overarching driver of Tau pathology and provide a potential target for therapeutic intervention. Teaser-IgLON5 autoantibodies induce clustering of neuronal cell surface proteins, leading to acute neuronal hyperactivity and Tau missorting.

neuroscience↗

bioRxiv2023

Broadly neutralizing antibody induction by non-stabilized SARS-CoV-2 Spike mRNA vaccination in nonhuman primates

Immunization with mRNA or viral vectors encoding spike with diproline substitutions (S-2P) has provided protective immunity against severe COVID-19 disease. How immunization with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike elicits neutralizing antibodies (nAbs) against difficult-to-neutralize variants of concern (VOCs) remains an area of great interest. Here, we compare immunization of macaques with mRNA vaccines expressing ancestral spike either including or lacking diproline substitutions, and show the diproline substitutions were not required for protection against SARS-CoV-2 challenge or induction of broadly neutralizing B cell lineages. One group of nAbs elicited by the ancestral spike lacking diproline substitutions targeted the outer face of the receptor binding domain (RBD), neutralized all tested SARS-CoV-2 VOCs including Omicron XBB.1.5, but lacked cross-Sarbecovirus neutralization. Structural analysis showed that the macaque broad SARS-CoV-2 VOC nAbs bound to the same epitope as a human broad SARS-CoV-2 VOC nAb, DH1193. Vaccine-induced antibodies that targeted the RBD inner face neutralized multiple Sarbecoviruses, protected mice from bat CoV RsSHC014 challenge, but lacked Omicron variant neutralization. Thus, ancestral SARS-CoV-2 spike lacking proline substitutions encoded by nucleoside-modified mRNA can induce B cell lineages binding to distinct RBD sites that either broadly neutralize animal and human Sarbecoviruses or recent Omicron VOCs. One Sentence SummaryNon-stabilized SARS-CoV-2 Spike mRNA vaccination activated B cells that target either conserved epitopes on SARS-CoV-2 Omicron variants of concern, or cross-neutralizing epitopes on pre-emergent Sarbecoviruses.

immunology↗

bioRxiv2023

Engineering immunogens that select for specific mutations in HIV broadly neutralizing antibodies

Vaccine development targeting rapidly evolving pathogens such as HIV-1 requires induction of broadly neutralizing antibodies (bnAbs) with conserved paratopes and mutations, and, in some cases, the same Ig-heavy chains. The current trial-and-error search for immunogen modifications that improve selection for specific bnAb mutations is imprecise. To precisely engineer bnAb boosting immunogens, we used molecular dynamics simulations to examine encounter states that form when antibodies collide with the HIV-1 Envelope (Env). By mapping how bnAbs use encounter states to find their bound states, we identified Env mutations that were predicted to select for specific antibody mutations in two HIV-1 bnAb B cell lineages. The Env mutations encoded antibody affinity gains and selected for desired antibody mutations in vivo. These results demonstrate proof-of-concept that Env immunogens can be designed to directly select for specific antibody mutations at residue-level precision by vaccination, thus demonstrating the feasibility of sequential bnAb-inducing HIV-1 vaccine design.

immunology↗