Biology subjects

Nguyen, B.

Publications and source records attributed to Nguyen, B..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

CD11b suppresses TLR7-driven inflammatory signaling to protect against lupus nephritis

Lupus Nephritis (LN) is a severe complication of systemic lupus erythematosus (SLE) that affects kidney function. Here, we investigated the role of CD11b, a protein encoded by the ITGAM gene, in the development of LN and its functional activation as a therapeutic strategy. Genetic coding variants of ITGAM significantly increase the risk for SLE and LN by producing a less active CD11b and leading to elevated levels of type I interferon (IFN I). However, a molecular mechanism for how these variants increase LN risk has been unclear. Here, we determined that these variants also significantly associate with elevations in soluble urokinase plasminogen activator receptor (suPAR), a known biomarker linked to kidney disease, suggesting a novel molecular connection. Pharmacologic activation of CD11b with a novel, clinical-stage agonist ONT01 significantly suppressed suPAR production in myeloid cells and reduced systemic inflammation and kidney damage in multiple experimental models of LN. Importantly, delaying treatment with ONT01 until after disease onset also significantly reduced serum suPAR and inflammatory cytokines, and decreased immune complex deposition in the glomerulus, glomerulonephritis and albuminuria, suggesting that CD11b activation is therapeutic for LN. Genetic activation of CD11b via a gain-of-function CD11b mutation also showed complete protection from LN, whereas genetic deletion of CD11b worsened the disease in mice, providing further evidence of the role of CD11b activation in regulating LN. Finally, transfer of human LN PBMCs generated human LN like disease in mice that was significantly reduced by ONT01. Together, these data provide strong evidence that ONT01 mediated CD11b activation can therapeutically modulate TLR7-driven inflammation and protect against LN. These findings support clinical development of CD11b agonists as novel therapeutics for treating lupus nephritis in human patients.

immunology↗

bioRxiv2024

Dietary resilience of coral reef fishes to habitat degradation

The ability of consumers to adjust their diet in response to resource shifts is a key mechanism allowing the persistence of populations and underlying species adaptive capacity. Yet on coral reefs, one of the marine habitats most vulnerable to global change, the extent to which species alter their diet remains poorly understood. Here, we integrated DNA-based gut content analyses (metabarcoding), otolith analysis, body condition, and field surveys to test how diet can mediate effects of habitat degradation on two invertivorous fishes: Chaetodon capistratus, a browser, and Hypoplectrus puella, an active predator. Metabarcoding revealed significant dietary variation in both species across a habitat gradient. However, the response was more pronounced in the browser, whose diet was anthozoan-dominated on healthy reefs, whereas annelid-dominated on degraded reefs. We found reduced growth and body condition on degraded reefs in the browser but not the active predator. Our results reveal that dietary versatility can serve as a mechanism to cope with degraded environments, but that species differ as to whether these changes are sufficient to buffer from changes in habitat. We detected intraspecific dietary variation across sites that suggests food webs and energy flow differ at relatively small scales between healthy and degraded reefs.

ecology↗

bioRxiv2024

AHR activation accelerates the resolution of TGF-β1 induced fibroblast activation and promotes alveolar type 1 cell regeneration in alveolar organoids

Regeneration of the alveolar epithelium is necessary to restore tissue architecture and gas exchange capabilities in chronic pulmonary diseases such as fibrosing interstitial lung disease. While it is known alveolar type 2 (AT2) cells give rise to alveolar type 1 (AT1) cells to repair the alveolar epithelium after injury, methods to promote this process under pathological settings are poorly understood. Here, using a complex 3D organoid culture with TGF-{beta}1 dependent impaired AT1 spheroid formation, we performed a high-throughput screen (HTS) with [~]16,800 compounds to identify small molecules that increase number of AT1 spheroids. Longitudinal single cell RNA sequencing (scRNA-seq) revealed that DB-11-BE87 increased AT1 regeneration by reducing TGF-{beta}1 induced fibroblast activation, concurrently with AHR activation in those cells. These studies highlight a novel HTS system to identify factors that can promote AT1 differentiation and suggest AHR activation as a method to counteract pathological TGF-{beta}1 signaling in pulmonary disease.

cell biology↗

bioRxiv2024

A stress-dependent TDP-43 SUMOylation program preserves neuronal function

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are overwhelmingly linked to TDP-43 dysfunction. Mutations in TDP-43 are rare, indicating that the progressive accumulation of exogenous factors - such as cellular stressors - converge on TDP-43 to play a key role in disease pathogenesis. Post translational modifications such as SUMOylation play essential roles in response to such exogenous stressors. We therefore set out to understand how SUMOylation may regulate TDP-43 in health and disease. We find that TDP-43 is regulated dynamically via SUMOylation in response to cellular stressors. When this process is blocked in vivo, we note age-dependent TDP-43 pathology and sex-specific behavioral deficits linking TDP-43 SUMOylation with aging and disease. We further find that SUMOylation is correlated with human aging and disease states. Collectively, this work presents TDP-43 SUMOylation as an early physiological response to cellular stress, disruption of which may confer a risk for TDP-43 proteinopathy.

neuroscience↗

bioRxiv2024

WISH-BONE: Whole-mount In Situ Histology, to label osteocyte mRNA and protein in 3D adult mouse bones

Bone is a three dimensional and highly dynamic tissue under constant remodeling. Commonly used tools to investigate bone biology require sample digestion for biomolecule extraction or only provide two-dimensional spatial information. There is a need for 3D tools to investigate spatially preserved biomarker expression in osteocytes. In this work, we present a new method, WISH-BONE, to label osteocyte mRNA and protein in whole-mount mouse bone. For mRNA labeling, we used Hybridization Chain Reaction - Fluorescence In Situ Hybridization to label genes of interest in osteocytes. For protein labeling, samples were preserved using an epoxy-based solution that protects tissue structure and biomolecular components. Then an enzymatic matrix permeabilization step was performed to enable antibody penetration. Immunostaining was used to label various proteins involved in bone homeostasis. We also demonstrate the use of customized fluorescent nanobodies to target and label proteins in the cortical bone. However, the relatively dim signal observed from nanobodies staining limited detection. In this study, we share protocols, highlight opportunities, and identify the challenges of this novel 3D labeling method. They are the first protocols for whole-mount osteocyte 3D labeling of mRNA and protein in mature mouse bones. WISH-BONE will allow the investigation of molecular signaling in bone cells in their 3D environment and could be applied to various bone-related fields of research.

bioengineering↗

bioRxiv2024

Structure-based probe reveals the presence of large transthyretin aggregates in plasma of ATTR amyloidosis patients

ATTR amyloidosis is a relentlessly progressive disease caused by the misfolding and systemic accumulation of amyloidogenic transthyretin into amyloid fibrils. These fibrils cause diverse clinical phenotypes, mainly cardiomyopathy and/or polyneuropathy. Little is known about the aggregation of transthyretin during disease development and whether this has implications for diagnosis and treatment. Using the cryogenic electron microscopy structures of mature ATTR fibrils, we developed a peptide probe for fibril detection. With this probe, we have identified previously unknown aggregated transthyretin species in plasma of patients with ATTR amyloidosis. These species are large, non-native, and distinct from monomeric and tetrameric transthyretin. Observations from our study open many questions about the biology of ATTR amyloidosis and reveals a potential diagnostic and therapeutic target.

molecular biology↗

bioRxiv2024

Subunit Communication within Dimeric SF1 DNA Helicases

Monomers of the Superfamily (SF) 1 helicases, E. coli Rep and UvrD, can translocate directionally along single stranded (ss) DNA, but must be activated to function as helicases. In the absence of accessory factors, helicase activity requires Rep and UvrD homo-dimerization. The ssDNA binding sites of SF1 helicases contain a conserved aromatic amino acid (Trp250 in Rep and Trp256 in UvrD) that stacks with the DNA bases. Here we show that mutation of this Trp to Ala eliminates helicase activity in both Rep and UvrD. Rep(W250A) and UvrD(W256A) can still dimerize, bind DNA, and monomers still retain ATP-dependent ssDNA translocase activity, although with lower processivity than wild type monomers. Although neither wtRep monomers nor Rep(W250A) monomers possess helicase activity by themselves, using both ensemble and single molecule methods, we show that helicase activity is achieved upon formation of a Rep(W250A)/wtRep hetero-dimer. An ATPase deficient Rep monomer is unable to activate a wtRep monomer indicating that ATPase activity is needed in both subunits of the Rep hetero-dimer. We find the same results with E. coli UvrD and its equivalent mutant (UvrD(W256A)). Importantly, Rep(W250A) is unable to activate a wtUvrD monomer and UvrD(W256A) is unable to activate a wtRep monomer indicating that specific dimer interactions are required for helicase activity. We also demonstrate subunit communication within the dimer by virtue of Trp fluorescence signals that only are present within the Rep dimer, but not the monomers. These results bear on proposed subunit switching mechanisms for dimeric helicase activity.

biochemistry↗

bioRxiv2023

Mycobacterium tuberculosis Ku stimulates multi-round DNA unwinding by UvrD1 monomers

Mycobacterium tuberculosis is the causative agent of Tuberculosis. During the host response to infection, the bacterium is exposed to both reactive oxygen species and nitrogen intermediates that can cause DNA damage. It is becoming clear that the DNA damage response in Mtb and related actinobacteria function via distinct pathways as compared to well-studied model bacteria. For example, we have previously shown that the DNA repair helicase UvrD1 is activated for processive unwinding via redox-dependent dimerization. In addition, mycobacteria contain a homo-dimeric Ku protein, homologous to the eukaryotic Ku70/Ku80 dimer, that plays roles in double-stranded break repair via non-homologous end-joining. Ku has been shown to stimulate the helicase activity of UvrD1, but the molecular mechanism, as well as which redox form of UvrD1 is activated, is unknown. We show here that Ku specifically stimulates multi-round unwinding by UvrD1 monomers which are able to slowly unwind DNA, but at rates 100-fold slower than the dimer. We also demonstrate that the UvrD1 C-terminal Tudor domain is required for the formation of a Ku-UvrD1 protein complex and activation. We show that Mtb Ku dimers bind with high nearest neighbor cooperativity to duplex DNA and that UvrD1 activation is observed when the DNA substrate is bound with two or three Ku dimers. Our observations reveal aspects of the interactions between DNA, Mtb Ku, and UvrD1 and highlight the potential role of UvrD1 in multiple DNA repair pathways through different mechanisms of activation. Declaration of Interests None

biophysics↗

bioRxiv2023

Disease-associated patterns of acetylation stabilize tau fibril formation

Assembly of the microtubule-associated protein into tauopathy fibril conformations dictates the pathology of a diversity of diseases. Recent cryogenic Electron Microscopy (cryo-EM) structures have uncovered distinct fibril conformations in different tauopathies but it remains unknown how these structures fold from a single protein sequence. It has been proposed that post-translational modifications may drive tau assembly but no direct mechanism for how modifications drive assembly has emerged. Leveraging established aggregation-regulating tau fragments that are normally inert, we tested the effect of chemical modification of lysines with acetyl groups on tau fragment conversion into amyloid aggregates. We identify specific patterns of acetylation that flank amyloidogenic motifs on the tau fragments that drive rapid fibril assembly. To understand how this pattern of acetylation may drive assembly, we determined a 3.9 [A] cryo-EM structure of an amyloid fibril assembled from an acetylated tau fragment. The structure uncovers how lysine acetylation patterns mediate gain-of-function interactions to promote amyloid assembly. Comparison of the structure to an ex vivo tau fibril conformation from Picks Disease reveals regions of high structural similarity. Finally, we show that our lysine- acetylated sequences exhibit fibril assembly activity in cell-based tau aggregation assays. Our data uncover the dual role of lysine residues in limiting aggregation while their acetylation leads to stabilizing pro-aggregation interactions. Design of tau sequence with specific acetylation patterns may lead to controllable tau aggregation to direct folding of tau into distinct folds.

biophysics↗

bioRxiv2022

Helicase Activity Promoted Through Dynamic Interactions Between a ssDNA Translocase and a Diffusing SSB Protein

Replication protein A (RPA) is a eukaryotic single stranded (ss) DNA binding (SSB) protein that is essential for all aspects of genome maintenance. RPA binds ssDNA with high affinity but can also diffuse along ssDNA. By itself, RPA is capable of transiently disrupting short regions of duplex DNA by diffusing from a ssDNA that flanks the duplex DNA. Using single molecule total internal reflection fluorescence and optical trapping combined with fluorescence approaches we show that S. cerevisiae Pif1 can use its ATP-dependent 5 to 3 translocase activity to chemo-mechanically push a single human RPA (hRPA) directionally along ssDNA at rates comparable to those of Pif1 translocation alone. We further show that using its translocation activity Pif1 can push hRPA from a ssDNA loading site into a duplex DNA causing stable disruption of at least 9 bp of duplex DNA. These results highlight the dynamic nature of hRPA enabling it to be readily reorganized even when bound tightly to ssDNA and demonstrate a new mechanism by which directional DNA unwinding can be achieved through the combined action of a ssDNA translocase that pushes an SSB protein.

biophysics↗

bioRxiv2022

Single-strain behavior predicts responses to environmental pH and osmolality in the gut microbiota.

Changes to gut environmental factors such as pH and osmolality due to disease or drugs correlate with major shifts in microbiome composition; however, we currently cannot predict which species can tolerate such changes or how the community will be affected. Here, we assessed the growth of 92 representative human gut bacterial strains spanning 28 families across multiple pH values and osmolalities in vitro. The ability to grow in extreme pH or osmolality conditions correlated with the availability of known stress response genes in many cases, but not all, indicating that novel pathways may participate in protecting against acid or osmotic stresses. Machine learning analysis uncovered genes or subsystems that are predictive of differential tolerance in either acid or osmotic stress. For osmotic stress, we corroborated the increased abundance of these genes in vivo during osmotic perturbation. The growth of specific taxa in limiting conditions in isolation in vitro correlated with survival in complex communities in vitro and in an in vivo mouse model of diet-induced intestinal acidification. Our data show that in vitro stress tolerance results are generalizable and that physical parameters may supersede interspecies interactions in determining the relative abundance of community members. Importantly, we provide an extensive resource for predicting shifts in microbial composition and gene abundance in complex perturbations. Furthermore, this work highlights the physical environment as a major driver of bacterial composition and the importance of performing physical measurements in animal and clinical studies to elucidate the drivers of shifts in microbiota abundance. Significance StatementChanges in pH and particle concentration (osmolality) commonly result from gut disease or the ingestion of common drugs, causing changes in bacterial growth and microbiota composition within the intestine. Thus far, the effects of physical parameters on the growth of intestinal bacterial taxa have not been well documented in the context of predicting microbiota community composition. To address this gap, we examined the growth of 92 bacterial species under varying pH and osmolality conditions. We found that physical parameters are key predictors of bacterial abundance in individual-strain cultures and in complex bacterial communities. Moreover, our results identified specific genes and pathways that are predictive of growth in specific environments. Together, these findings can aid in determining the effectiveness of microbiota therapies in gut environments subjected to various perturbations.

microbiology↗

bioRxiv2022

Modifying the trajectory of epicardial leads can substantially reduce MRI-induced RF heating in pediatric patients with a cardiac implantable electronic device

PurposeInfants and children with congenital heart defects, inherited arrhythmia syndromes, and congenital cardiac conduction disorders often receive epicardial implantable electronic devices. Unfortunately, once an epicardial device is implanted, the patient is no longer eligible to receive MRI exams due to an elevated risk of RF heating. Here we show that a simple modification in the trajectory of epicardial leads can substantially and reliably reduce RF heating during MRI at 1.5 T, with benefits extending to abandoned leads. MethodsElectromagnetic simulations were performed to assess RF heating of two common epicardial lead trajectories exhibiting different degrees of coupling with MRI incident electric fields. Experiments in anthropomorphic phantoms implanted with commercial cardiac implantable electronic devices (CIEDs) confirmed the findings. ResultsSimulations of an epicardial lead with a trajectory where the excess length of the lead was looped and placed on the anterior surface of the heart showed a 9-fold reduction in 0.1g-averaged SAR compared to the lead with excess length looped on the inferior surface of the heart. Repeated experiments with a commercial epicardial device confirmed the results, showing a 16-fold reduction in the average temperature rise for fully implanted systems with leads following low-SAR trajectories, and a 20-fold reduction in RF heating on an abandoned lead. ConclusionSurgical modification of epicardial lead trajectory can substantially reduce RF heating at 1.5 T, with benefits extending to abandoned leads.

biophysics↗

bioRxiv2022

Genetic and pharmacological reduction of CDK14 mitigates synucleinopathy

Parkinsonas disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of -Synuclein (-Syn) protein. Currently, no treatment can slow nor halt the progression of PD. Multiplications and mutations of the -Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress -Syn replicate several features of PD. Decreasing total -Syn levels, therefore, is an attractive approach to slow down neurodegeneration in patients with synucleinopathy. We previously performed a genetic screen for modifiers of -Syn levels and identified CDK14, a kinase of largely unknown function as a regulator of -Syn. To test the potential therapeutic effects of CDK14 reduction in PD, we ablated Cdk14 in the -Syn preformed fibrils (PFF)-induced PD mouse model. We found that loss of Cdk14 mitigates the grip strength deficit of PFF-treated mice and ameliorates PFF-induced cortical -Syn pathology, indicated by reduced numbers of pS129 -Syn-containing cells. In primary neurons, we found that Cdk14 depletion protects against the propagation of toxic -Syn species. We further validated these findings on pS129 -Syn levels in PD patient neurons. Finally, we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable in vitro and in vivo. We found that CDK14 inhibition decreases total and pathologically aggregated -Syn in human neurons, in PFF- challenged rat neurons and in the brains of -Syn-humanized mice. In summary, we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy.

neuroscience↗

bioRxiv2022

The impact of multigenerational high-fat diet feeding on the gut microbiome and host metabolism

Although human genetics partially explain the heritability of obesity and type 2 diabetes (T2D), the human gut microbiome also plays a significant role. While changes in human genetics at the population level occur only after many generations, the gut microbiome evolves over a shorter time span. The gut microbiome is also vertically transmitted across generations, therefore, changes in one generation can be passed on to subsequent generations. However, it is unknown whether the gut microbiome influences natural selection of its host under obesogenic pressure. Here, we show that C57BL/6 mice fed a high-fat diet (60% fat, HFD) over four generations develop resistance to obesity and the metabolic syndrome (MetS). Unexpectedly, the mice were increasingly leaner as well as more glucose tolerant and insulin sensitive across generations. This phenomenon was attributed to the most obese mice not yielding progenies, whereas the leanest mice successfully reproduced, and their offspring were also resistant to obesity. In other words, a population bottleneck was observed. Because all the mice were nearly genetically identical inbred C57BL/6J mice, the large variation in body weight gain in response to HFD feeding was likely independent of genetics. We explored whether microbial factors enriched in obesity-resistant mice promote healthier host metabolic phenotypes under HFD feeding, thereby contributing to the heterogeneity in body weight gain and providing an adaptive advantage to the host. Pearson correlation analysis revealed that body weight gain was positively correlated with Lactococcus lactis, as well as negatively correlated with Lactobacillus johnsonii and pathways for coenzyme A biosynthesis, amino acid biosynthesis (lysine, isoleucine, valine), and nucleotide biosynthesis (adenosine, guanosine). Overall, we observed multigenerational adaptation in the gut microbiome correlated with improved metabolism, yet further studies are needed to validate that these adaptations drive metabolic health.

microbiology↗

bioRxiv2022

How Glutamate Promotes Liquid-liquid Phase Separation and DNA Binding Cooperativity of E. coli SSB Protein

E. coli single-stranded-DNA binding protein (EcSSB) displays nearest-neighbor (NN) and non-nearest-neighbor (NNN)) cooperativity in binding ssDNA during genome maintenance. NNN cooperativity requires the intrinsically-disordered linkers (IDL) of the C-terminal tails. Potassium glutamate (KGlu), the primary E. coli salt, promotes NNN-cooperativity, while KCl inhibits it. We find that KGlu promotes compaction of a single polymeric SSB-coated ssDNA beyond what occurs in KCl, indicating a link of compaction to NNN-cooperativity. EcSSB also undergoes liquid-liquid phase separation (LLPS), inhibited by ssDNA binding. We find that LLPS, like NNN-cooperativity, is promoted by increasing [KGlu] in the physiological range, while increasing [KCl] and/or deletion of the IDL eliminate LLPS, indicating similar interactions in both processes. From quantitative determinations of interactions of KGlu and KCl with protein model compounds, we deduce that the opposing effects of KGlu and KCl on SSB LLPS and cooperativity arise from their opposite interactions with amide groups. KGlu interacts unfavorably with the backbone (especially Gly) and side chain amide groups of the IDL and therefore promotes amide-amide interactions in LLPS and NNN-cooperativity. By contrast, KCl interacts favorably with these amide groups and therefore inhibits LLPS and NNN-cooperativity. These results highlight the importance of salt interactions in regulating the propensity of proteins to undergo LLPS.

biochemistry↗

bioRxiv2021

Density-by-diet interactions during larval development shape adult life-history trait expression and fitness in a polyphagous fly

Habitat quality early in life determines individual fitness, with possible long-term evolutionary effects on groups and populations. In holometabolous insects, larval ecology plays a major role in determining the expression of traits in adulthood, but how ecological conditions during larval stage interact to shape adult life-history and fitness, particularly in non-model organisms, remains subject to scrutiny. Consequently, our knowledge of the interactive effects of ecological factors on insect development is limited. Here, using the polyphagous fly Bactrocera tryoni, we conducted a fully-factorial design where we manipulated larval density and larval diet (protein-rich, standard, and sugar-rich) to gain insights into how these ecological factors interact to modulate adult fitness. As expected, a protein-rich diet resulted in faster larval development, heavier and leaner adults that were more fecund compared with standard and sugar-rich diets, irrespective of larval density. Females from the protein-rich larval diet had overall higher reproductive rate (i.e., eggs per day) than females from other diets, and reproductive rate decreased linearly with density for females from the protein-rich but non-linearly for females from the standard and sugar-rich diets over time. Surprisingly, adult lipid reserve increased with larval density for adults from the sugar-rich diet (as opposed to decreasing, as in other diets), possibly due to a stress-response to an extremely adverse condition during development (i.e., high intraspecific competition and poor nutrition). Together, our results provide insights into how ecological factors early in life interact and shape the fate of individuals through life-stages in holometabolous insects.

zoology↗

bioRxiv2021

Yersinia pseudotuberculosis doxycycline tolerance strategies include modulating expression of genes involved in cell permeability and tRNA modifications

Antibiotic tolerance is typically associated with a phenotypic change within a bacterial population, resulting in a transient decrease in antibiotic susceptibility that can contribute to treatment failure and recurrent infections. Although tolerant cells may emerge prior to treatment, the stress of prolonged antibiotic exposure can also promote tolerance. Here, we sought to determine how Yersinia pseudotuberculosis responds to doxycycline exposure, to then verify if these gene expression changes could promote doxycycline tolerance in culture and in our mouse model of infection. Only four genes were differentially regulated in response to a physiologically-relevant dose of doxycycline: osmB and ompF were upregulated, tusB and cnfy were downregulated; differential expression also occurred during doxycycline treatment in the mouse. ompF, tusB and cnfy were also differentially regulated in response to chloramphenicol, indicating these could be general responses to ribosomal inhibition. cnfy has previously been associated with persistence and was not a major focus here. We found deletion of the OmpF porin resulted in increased antibiotic accumulation, suggesting expression may promote diffusion of doxycycline out of the cell, while OsmB lipoprotein had a minor impact on antibiotic permeability. Overexpression of tusB significantly impaired bacterial survival in culture and in the mouse, suggesting that tRNA modification by tusB, and the resulting impacts on translational machinery, may play an important role in promoting tolerance. We believe this is the first observation of bactericidal activity of doxycycline, which was revealed by reversing tusB downregulation.

microbiology↗

bioRxiv2021

Prime-boost vaccination regimens with INO-4800 and INO-4802 augment and broaden immune responses against SARS-CoV-2 in nonhuman primates

The enhanced transmissibility and immune evasion associated with emerging SARS-CoV-2 variants demands the development of next-generation vaccines capable of inducing superior protection amid a shifting pandemic landscape. Since a portion of the global population harbors some level of immunity from vaccines based on the original Wuhan-Hu-1 SARS-CoV-2 sequence or natural infection, an important question going forward is whether this immunity can be boosted by next-generation vaccines that target emerging variants while simultaneously maintaining long-term protection against existing strains. Here, we evaluated the immunogenicity of INO-4800, our synthetic DNA vaccine candidate for COVID-19 currently in clinical evaluation, and INO-4802, a next-generation DNA vaccine designed to broadly target emerging SARS-CoV-2 variants, as booster vaccines in nonhuman primates. Rhesus macaques primed over one year prior with the first-generation INO-4800 vaccine were boosted with either INO-4800 or INO-4802 in homologous or heterologous prime-boost regimens. Both boosting schedules led to an expansion of antibody responses which were characterized by improved neutralizing and ACE2 blocking activity across wild-type SARS-CoV-2 as well as multiple variants of concern. These data illustrate the durability of immunity following vaccination with INO-4800 and additionally support the use of either INO-4800 or INO-4802 in prime-boost regimens.

immunology↗