Biology subjects

Cao, L.

Publications and source records attributed to Cao, L..

At least 127 records · Page 7Linked to original sources

bioRxiv2021

Multivalent designed proteins protect against SARS-CoV-2 variants of concern

Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness. One-Sentence SummaryWe designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.

synthetic biology↗

bioRxiv2021

The molecular mechanism of cytoadherence to placenta or tumor cells through VAR2CSA from Plasmodium falciparum

Pregnancy Associated Malaria (PAM) threatens more than one million pregnant women and their infants in endemic regions due to poor outcomes. VAR2CSA plays a vital role in the cytoadherence of infected erythrocytes (IEs) to placenta against immuno-clearance via binding to Chondroitin sulfate A (CSA), which is displayed mostly on the surface of placental or tumor cells. In this study, we determined the cryo-EM structures of VAR2CSA ectodomain and its complex with CSA at a resolution of 3.6 [A] and 3.4 [A], respectively and revealed that CSA binding induces significant conformational change for ligand accommodation. Beyond structural studies, we generated VAR2CSA fragments and mutation and evaluated their binding activity to either isolated CSA or placental and tumor cell line using multi-disciplinary techniques. The results showed that 9-site mutation in DBL2X abolished the CSA binding activity and also disrupted its binding to both placental and tumor cells. Overall, our work clearly elucidated the molecular mechanism of cytoadherence to placental or tumor cells through VAR2CSA. Which may facilitate new PAM vaccine development and improve the drug delivery systems targeting both placenta and tumors.

biophysics↗

bioRxiv2021

Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-Translational Modification

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with protein L-isoaspartyl methyltransferase (PIMT) homologs. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an L-aspartate sidechain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/444711v1_ufig1.gif" ALT="Figure 1"> View larger version (9K): org.highwire.dtl.DTLVardef@158f0d2org.highwire.dtl.DTLVardef@2a6baeorg.highwire.dtl.DTLVardef@103a91dorg.highwire.dtl.DTLVardef@153dd99_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Fuscimiditide: a RiPP with {Omega}-Ester and Aspartimide Post-translational Modifications

Microviridins and other {omega}-ester linked peptides (OEPs) are characterized by sidechain-sidechain linkages installed by ATP-grasp enzymes. Here we describe the discovery of a new family of OEPs, the gene clusters of which also encode an O-methyltransferase with homology to the protein repair catalyst protein L-isoaspartyl methyltransferase (PIMT). We produced the first example of this new ribosomally synthesized and post-translationally modified peptide (RiPP), fuscimiditide, via heterologous expression. NMR analysis of fuscimiditide revealed that the peptide contains two ester crosslinks forming a stem-loop macrocycle. Furthermore, an unusually stable aspartimide moiety is found within the loop macrocycle. We have also fully reconstituted fuscimiditide biosynthesis in vitro establishing that ester formation catalyzed by the ATP-grasp enzyme is an obligate, rate-limiting first biosynthetic step. Aspartimide formation from aspartate is catalyzed by the PIMT homolog in the second step. The aspartimide moiety embedded in fuscimiditide hydrolyzes regioselectively to isoaspartate (isoAsp). Surprisingly, this isoAsp-containing protein is also a substrate for the PIMT homolog, thus driving any hydrolysis products back to the aspartimide form. Whereas aspartimide is often considered a nuisance product in protein formulations, our data here suggest that some RiPPs have aspartimide residues intentionally installed via enzymatic activity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/444834v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1c67f36org.highwire.dtl.DTLVardef@c9e774org.highwire.dtl.DTLVardef@17a7d62org.highwire.dtl.DTLVardef@bde61b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Transcriptomes of peripheral blood mononuclear cells from juvenile dermatomyositis patients show elevated inflammation even when clinically inactive

In juvenile dermatomyositis (JDM), the most common pediatric inflammatory myopathy, weakness is accompanied by a characteristic rash that often becomes chronic, and is associated with vascular damage. We hoped to understand the molecular underpinnings of JDM, particularly in untreated disease, which would facilitate the identification of novel mechanisms and clinical targets that might disrupt disease progression. We studied the RNA-Seq data from untreated JDM peripheral blood mononuclear cells (PBMCs; n=11), PBMCs from a subset of the same patients when clinically inactive (n=8/11), and separate samples of untreated JDM skin and muscle (n=4 each). All JDM samples were compared to non-inflammatory control tissues. The untreated JDM PBMCs showed a strong signature for type1 interferon response, along with IL-1, IL-10, and NF-{kappa}B. Surprisingly, PBMCs from clinically inactive JDM individuals had persistent immune activation that was enriched for IL-1 signaling. JDM skin and muscle both showed evidence for type 1 interferon activation and genes related to antigen presentation, and decreased expression of genes related for cellular respiration. Additionally we found that PBMC gene expression correlates with disease activity scores (DAS; skin, muscle, and total domains) and with nailfold capillary end row loop number (an indicator of microvascular damage). This included otoferlin, which was significantly increased in untreated JDM PBMCs and correlated with all 3 DAS domains. Overall, these data demonstrate that PBMC transcriptomes are informative of molecular disruptions in JDM and provide transcriptional evidence of chronic inflammation despite clinical quiescence.

genomics↗

bioRxiv2021

Biomass, microbial composition and functions are responsible for the differential removal of trace organic chemicals in biofiltration systems

Biofiltration processes help to remove trace organic chemicals (TOrCs) both in wastewater and drinking water treatment systems. However, the detailed TOrCs biotransformation mechanisms as well as the underlying drivers behind the variability of site specific transformation processes remain elusive. In this study, we used laboratory batch incubations to investigate the biotransformation of 51 TOrCs in eight bioactive filter materials of different origins treating a range of waters, from wastewater effluents to drinking water. Microscopy, 16S rRNA amplicon and whole metagenome sequencing for assessing associations between the biotransformation rate constants, microbial composition and genetic potential complemented chemical analysis. We observed strong differences in the mean global removal of TOrCs between the individual sand filters (-1.4% to 58%), which were mirrored in overall biomass, microbial community composition, and enzyme encoding genes. From the six investigated biomass markers, ATP turned out to be a major predictor of the mean global biotransformation rate, while compound specific biotransformations were correlated with the microbial community composition. High biomass ecosystems were indicated in our systems by a dominance of Nitrospirae, but individual TOrC biotransformation was statistically connected to rare taxa (< 2%) such as Hydrogenophaga, or indiviudal functions such as the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase encoding genes. In general, this study provides new insights into so far rarely addressed variability of TOrCs biotransformation. We propose novel biological indicators for the removal performance of TOrCs in biofiltration systems, highlighting the role of living biomass in predicting and normalizing the global transformation, and the role of the microbial community for the individual transformation of TOrCs in engineered and natural systems. Contribution to the Field StatementTrace organic chemicals (TOrCs) are an emerging problem in the aquatic environment that has attracted global attention over the last decade. Recent research efforts on this topic have increased our knowledge on the transformation of TOrCs and various technologies have been developed to improve their removal. In this study, we investigated a wide range of biotransformation of TOrCs by eight sand filter materials from wastewater and water treatment plants. Biotransformation rate constants were calculated using first-order kinetics to evaluate TOrC removal performance. We reevaluated the role of biomass and could thus explain a greater part of the global TOrC removal performance. The remaining variation in removal rates of individual compounds correlated with the microbiome of the biofilter. Rare biosphere lineages and specific enzyme categories genes were correlated with the removal of certain compounds. In summary, our research identified future indicators for successful biotransformation of TOrCs in biofilter systems.

microbiology↗

bioRxiv2021

Sentinel cells enable genetic detection of SARS-CoV-2 Spike protein

The COVID-19 pandemic has demonstrated the need for exploring different diagnostic and therapeutic modalities to tackle future viral threats. In this vein, we propose the idea of sentinel cells, cellular biosensors capable of detecting viral antigens and responding to them with customizable responses. Using SARS-CoV-2 as a test case, we developed a live cell sensor (SARSNotch) using a de novo-designed protein binder against the SARS-CoV-2 Spike protein. SARSNotch is capable of driving custom genetically-encoded payloads in immortalized cell lines or in primary T lymphocytes in response to purified SARS-CoV-2 Spike or in the presence of Spike-expressing cells. Furthermore, SARSNotch is functional in a cellular system used in directed evolution platforms for development of better binders or therapeutics. In keeping with the rapid dissemination of scientific knowledge that has characterized the incredible scientific response to the ongoing pandemic, we extend an open invitation for others to make use of and improve SARSNotch sentinel cells in the hopes of unlocking the potential of the next generation of smart antiviral therapeutics.

synthetic biology↗

bioRxiv2021

An Immune Cell Atlas Reveals Dynamic COVID-19 Specific Neutrophil Programming Amendable to Dexamethasone Therapy

SARS-CoV-2 is a novel coronavirus that causes acute respiratory distress syndrome (ARDS), death and long-term sequelae. Innate immune cells are critical for host defense but are also the primary drivers of ARDS. The relationships between innate cellular responses in ARDS resulting from COVID-19 compared to other causes of ARDS, such as bacterial sepsis is unclear. Moreover, the beneficial effects of dexamethasone therapy during severe COVID-19 remain speculative, but understanding the mechanistic effects could improve evidence-based therapeutic interventions. To interrogate these relationships, we developed an scRNA-Seq and plasma proteomics atlas (biernaskielab.ca/COVID_neutrophil). We discovered that compared to bacterial ARDS, COVID-19 was associated with distinct neutrophil polarization characterized by either interferon (IFN) or prostaglandin (PG) active states. Neutrophils from bacterial ARDS had higher expression of antibacterial molecules such as PLAC8 and CD83. Dexamethasone therapy in COVID patients rapidly altered the IFNactive state, downregulated interferon responsive genes, and activated IL1R2+ve neutrophils. Dexamethasone also induced the emergence of immature neutrophils expressing immunosuppressive molecules ARG1 and ANXA1, which were not present in healthy controls. Moreover, dexamethasone remodeled global cellular interactions by changing neutrophils from information receivers into information providers. Importantly, male patients had higher proportions of IFNactive neutrophils, a greater degree of steroid-induced immature neutrophil expansion, and increased mortality benefit compared to females in the dexamethasone era. Indeed, the highest proportion of IFNactive neutrophils was associated with mortality. These results define neutrophil states unique to COVID-19 when contextualized to other life-threatening infections, thereby enhancing the relevance of our findings at the bedside. Furthermore, the molecular benefits of dexamethasone therapy are also defined, and the identified pathways and plasma proteins can now be targeted to develop improved therapeutics.

immunology↗

bioRxiv2021

Large-scale design and refinement of stable proteins using sequence-only models

Engineered proteins generally must possess a stable structure in order to achieve their designed function. Stable designs, however, are astronomically rare within the space of all possible amino acid sequences. As a consequence, many designs must be tested computationally and experimentally in order to find stable ones, which is expensive in terms of time and resources. Here we report a neural network model that predicts protein stability based only on sequences of amino acids, and demonstrate its performance by evaluating the stability of almost 200,000 novel proteins. These include a wide range of sequence perturbations, providing a baseline for future work in the field. We also report a second neural network model that is able to generate novel stable proteins. Finally, we show that the predictive model can be used to substantially increase the stability of both expert-designed and model-generated proteins.

biophysics↗

bioRxiv2021

Ultrapotent miniproteins targeting the receptor-binding domain protect against SARS-CoV-2 infection and disease in mice

Despite the introduction of public health measures and spike protein-based vaccines to mitigate the COVID-19 pandemic, SARS-CoV-2 infections and deaths continue to rise. Previously, we used a structural design approach to develop picomolar range miniproteins targeting the SARS-CoV-2 receptor binding domain. Here, we investigated the capacity of modified versions of one lead binder, LCB1, to protect against SARS-CoV-2-mediated lung disease in human ACE2-expressing transgenic mice. Systemic administration of LCB1-Fc reduced viral burden, diminished immune cell infiltration and inflammation, and completely prevented lung disease and pathology. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lung even when given as many as five days before or two days after virus inoculation. Importantly, LCB1v1.3 protected in vivo against a historical strain (WA1/2020), an emerging B.1.1.7 strain, and a strain encoding key E484K and N501Y spike protein substitutions. These data support development of LCB1v1.3 for prevention or treatment of SARS-CoV-2 infection.

microbiology↗

bioRxiv2021

Surface GluA1 and glutamate transmission are increased in VPS35 D620N neurons and altered by LRRK2 kinase inhibition

Vacuolar protein sorting 35 (VPS35) regulates receptor recycling from endosomes. A missense mutation in VPS35 (D620N) leads to autosomal-dominant, late-onset Parkinsons disease. Here, we use a VPS35 D620N knock-in mouse to study the neurobiology of this mutation. In brain tissue, we confirm previous findings that the mutation results in reduced binding of VPS35 with WASH-complex member FAM21, and robustly elevated phosphorylation of the LRRK2 kinase substrate Rab10. In cultured cortical neurons, the mutation results in increased endosomal recycling protein density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate release, and GluA1 surface expression. LRRK2 kinase inhibition exerted genotype-specific effects on GluA1 surface expression, but did not impact glutamate release phenotypes. These results improve our understanding of the early effects of the D620N mutation on cellular functions that are specific to neurons. These observations provide candidate pathophysiological pathways that may drive eventual transition to late-stage parkinsonism in VPS35 families, and support a synaptopathy model of neurodegeneration.

neuroscience↗

bioRxiv2020

MolDiscovery: Learning Mass Spectrometry Fragmentation of Small Molecules

Identification of small molecules is a critical task in various areas of life science. Recent advances in mass spectrometry have enabled the collection of tandem mass spectra of small molecules from hundreds of thousands of environments. To identify which molecules are present in a sample, one can search mass spectra collected from the sample against millions of molecular structures in small molecule databases. This is a challenging task as currently it is not clear how small molecules are fragmented in mass spectrometry. The existing approaches use the domain knowledge from chemistry to predict fragmentation of molecules. However, these rule-based methods fail to explain many of the peaks in mass spectra of small molecules. Recently, spectral libraries with tens of thousands of labelled mass spectra of small molecules have emerged, paving the path for learning more accurate fragmentation models for mass spectral database search. We present molDiscovery, a mass spectral database search method that improves both efficiency and accuracy of small molecule identification by (i) utilizing an efficient algorithm to generate mass spectrometry fragmentations, and (ii) learning a probabilistic model to match small molecules with their mass spectra. We show our database search is an order of magnitude more efficient than the state-of-the-art methods, which enables searching against databases with millions of molecules. A search of over 8 million spectra from the Global Natural Product Social molecular networking infrastructure shows that our probabilistic model can correctly identify nearly six times more unique small molecules than previous methods. Moreover, by applying molDiscovery on microbial datasets with both mass spectral and genomics data we successfully discovered the novel biosynthetic gene clusters of three families of small molecules. AvailabilityThe command-line version of molDiscovery and its online web service through the GNPS infrastructure are available at https://github.com/mohimanilab/molDiscovery.

bioinformatics↗

bioRxiv2020

Double Lock of a Potent Human Monoclonal Antibody against SARS-CoV-2

Receptor recognition and subsequent membrane fusion are essential for the establishment of successful infection by SARS-CoV-2. Halting these steps can cure COVID-19. Here we have identified and characterized a potent human monoclonal antibody, HB27, that blocks SARS-CoV-2 attachment to its cellular receptor at sub-nM concentrations. Remarkably, HB27 can also prevent SARS-CoV-2 membrane fusion. Consequently, a single dose of HB27 conferred effective protection against SARS-CoV-2 in two established mouse models. Rhesus macaques showed no obvious adverse events when administrated with 10-fold of effective dose of HB27. Cryo-EM studies on complex of SARS-CoV-2 trimeric S with HB27 Fab reveal that three Fab fragments work synergistically to occlude SARS-CoV-2 from binding to ACE2 receptor. Binding of the antibody also restrains any further conformational changes of the RBD, possibly interfering with progression from the prefusion to the postfusion stage. These results suggest that HB27 is a promising candidate for immuno-therapies against COVID-19. HighlightsO_LISARS-CoV-2 specific antibody, HB27, blocks viral receptor binding and membrane fusion C_LIO_LIHB27 confers prophylactic and therapeutic protection against SARS-CoV-2 in mice models C_LIO_LIRhesus macaques showed no adverse side effects when administered with HB27 C_LIO_LICryo-EM studies suggest that HB27 sterically occludes SARS-CoV-2 from its receptor C_LI

biophysics↗

bioRxiv2020

Characterization and structural basis of a lethal mouse-adapted SARS-CoV-2

The ongoing SARS-CoV-2 pandemic has brought an urgent need for animal models to study the pathogenicity of the virus. Herein, we generated and characterized a novel mouse-adapted SARS-CoV-2 strain, named MASCp36, that causes severe acute respiratory symptoms and mortality in standard laboratory mice. Particularly, this model exhibits age and gender related skewed distribution of mortality akin to severe COVID-19, and the 50% lethal dose (LD50) of MASCp36 was 58 PFU in 9-month-old, male BALB/c mice. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, subsequently emerged at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three mutations in RBD significantly enhanced the binding affinity to its endogenous receptor, mouse ACE2 (mACE2). Cryo-electron microscopy (cryo-EM) analysis of human ACE2 (hACE2) or mACE2 in complex with the RBD of MASCp36 at 3.1 to 3.7 angstrom resolution elucidates molecular basis for the receptor-binding switch driven by specific amino acid substitutions. Interestingly, N501Y and Q493H enhanced the binding affinity to human ACE2 (hACE2); while triple mutations N501Y/Q493H/K417N decreased affinity to hACE2, thus led to the reduced infectivity of MASCp36 to human cells. Our study not only provides a robust platform for studying the pathogenesis of severe COVID-19 and rapid evaluation of coutermeasures against SARS-CoV-2, but also unveils the molecular mechanism for the rapid adaption and evolution of SARS-CoV-2 in human and animals. One sentence summaryA mouse adapted SARS-CoV-2 strain that harbored specific amino acid substitutions in the RBD of S protein showed 100% mortality in aged, male BALB/c mice.

microbiology↗

bioRxiv2020

Temporal patterns in the evolutionary genetic distance of SARS-CoV-2 during the COVID-19 pandemic

BackgroundDuring the pandemic of coronavirus disease 2019 (COVID-19), the genetic mutations occurred in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cumulatively or sporadically. In this study, we employed a computational approach to identify and trace the emerging patterns of the SARS-CoV-2 mutations, and quantify accumulative genetic distance across different periods and proteins. MethodsFull-length human SARS-CoV-2 strains in United Kingdom were collected. We investigated the temporal variation in the evolutionary genetic distance defined by the Hamming distance since the start of COVID-19 pandemic. FindingsOur results showed that the SARS-CoV-2 was in the process of continuous evolution, mainly involved in spike protein (S protein), the RNA-dependent RNA polymerase (RdRp) region of open reading frame 1 (ORF1) and nucleocapsid protein (N protein). By contrast, mutations in other proteins were sporadic and genetic distance to the initial sequenced strain did not show an increasing trend.

bioinformatics↗

bioRxiv2020

Remdesivir Metabolite GS-441524 Efficiently Inhibits SARS-CoV-2 Infection in Mouse Model

The outbreak of coronavirus disease 2019 (COVID-19) rapidly spreads across worldwide and becomes a global pandemic. Remdesivir is the only COVID-19 treatment approved by U.S. Food and Drug Administration (FDA); however, its effectiveness is still under questioning as raised by the results of a large WHO Solidarity Trial. Herein, we report that the parent nucleotide of remdesivir, GS-441524, potently inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Vero E6 and other cells. It exhibits good plasma distribution and longer half-life (t1/2=4.8h) in rat PK study. GS-441524 is highly efficacious against SARS-CoV-2 in AAV-hACE2 transduced mice and murine hepatitis virus (MHV) in mice, reducing the viral titers in CoV-attacked organs, without noticeable toxicity. Given that GS-441524 was the predominant metabolite of remdesivir in the plasma, the anti-COVID-19 effect of remdesivir may partly come from the effect of GS-441524. Our results also supported that GS-441524 as a promising and inexpensive drug candidate in the treatment of COVID-19 and future emerging CoVs diseases.

pharmacology and toxicology↗

bioRxiv2020

Expression analysis of photosynthesis-related genes in albino Artocarpus heterophyllus seedlings leaves

Albino Artocarpus heterophyllus Seedlings (AAS) were found in the preliminary investigation by our group and were used as materials for researching. The phenotype of AAS leaves were observed and measured. In parallel, the photosynthetic physiological parameters were determined under different photosynthetically active radiations (PAR). The results suggested that the length, width, area and thickness of AAS leaves were less than normal seedings. Likewise, the net photosynthetic rate, intercellular CO2 concentration, stomatal conductance and transpiration rate of AAS leaves were not susceptible to PAR in contrast to normal individuals. Furthermore, the transcriptome sequencing technology was performed to clarify the expression of genes related to photosynthesis. It is as expected that numerous down-regulated genes were found in the synthesis of photosynthetic pigments, as well as the pathways of photosynthesis - antenna proteins, photoreaction and carbon fixation reaction of AAS leaves. Compared to other albino plants, AAS have a longer life span and more stable phenotypic traits with larger leaves, which could provide ideal materials for investigating photosynthesis of woody plants.

physiology↗

bioRxiv2020

A proof of concept for neutralizing antibody-guided vaccine design against SARS-CoV-2

Mutations and transient conformational movements of receptor binding domain (RBD) that make neutralizing epitopes momentarily unavailable, present immune escape routes to SARS-CoV-2. To mitigate viral escape, we developed a cocktail of neutralizing antibodies (NAbs) targeting epitopes located on different domains of spike (S) protein. Screening of a library of monoclonal antibodies generated from peripheral blood mononuclear cells of COVID-19 convalescent patients yielded potent NAbs, targeting N-terminal domain (NTD) and RBD domain of S, effective at nM concentrations. Remarkably, combination of RBD-targeting NAbs and NTD-binding NAb, FC05, dramatically enhanced the neutralization potency in cell-based assays and animal model. Results of competitive SPR assays and cryo-EM structures of Fabs bound to S unveil determinants of immunogenicity. Combinations of immunogens, identified in NTD and RBD of S, when immunized in rabbits elicited potent protective immune responses against SARS-CoV-2. These results provide a proof-of-concept for neutralization-based immunogen design targeting SARS-CoV-2 NTD and RBD. One sentence summaryImmunogens identified in the NTD and RBD of the SARS-CoV-2 spike protein using a cocktail of non-competing NAbs when injected in rabbits elicited a potent protective immune response against SARS-CoV-2.

microbiology↗