Biology subjects

Liang, Y.

Publications and source records attributed to Liang, Y..

At least 271 records · Page 15Linked to original sources

bioRxiv2022

ASGR1 is a candidate receptor for SARS-CoV-2 that promotes infection of liver cells

Backgroud & AimsCurrently, the COVID-19 pandemic, caused by SARS-CoV-2 infection, represents a serious public health problem worldwide. Although it has been shown that ACE2 serves as the main receptor for SARS-CoV-2 entry into host cells, studies have shown that ACE2 is expressed at extremely low levels in various tissues, especially in some organs where virus particles have been found, such as the heart and liver. Therefore, these organs potentially express additional SARS-CoV-2 receptors that have not yet been discovered. Methods & ResultsHere, by a genome-wide CRISPR-Cas9 activation library screening, we found that ASGR1 promoted SARS-CoV-2 infection of 293T cells. In Huh-7 and HepG2 cell lines, simultaneous knock out of ACE2 and ASGR1 prevented SARS-CoV-2 pseudovirus infection. In the immortalized THLE-2 hepatocyte cell line and primary liver parenchymal cells, both of which hardly express ACE2, SARS-CoV-2 could successfully establish an infection. After treatment with ASGR1 antibody, the infection rate significantly reduced. This suggests that SARS-CoV-2 infects liver cells mainly through an ASGR1-dependent mechanism. Finally, we also found that the soluble ASGR1 could not only prevent the SARS-CoV-2 pseudovirus, which binds to the ASGR1 receptors, from infecting host liver cells, but also had a protective effect on those expressing ACE2, indicating that administration of soluble ASGR1 protein may represent a new treatment approach. ConclusionsColletively, these findings indicate that ASGR1 is a candidate receptor for SARS-CoV-2 that promotes infection of liver cells. Lay SummaryWe show that ASGR1 is a candidate receptor for SARS-CoV-2 to infect liver cells.

microbiology↗

bioRxiv2022

Allogeneic gene-edited HIV-specific CAR-T cells secreting PD-1 blocking scFv enhance anti-HIV activity in vitro

HIV-specific chimeric antigen receptor (CAR) T cells have been developed to target latently infected CD4+ T cells that express virus either spontaneously or after intentional latency reversal. However, the T-cell exhaustion and the patient-specific autologous paradigm of CAR-T hurdled the clinical application. Here, we created HIV-specific CAR-T cells using human peripheral blood mononuclear cells and a 3BNC117-E27 CAR (3BE CAR) construct that enables the expression of PD-1 blocking scFv E27 and the single-chain variable fragment of the HIV-1-specific broadly neutralizing antibody 3BNC117 to target native HIV envelope glycoprotein (Env). In comparison with T cells expressing 3BNC117-CAR alone, 3BE CAR-T cells showed greater anti-HIV potency with stronger proliferation capability, higher killing efficiency (up to ~75%) and enhanced cytokine secretion in the presence of HIV envelope glycoprotein-expressing cells. Furthermore, our approach achieved high levels (over 97%) of the TCR-deficient 3BE CAR-T cells with the functional inactivation of endogenous TCR to avoid graft-versus-host disease without compromising their antiviral activity relative to standard anti-HIV CAR-T cells. These data suggest that we have provided a feasible approach to large-scale generation of "off-the-shelf" anti-HIV CAR-T cells in combination with antibody therapy of PD-1 blockade, which can be a powerful therapeutic candidate for the functional cure of HIV.

bioengineering↗

bioRxiv2021

Cryo-EM structure of an amyloid fibril formed by full-length human SOD1 reveals its pathological conformational conversion

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by the selective death of motor neurons. Misfolded Cu, Zn-superoxide dismutase (SOD1) has been linked to both familial ALS and sporadic ALS. SOD1 fibrils formed in vitro are able to incorporate into cells, transmit intercellularly, and share toxic properties with ALS inclusions. Here we produced amyloid fibrils in vitro from recombinant, full-length apo human SOD1 under semi-reducing conditions and determined the atomic structure using cryo-EM. The SOD1 fibril consists of a single protofibril with a left-handed helix. The fibril core exhibits a serpentine fold comprising N-terminal segment (residues 3 to 55) and C-terminal segment (residues 86 to 153) with a structural break. The two segments are zipped up by three salt bridge pairs. By comparison with the structure of apo SOD1 dimer, we propose that eight {beta}-strands (to form a {beta}-barrel) and one -helix in the subunit of apo SOD1 convert into thirteen {beta}-strands stabilized by five hydrophobic cavities in the SOD1 fibril. Our data provide insights into how SOD1 converts between structurally and functionally distinct states.

biochemistry↗

bioRxiv2021

Combinatorial mRNA vaccination enhances protection against SARS-CoV-2 delta variant

Emergence of SARS-CoV-2 variants of concern (VOC), including the highly transmissible delta strain, has posed challenges to current COVID-19 vaccines that principally target the viral spike protein (S). Here, we report a nucleoside-modified mRNA vaccine that expresses the more conserved viral nucleoprotein (mRNA-N). We show that mRNA-N alone was able to induce a modest but significant control of SARS-CoV-2 in mice and hamsters. Critically, by combining mRNA-N with the clinically approved S-expressing mRNA vaccine (mRNA-S-2P), we found that combinatorial mRNA vaccination (mRNA-S+N) led to markedly enhanced protection against the SARS-CoV-2 delta variant compared to mRNA-S. In a hamster model, we demonstrated that while mRNA-S alone elicited significant control of the delta strain in the lungs ([~]45-fold reduction in viral loads compared to un-vaccinated control), its effectiveness in the upper respiratory tract was weak, whereas combinatorial mRNA-S+N vaccination induced markedly more robust control of the delta variant infection in the lungs ([~]450-fold reduction) as well as in the upper respiratory tract ([~]20-fold reduction). Immune analyses indicated that induction of N-specific immunity as well as augmented S-specific T-cell response and neutralizing antibody activity were collectively associated the enhanced protection against SARS-CoV-2 delta strain by combinatorial mRNA vaccination. These findings suggest that the combined effects of protection in the lungs and upper respiratory tract could both reduce the risk of severe disease as well as of infection and transmission.

microbiology↗

bioRxiv2021

Whole transcriptome sequencing reveals drought resistance-related genes in upland cotton

China, especially the Xinjiang cotton area, is facing severe agricultural water shortages, which seriously restrain the development of the cotton industry. Discovering cotton drought resistance genes and cultivating high-quality and drought-resistant cotton materials through molecular breeding methods are of great significance to the development of the cotton industry. In this study, the drought-resistant cotton material Xinluzhong NO.82 and the drought-sensitive cotton material Kexin NO.1 were used to identify a batch of drought-resistant candidate genes through whole transcriptome sequencing. The main research results obtained were as follows: the ceRNA (competing endogenous RNAs) network was constructed using full transcriptional sequencing to screen the core genes in the core pathway; two drought-related candidate genes were obtained. Gohir.A11G156000 was upregulated at 0 h vs 12 h and downregulated at 12 h vs 24 h. Gohir.A07G220600 was downregulated at 0 h vs 12 h and upregulated at 12 h vs 24 h. The results for drought-resistant materials and drought-sensitive materials were similar. Gohir.A11G156000, encoding GABA-T, which is homologous to POP2 in Arabidopsis thaliana, affects the drought resistance of plants by regulating the GABA content. Gohir.A07G220600 encodes L-aspartate oxidase, which is homologous to AO in Arabidopsis thaliana, and is involved in the early steps of NAD biosynthesis and in plant antioxidant reactions. This study confirmed that the use of gene expression regulatory networks can quickly screen reliable drought-resistance genes and can be used for subsequent gene function verification.

genomics↗

bioRxiv2021

Fast and sensitive GCaMP calcium indicators for imaging neural populations

Calcium imaging with protein-based indicators is widely used to follow neural activity in intact nervous systems. The popular GCaMP indicators are based on the calcium-binding protein calmodulin and the RS20 peptide. These sensors report neural activity at timescales much slower than electrical signaling, limited by their biophysical properties and trade-offs between sensitivity and speed. We used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators. The resulting jGCaMP8 sensors, based on calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (rise times, 2 ms) and still feature the highest sensitivity for neural activity reported for any protein-based sensor. jGCaMP8 sensors will allow tracking of larger populations of neurons on timescales relevant to neural computation.

neuroscience↗

bioRxiv2021

High-Plex Multiomic Analysis in FFPE Tissue at Single-Cellular and Subcellular Resolution by Spatial Molecular Imaging

The Spatial Molecular Imaging platform (CosMxTM SMI, NanoString Technologies, Seattle, WA) utilizes high-plex in-situ imaging chemistry for both RNA and protein detection. This automated instrument provides 1000s of plex, at high sensitivity (1 to 2 copies/cell), very low error rate (0.0092 false calls/cell) and background ([~]0.04 counts/cell). The imaging system generates three-dimensional super-resolution localization of analytes at [~]2 million cells per sample, four samples per run. Cell segmentation is morphology-based using antibodies, compatible with FFPE samples. Multiomic data (980 RNAs, 108 proteins) were measured at subcellular resolution using FFPE tissues (non-small cell lung (NSCLC) and breast cancer) and allowed identification of over 18 distinct cell types, 10 unique tumor microenvironments, and 100 pairwise ligand-receptor interactions. Over 800,000 single cells and [~]260 million transcripts data are released into the public domain allowing extended data analysis by the entire spatial biology research community.

genomics↗

bioRxiv2021

Spatially resolved whole transcriptome profiling in human and mouse tissue using Digital Spatial Profiling

Emerging spatial profiling technology has enabled high-plex molecular profiling in biological tissues, preserving the spatial and morphological context of gene expression. Here we describe expanding the chemistry for the Digital Spatial Profiling platform to quantify whole transcriptomes in human and mouse tissues using a wide range of spatial profiling strategies and sample types. We designed multiplexed in situ hybridization probe pools targeting the protein-coding genes in the human and mouse transcriptomes, hereafter referred to as the human or mouse Whole Transcriptome Atlas (WTA). We validated the human and mouse WTA using cell lines to demonstrate concordance with orthogonal gene expression profiling methods in profiled region sizes ranging from ~10-500 cells. By benchmarking against bulk RNAseq and fluorescence in situ hybridization, we demonstrate robust transcript detection possible down to ~100 transcripts per region. To assess the performance of WTA across tissue and sample types, we applied WTA to biological questions in cancer, molecular pathology, and developmental biology. We show that spatial profiling with WTA can detect expected spatial gene expression differences between tumor and tumor microenvironment, identify spatial disease-specific heterogeneity in gene expression in histological structures of the human kidney, and comprehensively map transcriptional programs in anatomical substructures of nine organs in the developing mouse embryo. Digital Spatial Profiling technology with the WTA assays provides a flexible method for spatial whole transcriptome profiling applicable to diverse tissue types and biological contexts.

genomics↗

bioRxiv2021

The APC/CCCS52A2 E3-Ligase Complex targeted PKN1 Protein is a Plant Lineage-Specific Driver of Cell Division

The anaphase-promoting complex/cyclosome (APC/C) marks key cell cycle proteins for proteasomal breakdown, thereby ensuring unidirectional progression through the cell cycle. Its target recognition is temporally regulated by activating subunits, one of which is called CELL CYCLE SWITCH 52 A2 (CCS52A2). We sought to expand the knowledge of identified APC/C targets by using the severe growth phenotypes of CCS52A2-deficient Arabidopsis thaliana plants as a readout in a suppressor mutagenesis screen, resulting in the identification of the previously undescribed gene called PIKMIN1 (PKN1). PKN1 deficiency rescues the disorganized root stem cell phenotype of the ccs52a2-1 mutant, whereas an excess of PKN1 inhibits growth of ccs52a2-1 plants, indicating the importance of PKN1 abundance for proper development. Accordingly, the lack of PKN1 in a wild-type background negatively impacts cell division, while its ectopic expression promotes proliferation. PKN1 shows a cell cycle phase-dependent accumulation pattern, localizing to microtubular structures, including the preprophase band, the mitotic spindle, and phragmoplast. PKN1 is conserved throughout the plant kingdom, with its function in cell division being evolutionary conserved in the liverwort Marchantia polymorpha. Our data thus demonstrate that PKN1 represents a novel, plant-specific gene with a rate-limiting role in cell division, which is proteolytically controlled by the CCS52A2-activated APC/C. One-Sentence SummaryPKN1 is a conserved plant-specific protein that is rate-limiting for cell division, likely through its interaction with microtubuli, and is proteolytically controlled by APC/CCCS52A2.

plant biology↗

bioRxiv2021

Genome-wide association analysis of cotton salt stress response-related sites

Soil salinization is the main abiotic stress factor affecting agricultural production worldwide, and salt stress has a significant impact on plant growth and development. Cotton is one of the most salt-tolerant crops. Its salt tolerance varies greatly depending on the variety, growth stage, organs, and soil salt types. Therefore, the selection and utilization of excellent salt-tolerant germplasm resources and the excavation of excellent salt-tolerant salt and salt resistance genes play important roles in improving cotton production in saline-alkali soils. In this study, we analysed the population structure and genetic diversity of 144 elite Gossypium hirsutum cultivar accessions collected from around the world, and especially from China. Illumina Cotton SNP 70K was used to obtain genome-wide single-nucleotide polymorphism (SNP) data for 149 experimental materials, and 18,432 highly consistent SNP loci were obtained by filtering. PCA (principal component analysis) indicated that 149 upland cotton materials could be divided into 2 subgroups, including subgroup 1 with 78 materials and subgroup 2 with 71 materials. Using the obtained SNP and other marker genotype test results, under salt stress, the salt tolerance traits 3d_Germination_potential, 3d_Bud_length_drop_rate, 7d_Germination_rate, 7d_Bud_length_drop_rate, 7d_Germination_weight, 3d_Bud_length, 7d_Bud_length, relative_germination_potential, Relative_germination_rate, 7d_Bud_weight_drop_rate, Salt tolerance index 3d_Germination_potential_index, 3d_Bud_length_index, 7d_Bud_length_index, 7d_Bud_weight_index, and 7d_Germination_rate_index were evaluated by genome association analysis. A total of 27 SNP markers closely related to salt tolerance traits and 15 SNP markers closely related to salt tolerance index were detected. At the SNP locus associated with the traits of the bud length decline rate at 7 days, alleles Gh_A01G0034 and Gh_D01G0028 related to plant salt tolerance were detected, and they are related to intracellular transport, membrane microtubule formation and actin network. This study provides a theoretical basis for the selection and breeding of salt-tolerant upland cotton varieties.

genomics↗

bioRxiv2021

CRISPR activation screening identifies VGLL3 and GATA2 as transcriptional regulators of PD-L1

The PD-L1/2 - PD-1 immune checkpoint is essential for the proper induction of peripheral tolerance and limits autoimmunity, whereas tumor cells exploit their expression to promote immune evasion. Many different cell types express PD-L1/2, either constitutively or upon stimulation, but the factors driving this expression are often not defined. Here, using genome-wide CRISPR-activation screening, we identified three factors that upregulate PD-L1 expression; GATA2, MBD6, and VGLL3. GATA2 and VGLL3 act as transcriptional regulators and their expression induced PD-L1 in many different cell types. Conversely, loss of VGLL3 impaired IFN{gamma}-induced PD-L1/2 expression in keratinocytes. Mechanistically, by performing a second screen to identify proteins acting together with VGLL3, we found that VGLL3 forms a complex with TEAD1 and RUNX1/3 to drive expression of PD-L1/2. Collectively, our work identified a new transcriptional network controlling PD-L1/2 expression and suggests that VGLL3, in addition to its known role in the expression of pro-inflammatory genes, can balance inflammation by upregulating the anti-inflammatory factors PD-L1 and PD-L2.

immunology↗

bioRxiv2021

Reactivation of transposable elements following hybridization in fission yeast

Hybridization is thought to reactivate transposable elements (TEs) that were efficiently suppressed in the genomes of the parental hosts. Here, we provide evidence for this genomic shock hypothesis in the fission yeast Schizosaccharomyces pombe. In this species, two divergent lineages (Sp and Sk) have experienced recent, likely human induced, hybridization. We used long-read sequencing data to assemble genomes of 37 samples derived from 31 S. pombe strains spanning a wide range of ancestral admixture proportions. A comprehensive TE inventory revealed exclusive presence of long terminal repeat (LTR) retrotransposons. Sequence analysis of active full-length elements, as well as solo-LTRs, revealed a complex history of homologous recombination. Population genetic analyses of syntenic sequences placed insertion of many solo-LTRs prior to the split of the Sp and Sk lineages. Most full-length elements were inserted more recently after hybridization. With the exception of a single full-length element with signs of positive selection, both solo-LTRs, and in particular, full-length elements carried signatures of purifying selection indicating effective removal by the host. Consistent with reactivation upon hybridization, the number of full-length LTR retrotransposons, varying extensively from zero to 87 among strains, significantly increased with the degree of genomic admixture. This study gives a detailed account of global TE diversity in S. pombe, documents complex recombination histories within TE elements and provides evidence for the genomic shock hypothesis.

evolutionary biology↗

bioRxiv2021

Temporal variation in lymphocyte proteomics

Chronic Lymphocytic Leukemia (CLL) is a slow progressing disease, characterized by a long asymptomatic stage followed by a symptomatic stage during which patients receive treatment. While proteomic studies have discovered differential pathways in CLL, the proteomic evolution of CLL during the asymptomatic stage has not been studied. In this pilot study, we show that by using small sample sizes comprising ~145 cells, we can detect important features of CLL necessary for studying tumor evolution. Our small samples are collected at two time points and reveal large proteomic changes in healthy individuals over time. A meta-analysis of two CLL proteomic papers showed little commonality in differentially expressed proteins and demonstrates the need for larger control populations sampled over time. To account for proteomic variability between time points and individuals, large control populations sampled at multiple time points are necessary for understanding CLL progression. Data is available via ProteomeXchange with identifier PXD027429.

bioinformatics↗

bioRxiv2021

Prediction of DNA from context using neural networks

One way to better understand the structure in DNA is by learning to predict the sequence. Here, we train a model to predict the missing base at any given position, given its left and right flanking contexts. Our best-performing model is a neural network that obtains an accuracy close to 54% on the human genome, which is 2% points better than modelling the data using a Markov model. In likelihood-ratio tests, we show that the neural network is significantly better than any of the alternative models by a large margin. We report on where the accuracy is obtained, observing first that the performance appears to be uniform over the chromosomes. The models perform best in repetitive sequences, as expected, although they are far from random performance in the more difficult coding sections, the proportions being ~ 70:40%. Exploring further the sources of the accuracy, Fourier transforming the predictions reveals weak but clear periodic signals. In the human genome the characteristic periods hint at connections to nucleosome positioning. To understand this we find similar periodic signals in GC/AT content in the human genome, which to the best of our knowledge have not been reported before. On other large genomes similarly high accuracy is found, while lower predictive accuracy is observed on smaller genomes. Only in mouse did we see periodic signals in the same range as in human, though weaker and of different type. Interestingly, applying a model trained on the mouse genome to the human genome results in a performance far below that of the human model, except in the difficult coding regions. Despite the clear outcomes of the likelihood ratio tests, there is currently a limited superiority of the neural network methods over the Markov model. We expect, however, that there is great potential for better modelling DNA using different neural network architectures.

bioinformatics↗

bioRxiv2021

Context dependency of nucleotide probabilities and variants in human DNA

BackgroundGenomic DNA has been shaped by mutational processes through evolution. The cellular machinery for error correction and repair has left its marks in the nucleotide composition along with structural and functional constraints. Therefore, the probability of observing a base in a certain position in the human genome is highly context-dependent. ResultsHere we develop context-dependent nucleotide models. We first investigate models of nucleotides conditioned on sequence context. We develop a bidirectional Markov model that use an average of the probability from a Markov model applied to both strands of the sequence and thus depends on up to 14 bases to each side of the nucleotide. We show how the genome predictability varies across different types of genomic regions. Surprisingly, this model can predict a base from its context with an average of more than 50% accuracy. For somatic variants we show a tendency towards higher probability for the variant base than for the reference base. Inspired by DNA substitution models, we develop a model of mutability that estimates a mutation matrix (called the alpha matrix) on top of the nucleotide distribution. The alpha matrix can be estimated from a much smaller context than the nucleotide model, but the final model will still depend on the full context of the nucleotide model. With the bidirectional Markov model of order 14 and an alpha matrix dependent on just one base to each side, we obtain a model that compares well with a model of mutability that estimates mutation probabilities directly conditioned on three nucleotides to each side. For somatic variants in particular, our model fits better than the simpler model. Interestingly, the model is not very sensitive to the size of the context for the alpha matrix. ConclusionsOur study found strong context dependencies of nucleotides in the human genome. The best model uses a context of 14 nucleotides to each side. Based on these models, a substitution model was constructed that separates into the context model and a matrix dependent on a small context. The model fit somatic variants particularly well.

bioinformatics↗

bioRxiv2021

Nuclear hormone receptors promote gut and glia detoxifying enzyme induction and protect C. elegans from the mold P. brevicompactum

Animals encounter microorganisms in their habitats, adapting physiology and behavior accordingly. The nematode Caenorhabditis elegans is found in microbe-rich environments; however, its responses to fungi are not extensively studied. Here we describe interactions of C. elegans and Penicillium brevicompactum, an ecologically-relevant mold. Transcriptome studies reveal that co-culture upregulates stress-response genes, including xenobiotic metabolizing enzymes (XMEs), in C. elegans intestine and AMsh glial cells. The nuclear hormone receptors (NHR) NHR-45 and NHR-156 are key induction regulators, and mutants that cannot induce XMEs in the intestine when exposed to P. brevicompactum experience mitochondrial stress and exhibit developmental defects. Different C. elegans wild isolates harbor sequence polymorphisms in nhr-156, resulting in phenotypic diversity in AMsh glia responses to microbe exposure. We propose that P. brevicompactum mitochondria-targeting mycotoxins are deactivated by intestinal detoxification, allowing tolerance to moldy environments. Our studies support the idea that C. elegans NHR gene expansion/diversification underlies adaptation to microbial environments.

cell biology↗

bioRxiv2021

Temporal Analyses of Postnatal Liver Development and Maturation by Single Cell Transcriptomics

Liver is the major metabolic organ, although its postnatal development and maturation are inadequately understood. We analyzed 52,834 single cell transcriptomes and identified 31 cell types or states in mouse livers at postnatal day 1, 3, 7, 21 and 56. We observed unexpectedly high levels of hepatocyte heterogeneity in the developing liver and progressive construction of the zonated metabolic functions from pericentral to periportal hepatocytes, which was orchestrated with development of sinusoid endothelial, stellate and Kupffer cells. Trajectory and gene regulatory analyses captured 36 transcription factors, including a circadian regulator Bhlhe40, in programming liver development. Remarkably, we identified a special group of macrophages enriched at day 7 with a hybrid phenotype of macrophages and endothelial cells, which may regulate sinusoidal construction and Treg cell function. This study provides a comprehensive atlas that covers all hepatic cell types instrumental for further dissection of liver development, metabolic functions and diseases. In BriefO_LISingle cell transcriptomics of all hepatic cell types in neonatal and adult livers C_LIO_LIConcerted development of zonated metabolic functions in hepatocytes and NPCs C_LIO_LITransient emergence of a distinct group of macrophages at postnatal day 7 C_LIO_LIHepatic cell-cell communications that program postnatal liver development C_LI

developmental biology↗

bioRxiv2021

Structure and computation-guided design of a mutation-integrated trimeric RBD candidate vaccine with broad neutralization against SARS-CoV-2

The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 is an attractive target for COVID-19 vaccine developments, which naturally exists in a trimeric form. Here, guided by structural and computational analyses, we present a mutation-integrated trimeric form of RBD (mutI tri-RBD) as a broadly protective vaccine candidate, in which three RBDs were individually grafted from three different circulating SARS-CoV-2 strains including the prototype, Beta (B.1.351) and Kappa (B.1.617). The three RBDs were then connected end-to-end and co-assembled to possibly mimic the native trimeric arrangements in the natural S protein trimer. The recombinant expression of the mutI tri-RBD, as well as the homo-tri-RBD where the three RBDs were all truncated from the prototype strain, by mammalian cell exhibited correct folding, strong bio-activities, and high stability. The immunization of both the mutI tri-RBD and homo-tri-RBD plus aluminum adjuvant induced high levels of specific IgG and neutralizing antibodies against the SARS-CoV-2 prototype strain in mice. Notably, regarding to the "immune-escape" Beta (B.1.351) variant, mutI tri-RBD elicited significantly higher neutralizing antibody titers than homo-tri-RBD. Furthermore, due to harboring the immune-resistant mutations as well as the evolutionarily convergent hotspots, the designed mutI tri-RBD also induced strong broadly neutralizing activities against various SARS-CoV-2 variants, especially the variants partially resistant to homo-tri-RBD. Homo-tri-RBD has been approved by the China National Medical Products Administration to enter clinical trial (No. NCT04869592), and the superior broad neutralization performances against SARS-CoV-2 support the mutI tri-RBD as a more promising vaccine candidate for further clinical developments.

immunology↗