Biology subjects

Huang, J.

Publications and source records attributed to Huang, J..

At least 469 records · Page 26Linked to original sources

bioRxiv2021

Lung-selective Cas13d-based nanotherapy inhibits lethal SARS-CoV-2 infection by targeting host protease Ctsl

The COVID-19 pandemic persists as a global health crisis for which curative treatment has been elusive. Development of effective and safe anti-SARS-CoV-2 therapies remains an urgent need. SARS-CoV-2 entry into cells requires specific host proteases including TMPRSS2 and Cathepsin L (Ctsl)1-3, but there has been no reported success in inhibiting host proteases for treatment of SARS-CoV-2 pathogenesis in vivo. Here we have developed a lung Ctsl mRNA-targeted, CRISPR/Cas13d-based nanoparticle therapy to curb fatal SARS-CoV-2 infection in a mouse model. We show that this nanotherapy can decrease lung Ctsl expression in normal mice efficiently, specifically, and safely. Importantly, this lung-selective Ctsl-targeted nanotherapy significantly extended the survival of lethally SARS-CoV-2 infected mice by decreasing lung virus burden, reducing expression of pro-inflammatory cytokines/chemokines, and diminishing the severity of pulmonary interstitial inflammation. Additional in vitro analyses demonstrated that Cas13d-mediated Ctsl knockdown inhibited infection mediated by the spike protein of SARS-CoV-1, SARS-CoV-2, and more importantly, the authentic SARS-CoV-2 B.1.617.2 Delta variant, regardless of TMPRSS2 expression status. Our results demonstrate the efficacy and safety of a lung-selective, Ctsl-targeted nanotherapy against infection by SARS-CoV-2 and likely other emerging coronaviruses, forming a basis for investigation of this approach in clinical trials.

microbiology↗

bioRxiv2021

Data-driven approaches for genetic characterization of SARS-CoV-2 lineages

The genome of the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has been sequenced at an unprecedented scale, leading to a tremendous amount of viral genome sequencing data. To understand the evolution of this virus in humans, and to assist in tracing infection pathways and designing preventive strategies, we present a set of computational tools that span phylogenomics, population genetics and machine learning approaches. To illustrate the utility of this toolbox, we detail an in depth analysis of the genetic diversity of SARS-CoV-2 in first year of the COVID-19 pandemic, using 329,854 high-quality consensus sequences published in the GISAID database during the pre-vaccination phase. We demonstrate that, compared to standard phylogenetic approaches, haplotype networks can be computed efficiently on much larger datasets, enabling real-time analyses. Furthermore, time series change of Tajimas D provides a powerful metric of population expansion. Unsupervised learning techniques further highlight key steps in variant detection and facilitate the study of the role of this genomic variation in the context of SARS-CoV-2 infection, with Multiscale PHATE methodology identifying fine-scale structure in the SARS-CoV-2 genetic data that underlies the emergence of key lineages. The computational framework presented here is useful for real-time genomic surveillance of SARS-CoV-2 and could be applied to any pathogen that threatens the health of worldwide populations of humans and other organisms.

bioinformatics↗

bioRxiv2021

Aberrant epigenetic and transcriptional events associated with breast cancer risk

BackgroundGenome-wide association studies have identified several breast cancer susceptibility loci. However, biomarkers for risk assessment are still missing. Here, we investigated cancer-related molecular changes detected in tissues from women at high risk for breast cancer prior to disease manifestation. Disease-free breast tissue cores donated by healthy women (N=146, median age=39 years) were processed for both methylome (MethylCap) and transcriptome (Illuminas HiSeq4000) sequencing. Analysis of tissue microarray and primary breast epithelial cells was used to confirm gene expression dysregulation. ResultsTranscriptomic analysis identified 69 differentially expressed genes between women at either high and those at average risk of breast cancer (Tyrer-Cuzick model) at FDR<0.05 and fold change[≥]2. The majority of the identified genes were involved in DNA damage checkpoint, cell cycle, and cell adhesion. Two genes, FAM83A and NEK2, were overexpressed in tissue sections (FDR<0.01) and primary epithelial cells (p<0.05) from high-risk breasts. Moreover, 1698 DNA methylation aberrations were identified in high-risk breast tissues (FDR<0.05), partially overlapped with cancer-related signatures and correlated with transcriptional changes (p<0.05, r[≤]0.5). Finally, among the participants, 35 women donated breast biopsies at two time points, and age-related molecular alterations enhanced in high-risk subjects were identified. ConclusionsNormal breast tissue from women at high risk of breast cancer bears molecular aberrations that may contribute to breast cancer susceptibility. This study is the first molecular characterization of the true normal breast tissues and provides an opportunity to investigate molecular markers of breast cancer risk, which may lead to new preventive approaches.

cancer biology↗

bioRxiv2021

CRISPR-Cas12a induced DNA double-strand breaks are repaired by locus-dependent and error-prone pathways in a fungal pathogen

CRISPR-Cas mediated genome engineering has revolutionized functional genomics. However, basic questions remain regarding the mechanisms of DNA repair following Cas-mediated DNA cleavage. We developed CRISPR-Cas12a ribonucleoprotein genome editing in the fungal plant pathogen, Magnaporthe oryzae, and found frequent donor DNA integration despite the absence of long sequence homology. Interestingly, genotyping from hundreds of transformants showed that frequent non-canonical DNA repair outcomes predominated the recovered genome edited strains. Detailed analysis using sanger and nanopore long-read sequencing revealed five classes of DNA repair mutations, including single donor DNA insertions, concatemer donor DNA insertions, large DNA deletions, deletions plus donor DNA insertions, and infrequently we observed INDELs. Our results show that different error-prone DNA repair pathways resolved the Cas12a-mediated double-strand breaks (DSBs) based on the DNA sequence of edited strains. Furthermore, we found that the frequency of the different DNA repair outcomes varied across the genome, with some tested loci resulting in more frequent large-scale mutations. These results suggest that DNA repair pathways provide preferential repair across the genome that could create biased genome variation, which has significant implications for genome engineering and the genome evolution in natural populations.

microbiology↗

bioRxiv2021

Epigenetic reprogramming of human macrophages by an intestinal pathogen

Various pathogens systematically reprogram gene expression in macrophages, but the underlying mechanisms are largely unknown. We investigated whether the enteropathogen Yersinia enterocolitica alters chromatin states to reprogram gene expression in primary human macrophages. Genome-wide chromatin immunoprecipitation (ChIP) seq analyses showed that pathogen-associated molecular patterns (PAMPs) induced up- or down-regulation of histone modifications (HMod) at approximately 14500 loci in promoters and enhancers. Effectors of Y. enterocolitica reorganized about half of these dynamic HMod, with the effector YopP being responsible for about half of these modulatory activities. The reorganized HMod were associated with genes involved in immune response and metabolism. Remarkably, the altered HMod also associated with 61 % of all 534 known Rho GTPase pathway genes, revealing a new level in Rho GTPase regulation and a new aspect of bacterial pathogenicity. Changes in HMod were associated to varying degrees with corresponding gene expression, e. g. depending on chromatin localization and cooperation of the HMod. In summary, infection with Y. enterocolitica remodels HMod in human macrophages to modulate key gene expression programs of the innate immune response. Author SummaryHuman pathogenic bacteria can affect epigenetic histone modifications to modulate gene expression in host cells. However, a systems biology analysis of this bacterial virulence mechanism in immune cells has not been performed. Here we analyzed genome-wide epigenetic histone modifications and associated gene expression changes in primary human macrophages infected with enteropathogenic Yersinia enterocolitica. We demonstrate that Yersinia virulence factors extensively modulate histone modifications and associated gene expression triggered by the pathogen-associated molecular patterns (PAMPs) of the bacteria. The epigenetically modulated genes are involved in several key pathways of the macrophage immune response, including the Rho GTPase pathway, revealing a novel level of Rho GTPase regulation by a bacterial pathogen. Overall, our findings provide an in-depth view of epigenetic and gene expression changes during host-pathogen interaction and might have further implications for understanding of the innate immune memory in macrophages.

microbiology↗

bioRxiv2021

Rational Discovery of New Folds for Human Solute Carrier Proteins

Solute carrier (SLC) superfamily is the largest group responsible for transmembrane transport of substances in human cells. It includes more than 400 members which are organized into 65 families according to their physiological function and sequence similarity. Different families of solute transporters can adopt the same or different folds that determine the working mechanism and reflect the evolutionary relationship between SLC members. Analysis of structural data in literatures shows that there are 13 different folds in the solute carrier superfamily covering 40 families and 343 members. To further study their working mechanism, we systematically explored the SLC superfamily to look for more folds. Based on our results, at least three new folds were found for SLC superfamily, and one was experimentally verified in the SLC44 family. Our work has laid a foundation and provided important insights for the systematic and comprehensive study on the structure and function of solute carriers.

biophysics↗

bioRxiv2021

The evolution of fluoroquinolone resistance in Salmonella under exposure to sub-inhibitory concentration of enrofloxacin

The evolution of resistance in Salmonella to fluoroquinolones (FQs) under a broad range of sub-inhibitory concentrations (sub-MICs) has not been systematically studied. This study investigated the mechanism of resistance development in Salmonella enterica serovar Enteritidis (S. Enteritidis) under sub-MICs of 1/128xMIC to 1/2xMIC of enrofloxacin (ENR), a widely used veterinary FQ. It was shown that the resistance rate and resistance level of S. Enteritidis varied with the increase of ENR concentration and duration of selection. qRT-PCR results demonstrated that the expression of outer membrane porin (OMP) genes, ompF, ompC and ompD, were down-regulated first to rapidly adapt and develop resistance of [≤] 4xMIC, and as the resistance level increased ([≥]8xMIC), the up-regulated expression of efflux pump genes, acrB, emrB amd mdfA, along with mutations in quinolone resistance-determining region (QRDR) gradually played a decisive role. Cytohubba analysis based on transcriptomic profiles demonstrated that purB, purC, purD, purF, purH, purK, purL, purM, purN and purT were the hub genes for the FQs resistance. de novo IMP biosynthetic process, purine ribonucleoside monophosphate biosynthetic process and purine ribonucleotide biosynthetic process were the top three biological processes screened by MCODE. This study first described the dynamics of FQ resistance evolution in Salmonella under a long-term selection of sub-MICs of ENR in vitro. In addition, this work offers greater insight into the transcriptome changes of S. Enteritidis under the selection of ENR and provides a framework for FQs resistance of Salmonella for further studies.

microbiology↗

bioRxiv2021

ITRAQ-based quantitative proteomic analysis of japonica rice seedling during cold stress

Low temperature is one of the important environmental factors that affect rice growth and yield. To better understand the japonica rice responses to cold stress, isobaric tags for relative and absolute quantification (iTRAQ) labeling based quantitative proteomics approach was used to detected changes in protein level. Two-week-old seedlings of the cold tolerance rice variety Kongyu131 were treated at 8{degrees}C for 24, 48 and 72 h, then the total proteins were extracted from tissues and used for quantitative proteomics analysis. A total of 5082 proteins were detected for quantitative analysis, of which 289 proteins were significantly regulated, consisting of 169 uniquely up-regulated proteins and 125 uniquely down-regulated proteins in cold stress groups relative to control group. Functional analysis revealed most of regulation proteins involved in photosynthesis, metabolic pathway, biosynthesis of secondary metabolites and carbon metabolism. Western blot analysis showed that protein regulation was consistent with the iTRAQ data. The corresponding genes of 25 regulation proteins were used for quantitative real time PCR analysis, and the results showed that the mRNA level was not always parallel to the corresponding protein level. The importance of our study is providing new insights into cold stress responses in rice on proteomic aspect.

plant biology↗

bioRxiv2021

Kindlin-2 preserves integrity of the articular cartilage to protect against osteoarthritis

Osteoarthritis (OA) is an aging-related degenerative joint disease, which has no cure partly due to limited understanding of its pathological mechanism(s). Here we report that the focal adhesion protein Kindlin-2, but not Kindlin-1 or -3, is highly expressed in articular chondrocytes of the hyaline cartilage, which is dramatically decreased in the degenerated articular cartilage of aged mice and patients with OA. Inducible deletion of Kindlin-2 in chondrocytes at adult stage leads to spontaneous OA and much severe OA lesions in the mice receiving the surgery of destabilization of the medial meniscus. Mechanistically, Kindlin-2 deficiency promotes mitochondrial oxidative stress and activates Stat3 in articular chondrocytes, leading to Runx2-mediated chondrocyte hypertrophic differentiation and catabolism. In vivo, systemic pharmacological blockade of Stat3 activation or genetic ablation of Stat3 in chondrocytes reverses aberrant accumulation of Runx2 and ECM-degrading enzymes and limits OA deteriorations caused by Kindlin-2 deficiency. Furthermore, genetic inactivation of Runx2 in chondrocytes reverses structural changes and OA lesions caused by Kindlin-2 deletion without down-regulating p-Stat3 in articular chondrocytes. Of translational significance, intraarticular injection of Kindlin-2-expressing adeno-associated virus decelerates progression of aging- and instability-induced knee joint OA in mice. Collectively, we identify a novel pathway comprising of Kindlin-2, Stat3 and Runx2 in articular chondrocytes responsible for maintaining integrity of the articular cartilage and define a potential therapeutic target for OA.

pathology↗

bioRxiv2021

The synergistic effect of circRNA methylation promotes pulmonary fibrosis

RationaleN6-Methyladenosine (m6A) is the most common type of RNA methylation modification, mainly occurring on mRNA. Whether m6A-modified circRNAs are involved in different settings of pulmonary fibrosis remains unclear. Methods and ResultsUsing an m6A-circRNA epitranscriptomic chip, candidate circRNAs were selected, in which hsa_circ_0000672 and hsa_circ_0005654 were specifically involved in SiO2-induced pulmonary fibrosis by targeting the same protein, eIF4A3, indicating that the m6A modification of these two circRNAs has a synergistic effect on fibroblast dysfunction induced by SiO2. A mechanistic study revealed that the m6A modification of circRNAs was mainly mediated by the methyltransferase METTL3. Furthermore, METTL3 promoted the activation, migration and activity of pulmonary fibroblasts and participated in SiO2-induced pulmonary fibrosis via circRNA m6A modification. Conclusionm6A methylation of circRNAs mediates silica-induced fibrosis via synergistic effects, enriching the understanding of circRNAs and uncovering a potential new target to treat fibrosis-related diseases.

pharmacology and toxicology↗

bioRxiv2021

Recombinant Lloviu virus as a model to study inaccessible zoonotic viruses

Next generation sequencing has revealed the presence of many RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, many of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Recombinant LLOV (rLLOV) displays typical filovirus features, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal against pandemic preparedness.

microbiology↗

bioRxiv2021

Human airway lineages derived from pluripotent stem cells reveal the epithelial responses to SARS-CoV-2 infection

There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors ACE2 and TMPRSS2. Multiciliated cells are the primary initial target of SARS-CoV-2 infection. Upon infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses and treatment with remdesivir or camostat methylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia. Highlights and eTOC blurbO_LISubsets of human iPSC-airway epithelial cells express SARS-Co-V entry factors ACE2 and TMPRSS2. C_LIO_LIiPSC-airway cells are permissive to SARS-CoV-2 infection via multiciliated cells. C_LIO_LISARS-CoV-2 infection of iPSC-airway leads to a robust interferon and inflammatory response. C_LIO_LIiPSC-airway is a physiologically relevant model to study SARS-CoV-2 infection. C_LI

cell biology↗

bioRxiv2021

Genome-wide identification and characterization of DNA/RNA differences associated with Fusarium graminearum infection in wheat

RNA editing (DNA/RNA differences) as a post-transcriptional modification approach to enrich genetic information, plays the crucial role in regulating diverse biological processes in eukaryotes. Although it has been extensively studied in plant chloroplast and mitochondria genome, RNA editing in plant nuclear genome, especially those associated with Fusarium head blight (FHB), is not well studied at present. Here, we investigated the DNA/RNA differences associated with FHB through a novel method by comparing the RNA-seq data from Fusarium-infected and control samples from 4 wheat genotypes. A total of 187 DNA/RNA differences were identified in 36 wheat genes, representing the first landscape of the FHB-responsive RNA editome in wheat. Furthermore, all of these 36 edited genes were located in the FHB related co-expression gene modules, which may involve in regulating FHB response. Finally, the effects of DNA/RNA differences were systematically investigated to show that they could cause the change of RNA structure and protein structure in edited genes. In particular, the G to C editing (chr3A_487854715) in TraesCS3A02G263900, which is the orthology of OsRACK1, resulted that it was targeted by tae-miR9664-3p to control its expression in different genotype through different editing efficiency, suggesting RNA editing could mediate miRNA to participate in the regulation network of FHB tolerance. This study reported the first wheat DNA/RNA differences associated with FHB, which not only contribute to better understand the molecular basis underlying FHB tolerance, but also shed light on improving FHB tolerance through epigenetic method in wheat and beyond.

plant biology↗

bioRxiv2021

SARS-CoV-2 mRNA Vaccine Induces Robust Specific and Cross-reactive IgG and Unequal Strain-specific Neutralizing Antibodies in Naïve and Previously Infected Recipients

With the advance of SARS-CoV-2 vaccines, the outlook for overcoming the global COVID-19 pandemic has improved. However, understanding of immunity and protection offered by the SARS-CoV-2 vaccines against circulating variants of concern (VOC) is rapidly evolving. We investigated the mRNA vaccine-induced antibody responses against the referent WIV04 (Wuhan) strain, circulating variants, and human endemic coronaviruses in 168 naive and previously infected people at three-time points. Samples were collected prior to vaccination, after the first and after the second doses of one of the two available mRNA-based vaccines. After full vaccination, both naive and previously infected participants developed comparable robust SARS-CoV-2 specific spike IgG levels, modest IgM and IgA binding antibodies, and varying degrees of HCoV cross-reactive antibodies. However, the strength and frequency of neutralizing antibodies produced in naive people were significantly lower than in the previously infected group. We also found that 1/3rd of previously infected people had undetectable neutralizing antibodies after the first vaccine dose; 40% of this group developed neutralizing antibodies after the second dose. In all subjects neutralizing antibodies produced against the B.1.351 and P.1 variants were weaker than those produced against the reference and B.1.1.7 strains. Our findings provide support for future booster vaccinations modified to be active against the circulating variants.

immunology↗

bioRxiv2021

SE(3)-Equivariant Energy-based Models for End-to-End Protein Folding

Accurate prediction of protein structures is critical for understanding the biological function of proteins. Nevertheless, most structure optimization methods are built upon pre-defined statistical energy functions, which may be sub-optimal in formulating the conformation space. In this paper, we propose an end-to-end approach for protein structure optimization, powered by SE(3)-equivariant energy-based models. The conformation space is characterized by a SE(3)-equivariant graph neural network, with substantial modifications to embed the protein-specific domain knowledge. Furthermore, we introduce continuously-annealed Langevin dynamics as a novel sampling algorithm, and demonstrate that such process converges to native protein structures with theoretical guarantees. Extensive experiments indicate that SE(3)-Fold achieves comparable structure optimization accuracy, compared against state-of-the-art baselines, with over 1-2 orders of magnitude speed-up.

bioinformatics↗

bioRxiv2021

Distinct allosteric mechanisms of first-generation MsbA inhibitors

Present in all kingdoms of life, ATP-binding cassette (ABC) transporters couple ATP hydrolysis to mechanical force and facilitate trafficking of diverse substrates across biological membranes. Although many ABC transporters are promising drug targets, their mechanisms of regulation by small molecule inhibitors remain largely unknown. Herein, we used the lipopolysaccharide (LPS) flippase MsbA, a prototypical ABC exporter, as a model system to probe mechanisms of allosteric modulation by compounds binding to the transmembrane domains (TMDs). Recent chemical screens have identified intriguing LPS transport inhibitors targeting MsbA: the ATPase stimulator TBT1 and the ATPase inhibitor G247. Despite preliminary biochemical and structural data, it is unclear how TBT1 and G247 bind to the MsbA TMDs yet induce opposite allosteric effect in the nucleotide-binding domains (NBDs). Through single-particle EM, mutagenesis and activity assay, we show that TBT1 and G247 bind adjacent yet separate locations in the TMDs, inducing drastic changes in TMD conformation and NBD positioning. Two TBT1 molecules asymmetrically occupy the LPS binding site to break the symmetry of MsbA, resulting in disordered transmembrane helices and decreased NBD distance. In this novel inhibited ABC transporter state, decreased distance between the NBDs causes stimulation of ATP hydrolysis yet LPS transport blockage. In contrast, G247 acts as a TMDs wedge, symmetrically increasing NBD separation and preventing conformational transition of MsbA. Our study uncovers the distinct mechanisms of the first-generation MsbA-specific inhibitors and demonstrates that rational design of substrate-mimicking compounds can be exploited to develop useful ABC transporter modulators.

biochemistry↗

bioRxiv2021

RNA silencing by CRISPR in plants does not require Cas13

RNA-targeting CRISPR-Cas can provide potential advantages over DNA editing, such as avoiding pleiotropic effects of genome editing, providing precise spatiotemporal regulation and expanded function including anti-viral immunity. Here, we report the use of CRISPR-Cas13 in plants to reduce both viral and endogenous RNA. Unexpectedly, we discovered that crRNA designed to guide Cas13 could, in the absence of the Cas13 protein, cause substantial reduction in RNA levels as well. We demonstrate Cas13-independent guide-induced gene silencing (GIGS) in three plant species, including stable transgenic Arabidopsis. We determined that GIGS utilizes endogenous RNAi machinery despite the fact that crRNA are unlike canonical triggers of RNAi such as miRNA, hairpins or long double-stranded RNA. These results suggest that GIGS offers a novel and flexible approach to RNA reduction with potential benefits over existing technologies for crop improvement. Our results demonstrate that GIGS is active across a range of plant species, evidence similar to recent findings in an insect system, which suggests that GIGS is potentially active across many eukaryotes.

plant biology↗

bioRxiv2021

Structure, immunogenicity, and conformation-dependent receptor binding of the post-fusion human metapneumovirus F protein

Human metapneumovirus (hMPV) is an important cause of acute viral respiratory infection. As the only target of neutralizing antibodies, the hMPV fusion (F) protein has been a major focus for vaccine development and targeting by drugs and monoclonal antibodies (mAbs). While X-ray structures of trimeric pre-fusion and post-fusion hMPV F proteins from genotype A, and monomeric pre-fusion hMPV F protein from genotype B have been determined, structural data for the post-fusion conformation for genotype B is lacking. We determined the crystal structure of this protein and compared the structural differences of post-fusion hMPV F between hMPV A and B genotypes. We also assessed the receptor binding properties of the hMPV F protein to heparan sulfate. A library of heparan sulfate (HS) oligomers was used to verify the HS binding activity of hMPV F, and several compounds showed binding to predominantly pre-fusion hMPV F, but had limited binding to post-fusion hMPV F. Furthermore, mAbs to antigenic sites III and the 66-87 intratrimeric epitope block heparan binding. In addition, we evaluated the efficacy of post-fusion hMPV B2 F protein as a vaccine candidate in BALB/c mice. Mice immunized with hMPV B2 post-fusion F protein showed a balanced Th1/Th2 immune response and generated neutralizing antibodies against both subgroup A2 and B2 hMPV strains, which protected the mice from hMPV challenge. Antibody competition analysis revealed the antibodies generated by immunization target two known antigenic sites (III and IV) on hMPV F. Overall, this study provides new characteristics of the hMPV F protein informative for vaccine and therapy development. ImportanceHuman metapneumovirus (hMPV) is an important cause of viral respiratory disease. In this paper, we report the X-ray crystal structure of the hMPV fusion (F) protein in the post-fusion conformation from genotype B. We also assessed binding of the hMPV F protein to heparin and heparan sulfate, a previously reported receptor for the hMPV F protein. Furthermore, we determined the immunogenicity and protective efficacy of post-fusion hMPV B2 F protein, which is the first study using a homogenous conformation of the protein. Antibodies generated in response to vaccination give a balanced TH1/TH2 response and target two previously discovered neutralizing epitopes.

microbiology↗