Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking out viral receptor ACE2. The lead drug possessing an Fc tag (Nanosota-1C-Fc) bound to SARS-CoV-2 RBD with a Kd of 15.7picomolar (~3000 times more tightly than ACE2 did) and inhibited SARS-CoV-2 infection with an ND50 of 0.16microgram/milliliter (~6000 times more potently than ACE2 did). Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy in hamsters subjected to SARS-CoV-2 infection. Unlike conventional antibody drugs, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-Fc documented a greater than 10-day in vivo half-life efficacy and high tissue bioavailability. Nanosota-1C-Fc is a potentially effective and realistic solution to the COVID-19 pandemic.Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a novel series of single-domain antibodies ,  The novel coronavirus SARS-CoV-2 has led to the COVID-19 pandemic, devastating human health and the global economy , 4. A rein vivo.Nanobodies are unique antibodies derived from heavy chain-only antibodies found in members of the camelidae family   5, 6). , 6. 5, 6The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a prime target for therapeutic development . The spiNanosota-1. Identified by screening a camelid nanobody phage display library against the SARS-CoV-2 RBD, the Nanosota-1 series bound potently to the SARS-CoV-2 RBD and were effective at inhibiting SARS-CoV-2 infection in vitro. The best performing drug, Nanosota-1C-Fc, demonstrated preventative and therapeutic efficacy in a hamster model of SARS-CoV-2 infection. Nanosota-1C-Fc was produced at high yields easily scalable for mass production and was also found to have a pharmacologically relevant in vivo half-life and excellent bioavailability. Our data suggest that Nanosota-1c-Fc may provide an effective solution to the COVID-19 pandemic.Here, we report the development of a novel series of anti-SARS-CoV-2 nanobody therapeutics, Nanosota-1A and then subjected to two rounds of affinity maturation. For each round of affinity maturation, random mutations were introduced to the whole gene of Nanosota-1A through error-prone PCR, and mutant phages were selected for enhanced binding to SARS-CoV-2 RBD. Nanobodies contain four framework regions (FRs) as structural scaffolds and three complementarity-determining regions (CDRs) for antigen binding. The nanobody after the first round of affinity maturation, named Nanosota-1B, possessed one mutation in CDR3 and two other mutations in FR3 (near CDR3). Affinity maturation of Nanosota-1B resulted in Nanosota-1C, which possessed one mutation in CDR2 and another mutation in FR2. We next made an Fc-tagged version of Nanosota-1C, termed Nanosota-1C-Fc, to create a bivalent construct with increased molecular weight.For the rapid identification of virus-targeting nanobodies, we constructed a naïve nanobody phage display library using B cells isolated from the spleen, bone marrow, and blood of nearly a dozen non-immunized llamas and alpacas . RecombiNanosota-1 drugs to SARS-CoV-2 RBD, we determined the crystal structure of SARS-CoV-2 RBD complexed with Nanosota-1C. The structure showed that Nanosota-1C binds close to the center of the SARS-CoV-2 RBM , confirming success of the stepwise affinity maturation. Nanosota-1C-Fc had the highest RBD-binding affinity (Kd - 15.7 pM), which was ~3,000 times tighter than the RBD-binding affinity of ACE2. Moreover, compared with ACE2, Nanosota-1C-Fc bound to the RBD with a higher kon and a lower koff, demonstrating significantly faster binding and slower dissociation. Next, we investigated the competitive binding among Nanosota-1C, ACE2, and RBD using protein pull-down assay . Nanosota-1C-Fc had an ND50 for the SARS-CoV-2 pseudovirus of 0.27 μg/ml, which was ~10 times more potent than monovalent Nanosota-1C (2.52 μg/ml) and over 100 times more potent than ACE2 (44.8 μg/ml) . Nanosota-1C-Fc had an ND50 of 0.16 μg/ml, which was ~20 times more potent than monovalent Nanosota-1C (3.23 μg/ml) and ~6000 times more potent than ACE2 (980 μg/ml) (Nanosota-1C-Fc and Nanosota-1C are potent inhibitors of SARS-CoV-2 pseudovirus entry and authentic SARS-CoV-2 infection.The ability of the erformed . For the8 μg/ml) . Additio protein , which h protein . For the0 μg/ml) ; Fig. S6in vitro studies, we next evaluated the therapeutic efficacy of the lead drug Nanosota-1C-Fc in a hamster model challenged with SARS-CoV-2 via intranasal inoculation. In addition to an untreated control group, three groups of animals were injected with a single dose of Nanosota-1C-Fc: (i) 24 hours pre-challenge at 20 mg/kg body weight, (ii) 4 hours post-challenge at 20 mg/kg, and (iii) 4 hours post-challenge at 10 mg/kg. As previously validated in this model , as evidenced by the favorable therapeutic metrics  for one week and then measured the remaining SARS-CoV-2 RBD-binding capacity by ELISA , a single injected dose of Nanosota-1C-Fc can theoretically protect a person from SARS-CoV-2 infection for days or weeks in the outpatient setting, reducing the spread of SARS-CoV-2 in human populations. Second, we also learned from our in vivo study that Nanosota-1C-Fc can potentially be used to treat SARS-CoV-2 infections, thus, saving lives and alleviating symptoms in infected patients in the clinical setting. Third, though ephemeral in nature given its short half-life and rapid clearance from the blood, Nanosota-1C could be used as an inhaler to treat infections in the respiratory tracts  protocols of the University of Texas Medical Branch (protocol number 2007072) and of the University of Minnesota (protocol number 2009–38426A).E. coli (Lucigen), TG1 E. coli (Lucigen), SHuffle T7 E. coli (New England Biolabs) were grown in TB medium or 2YT medium with 100 mg/L ampicillin. Vero E6 cells (American Type Culture Collection) were grown in Eagle’s minimal essential medium (EMEM) supplemented with penicillin (100 units/ml), streptomycin (100 μg/ml), and 10% fetal bovine serum (FBS). SARS-CoV-2 spike (GenBank accession number QHD43416.1) and ACE2 (GenBank accession number NM_021804) were described previously  with a N-terminal PelB leader sequence and C-terminal His tag and HA tag. Nanosota-1C-Fc was cloned into pET42b vector (Novagen) with a C-terminal human IgG1 Fc tag. SARS-CoV-2 (US_WA-1 isolate) from CDC (Atlanta) was used throughout the study. All experiments involving infectious SARS-CoV-2 were conducted at the University of Texas Medical Branch and University of Iowa in approved biosafety level 3 laboratories.HEK293T cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin (Life Technologies). ss320 6 tag, and a HA tag. The PCR2 product was digested with SFI-I (New England Biolabs) and then was ligated with SFI-I-digested PADL22c vector. The ligated product was transformed via electroporation into TG1 E. coli (Lucigen). Aliquots of cells were spread onto 2YT agar plates supplemented with ampicillin and glucose, incubated at 30°C overnight, and then scraped into 2YT media. After centrifugation, the cell pellet was suspended into 50% glycerol and stored at −80°C. The library size was 7. 5 × 1010. To display nanobodies on phages, aliquots of the TG1 E. coli bank were inoculated into 2YT media, grown to early logarithmic phase, and infected with M13K07 helper phage.The camelid nanobody phage display library was constructed as previously described , 30. BriE. coli and rescued with M13K07 helper phage. The eluted phages from round 4 were used to infect ss320 E. coli. Single colonies were picked into 2YT media and nanobody expressions were induced with 1 mM IPTG. The supernatants were subjected to ELISA for selection of strong binders (described below). The strong binders were then expressed and purified (described below) and subjected to SARS-CoV-2 pseudovirus entry assay for selection of anti-SARS-CoV-2 efficacy (described below). The lead nanobody after initial screening was named Nanosota-1A.The above camelid nanobody phage display library was used in the bio-panning as previously described . BrieflyNanosota-1A was performed as previously described (Nanosota-1A using error-prone PCR. Two rounds of error-prone PCR were performed using the GeneMorph II Random Mutagenesis Kit (Agilent Technologies). The PCR product was cloned into the PADL22c vector and transformed via electroporation into the TG1 E. coli. The library size was 6 × 108. Three rounds of bio-panning were performed using 25 ng, 10 ng and 2 ng RBD-Fc, respectively. The strongest binder after affinity maturation was named Nanosota-1B. A second round of affinity maturation was performed in the same way as the first round, except that three rounds of bio-panning were performed using 10 ng, 2 ng and 0.5 ng RBD-Fc, respectively. The strongest binder after the second round of affinity maturation was named Nanosota-1C.Affinity maturation of escribed . BrieflyNanosota-1A, 1B and 1C were each purified from the periplasm of ss320 E. coli after the cells were induced by 1 mM IPTG. The cells were collected and re-suspended in 15 ml TES buffer , shaken on ice for 1 hour and then incubated with 40 ml TES buffer followed by shaking on ice for another hour. The protein in the supernatant was sequentially purified using a Ni-NTA column and a Superdex200 gel filtration column  as previously described . The protein in the supernatant was sequentially purified on protein A column and Superdex200 gel filtration column as previously described (http://www.addgene.org/protocols/plko/). The proteins were secreted to cell culture media, harvested, and purified on either Ni-NTA column (for His-tagged proteins) or protein A column (for Fc-tagged protein) and then on Superdex200 gel filtration column as previously described  or human ACE2 ectodomain  were made according to the E and F sections of the pLKO.1 Protocol from Addgene (Nanosota-1 drugs (either purified recombinant drugs or drugs in the mouse serum) as previously described (2SO4. The absorbance at 450 nm (A450) was measured using a Synergy LX Multi-Mode Reader (BioTek).ELISA was performed to detect the binding between SARS-CoV-2 RBD and escribed . BrieflyNanosota-1C complex for crystallization, the two proteins were mixed together in solution and purified using a Superdex200 gel filtration column . The complex was concentrated to 10 mg/ml in buffer 20 mM Tris pH 7.2 and 200 mM NaCl. Crystals were screened at High-Throughput Crystallization Screening Center  as previously described  PEG 4000. Crystals were soaked briefly in 50 mM MnCl2, 50 mM MES pH 6.0, 25% (W/V) PEG 4000 and 30% ethylene glycol before being flash-frozen in liquid nitrogen. X-ray diffraction data were collected at the Advanced Photon Source beamline 24-ID-E. The structure was determined by molecular replacement using the structures of SARS-CoV-2 RBD (PDB 6M0J) and another nanobody (PDB 6QX4) as the search templates. Structure data and refinement statistics are shown in To prepare the RBD/escribed , and werNanosota-1 drugs were injected at different concentrations: 320 nM – 10 nM for Nanosota-1A; 80 nM - 2.5 nM for Nanosota-1B and Nanosota-1C; 20 nM - 1.25 nM for Nanosota-1C-Fc. The resulting data were fit to a 1:1 binding model using Biacore Evaluation Software .Surface plasmon resonance assay using a Biacore S200 system  was carried out as previously described  and 4 μg human ACE2  were added to the RBD-bound beads. After one-hour incubation at room temperature, the bound proteins were eluted using elution buffer (0.1 M glycine pH 2.7). The samples were then subjected to SDS-PAGE and analyzed through Western blot using an anti-His antibody.Protein pull-down assay was performed using Immunoprecipitation kit (Invitrogen) as previously described . BrieflyNanosota-1C and 121 μg SARS-CoV-2 RBD were incubated together at room temperature for 30 min. The mixture was subjected to gel filtration chromatography. Samples from each peak off the column were then subjected to SDS-PAGE and analyzed through Coomassie blue staining.Gel filtration chromatography assay was performed on a Superdex200 column. 500 μg human ACE2, 109 μg Nanosota-1 drugs in neutralizing SARS-CoV-2 pseudovirus entry was evaluated as previously described (50).The potency of escribed , 22. BriNanosota-1 drugs in neutralizing authentic SARS-CoV-2 infections was evaluated using a SARS-CoV-2 plaque reduction neutralization test (PRNT) assay. Specifically, individual drugs were serially diluted in DMEM and mixed 1:1 with 80 pfu SARS-CoV-2 at 37°C for 1 hour. The mixtures were then added into Vero E6 cells at 37°C for an additional 45 minutes. After removing the culture medium, cells were overlaid with 0.6% agarose and cultured for 3 days. Plaques were visualized by 0.1% crystal violet staining. The efficacy of each drug was calculated and expressed as the concentration capable of reducing the number of virus plaques by 50% compared to control serum-exposed virus .The potency of 6 Median Tissue Culture Infectious Dose or TCID50) in 100 μL DMEM (50 μL per nare). Sample size was comparable to previous animal challenge studies  24 hours pre-challenge at 20 mg/kg body weight of hamsters; (2) 4 hours post-challenge at 20 mg/kg body weight of hamsters; (3) 4 hours post-challenge at 10 mg/kg body weight of hamsters. Hamsters in the control (negative) group were administered PBS buffer 24 hours pre-challenge. An additional group was tested for a different hypothesis and the data were not included in the current study. Body weights were collected daily beginning prior to challenge. Nasal swabs were collected prior to challenge and additionally 1 day, 2 days, 3 days, 5 days and 10 days post-challenge for quantitative real-time RT-PCR . Hamsters were humanely euthanized 10 days post-challenge via overexposure to CO2. The lungs and bronchial tubes were collected and fixed in formalin for histopathological analysis. This experiment was performed in accordance with the guidelines set by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch (UTMB).Equal sex Syrian hamsters (n=24) were obtained from Envigo (IN) and challenged via intranasal inoculation with SARS-CoV-2 (at a titer of 1 × 10 studies  and consNanosota-1C or Nanosota-1C-Fc (100 μg in 100 μl PBS buffer). At varying time points, mice were euthanized and whole blood was collected. Then sera were prepared through centrifugation of the whole blood at 1500×g for 10 min. The sera were then subjected to ELISA for evaluation of their SARS-CoV-2 RBD-binding capability.Male C57BL/6 mice (3 to 4 weeks old) (Envigo) were intravenously injected (tail-vein) with in vivo biodistribution of Nanosota-1C-Fc and Nanosota-1C, the nanobodies were labeled with Zirconium-89 [89Zr] and injected into male C57BL/6 mice (5 to 6 weeks old) (Envigo). Briefly, the nanobodies were first conjugated to the bifunctional chelator p-SCN-Bn-Deferoxamine  as previously described  was then conjugated as previously described  were intravenously injected (tail-vein). Mice were euthanized at different time points. Organs were collected and counted on an automatic gamma-counter (Hidex). The total number of counts per minute (cpm) for each organ or tissue was compared with a standard sample of known activity and mass. Count data were corrected to both background and decay. The percent injected dose per gram (%ID/g) was calculated by normalization to the total amount of activity injected into each mouse.To evaluate the escribed , and [89escribed . [89Zr]-1


However, this targeted approach has to date been unable to identify major regulators of cell-to-cell gene expression variability in humans. To address this, we have combined single-cell protein expression measurements in the human immune system using flow cytometry with a quantitative genetics analysis. For the majority of proteins whose variability in expression has a heritable component, we find that genetic variants act in trans, with notably fewer variants acting in cis. Furthermore, we highlight using Mendelian Randomization that these variability-Quantitative Trait Loci might be driven by the cis regulation of upstream genes. This indicates that natural selection may balance the impact of gene regulation in cis with downstream impacts on expression variability in trans.Identifying the factors that shape protein expression variability in complex multi-cellular organisms has primarily focused on promoter architecture and regulation of single-cell expression  cis) genetic factors minimally influence this variability. We have combined genetic measurements with flow cytometry single-cell protein levels to resolve the genetic control of gene expression variability in human immune cells. Importantly, we have demonstrated that whilst genetic variants near the target genes (cis) rarely influence variability, there is still an extensive genetic contribution from genetic loci faraway, or on a separate chromosome (trans). Furthermore, we have resolved that these trans genetic effects regulate the expression of other nearby genes, which leads to changes in gene expression variability of our target proteins. Our findings can be explained by an evolutionary balance between the cis regulation of gene expression levels, and the downstream consequences on gene expression variability.Genetic variation can change how much a gene is turned on or off in a tissue or a population of cells of the same type. However, this averaging of expression levels across a cell population masks an important aspect of gene expression regulation, namely its variability. Recent work in humans has indicated that nearby ( Intrinsic noise represents the differences in promoter output between two alleles of the same gene, whilst extrinsic noise represents all other sources of variability  (defined by the light grey box).Error bars represent bootstrapped standard errors from 100 permutations. Orange points are those with h(EPS)Click here for additional data file.S12 FigDistribution of empirical p-values of additive genetic variance components for expression variability traits.(EPS)Click here for additional data file.S13 FigVariance components estimates grouped by protein for mean (blue) and variability (orange) traits across cell types. Y-axis shows the % phenotypic variance, X-axis shows the variance components .(EPS)Click here for additional data file.S14 Fig10 association p-values (y-axis) between expression variability and genome-wide genetic variants (x-axis). The purple horizontal line represents genome-wide significance threshold (5x10-8), and the orange line represents the Bonferroni corrected threshold (8.47x10-10).Linear mixed model -log(EPS)Click here for additional data file.S15 Fig10 association p-values (y-axis) between expression variability and genome-wide genetic variants (x-axis). The purple horizontal line represents genome-wide significance threshold (5x10-8), and the orange line represents the Bonferroni corrected threshold (8.47x10-10).Linear mixed model -log(EPS)Click here for additional data file.S16 Fig10 association p-values (y-axis) between expression variability and genome-wide genetic variants (x-axis). The purple horizontal line represents genome-wide significance threshold (5x10-8), and the orange line represents the Bonferroni corrected threshold (8.47x10-10).Linear mixed model -log(EPS)Click here for additional data file.S17 Fig10 association p-values (y-axis) between mean expression levels and genome-wide genetic variants (x-axis). The purple horizontal line represents genome-wide significance threshold (5x10-8), and the orange line represents the Bonferroni corrected threshold (8.47x10-10).Linear mixed model -log(EPS)Click here for additional data file.S18 FigVenn diagrams showing the overlap of mean (a) and expression variability traits (b) between the Milieu Intérieur (red) and TwinsUK (blue).(EPS)Click here for additional data file.S19 FigShown are the MR regression estimate (β), error bars denote the 95% CI for relationships at FDR 10%. Y-axis labels show the vProtein and eGene. Points are coloured by the cell type in which the eGene and variability QTL are both present.(EPS)Click here for additional data file.S20 Fig(EPS)Click here for additional data file.S21 Fig(EPS)Click here for additional data file.S22 Fig(EPS)Click here for additional data file.S23 Fig(EPS)Click here for additional data file.S24 Fig(EPS)Click here for additional data file.S25 Fig(EPS)Click here for additional data file.S26 Fig(EPS)Click here for additional data file.S27 Fig(EPS)Click here for additional data file.S28 Fig(EPS)Click here for additional data file.S29 Fig(EPS)Click here for additional data file.S30 Fig(EPS)Click here for additional data file.S31 Fig(EPS)Click here for additional data file.S32 Fig(EPS)Click here for additional data file.S33 Fig(EPS)Click here for additional data file.S34 Fig(EPS)Click here for additional data file.S35 Fig10 association p-values (y-axis) between CD3 expression variability in granulocytes and genome-wide genetic variants (x-axis) without (top) and with (bottom) adjustment for FCGR2A genotype at rs4657041. The purple horizontal line represents genome-wide significance threshold (5x10-8), and the orange line represents the Bonferroni corrected threshold (8.47x10-10).Linear mixed model -log(EPS)Click here for additional data file.S36 Figtrans variability-pQTL and cis-eQTL. Point size is proportional to the -log10 p-value from a χ2 test.Shown are the Cochrane’s Q-values from the MR-Egger regression across SNPs (x-axis) for each pair of varSNP and eGene (y-axis). Points are coloured by the broad matching cell type between the (EPS)Click here for additional data file.S37 FigShown are the proportion of QTLs detected (y-axis) as a function of the sample size (x-axis) for both mean  and variability (turquoise) traits. (a) Cis-QTL mapping and (b) trans-QTL mapping were performed separately. Numbers denote the total number of QTLs detected with the largest sample size.(EPS)Click here for additional data file.


HER2 amplification greatly contributes to the tumorigenesis of multiple cancers. Intronic miR-4728-5p is transcribed along with its host gene HER2. However, little is known about the role of miR-4728-5p in cancer. This study aims to elucidate the potential role of miR-4728-5p and the underlying mechanism in breast cancer. Kaplan-Meier analysis showed that higher expression of HER2 led to worse survival outcomes in breast cancer patients. The TCGA dataset revealed that compared to normal breast tissues, HER2 and miR-4728-5p levels were significantly upregulated in breast cancer tissues with a positive correlation. In functional assays, miR-4728-5p was confirmed to promote the proliferation and migration in breast cancer cell BT474. EBP1 was identified as a direct target of miR-4728-5p via bioinformatics and luciferase reporter assays. miR-4728-5p was further demonstrated to increase HER2 expression and promote cell proliferation and migration by directly inhibiting EBP1 in breast cancer. Taken together, the HER2-intronic miR-4728-5p/EBP1/HER2 feedback loop plays an important role in promoting breast cancer cell proliferation and migration. Our study provides novel insights for targeted therapies of breast cancer. Breast cancer is the most common malignant tumor diagnosed among women, accounting for 30% of all new cancer cases in United States this year . DespitemicroRNAs (miRNAs) are single-stranded noncoding RNA molecules of approximately 19–25 nucleotides and ubiquitously expressed in all kinds of tissues. miRNAs directly target the 3’ untranslated regions (3’UTR) of message RNAs (mRNAs) by complementary base pairing, leading to silencing or degradation of targeted mRNAs . These dIntronic miRNAs are derived from the introns of protein-coding genes and transcribed along with their host genes , 6. miR-In this study, we demonstrated HER2-encoded miR-4728-5p could augment cell proliferation and migration by directly targeting ErbB3-binding protein 1 (EBP1) in breast cancer. Moreover, miR-4728-5p-mediated downregulation of EBP1 in turn enhanced HER2 expression, indicating a feedback loop consisting of HER2, miR4728-5p and EBP1. Our study suggests miR-4728-5p as a novel potential therapeutic target and provides new insights for the tumorigenesis in breast cancer.Breast cancer cells MCF-7 (HER2-) and BT474 (HER2+) were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences  and cultured under suggested conditions . MCF-7 c5’-AGCGACCAGGATTATATTCTCAAG-3’; EBP1 reverse: 5’-ATAACATCTGCTTTCCTCCCTG-3’; HER2 forward: 5’-AGCCTTGCCCCATCAACTG-3’; HER2 reverse: 5’-AATGCCAACCACCGCAGA-3’; GAPDH forward: 5’-GCACCGTCAAGGCTGAGAAC-3’; GAPDH reverse: 5’- TGGTGAAGACGCCAGTGGA -3’.Total RNA was extracted using the TRIzol . miRNA and mRNA quantifications were performed respectively using Taqman probes  or SYBR Green Dye  as previously shown . U6 and Total protein was isolated using RIPA Lysis buffer . Protein concentration was quantified with a Pierce BCA kit . Samples were separated by 10% SDS-PAGE gel . Antibodies against EBP1 (sc-393114), HER2 (sc-33684) and GAPDH (sc-166574) were purchased from Santa Cruz Biotechnology.CCCUCCC to GGGAGGG in order to generate a mutant luciferase vector. 293T cells were utilized to perform the luciferase report assay. miR-4728-5p mimics or inhibitor was transfected using Lipofectamine 2000  along with luciferase vectors and β-gal control plasmid. The fluorescence value was assayed using a luciferase assay kit . Relative luciferase activity was normalized to β-gal.The EBP1 3’UTR fragment containing miR-4728-5p binding site was inserted into the pMIR-reporter vector . The wild-type binding site was mutated from 5’-GTGAGGTGGAAAGGCGT-3’) was synthesized by GenePharma (China). The EBP1 expression plasmid and siRNA were transfected using Lipofectamine 2000 . Total RNA and protein were isolated 24 h post-transfection. Protein and mRNA levels were assessed by Western blot and quantitative RT-PCR.The full-length open reading frame of human EBP1 was cloned into a mammalian expression plasmid . An empty plasmid served as the negative control. EBP1 siRNA (sense: 3 cells per well) for the proliferation activity which was determined at 0, 6, 12, 18, and 24 h post-transfection using the Cell Counting Kit-8  according to the manufacturer’s instructions. For the migration assay, transfected BT474 cells were treated with mitomycin C  for 10 h to prevent proliferation, and equal number of cells were then seeded into the 24-Well Millicell plates  containing an 8-μm pore membrane as previously described [BT474 cells were transfected and seeded into 96-well plates . Experiments were repeated in triplicate. Student’s t-test was applied to analyze differences between two groups. P values were considered to be statistically significant when it is less than 0.05.Targetscan  was usedhttp://kmplot.com/analysis/) and found that higher-expressed HER2  and transwell assays. As shown, overexpression of miR-4728-5p remarkably promoted cell proliferation  and migrAmong the potential targets of miR-4728-5p predicted via bioinformatics, EBP1 drew our attention as it could suppress HER2 transcription by binding to the promoter region . Two preTo figure out whether miR-4728-5p promotes cell proliferation and migration by targeting EBP1, we constructed an EBP1 overexpression vector without the miR-4728-5p responsive 3’UTR to specifically restore EBP1 expression inhibited by miR-4728-5p. Subsequently, restoration of EBP1  completeThe EGFR family, crucially involved in the tumorigenesis of multiple cancers, comprises four receptors, namely, EGFR/HER1, HER2, HER3 and HER4. Dimerization between EGFR members is essentially required for the signaling activities and functions of these receptors . For exaEBP1 is a ligand of HER3 but can reduce HER2 transcriptions by mitigating the activity of HER2 promoter . StudiesIn the present study, we first focused on the potential function of HER2-encoded miR-4728-5p in breast cancer. Through analyzing gene expressions from a TCGA cohort of breast cancer patients, we found miR-4728-5p was highly expressed along with HER2 in the cancer tissues. Pearson analysis showed a positive correlation between miR-4728-5p and HER2 expression levels. Furthermore, overexpressing of miR-4728-5p dramatically accelerated the proliferation and migration activities of BT474 cells, while silencing of miR-4728-5p had the reverse effect, indicating miR-4728-5p plays an oncogenic role in breast cancer.When further predicting the potential target genes of miR-4728-5p using bioinformatics, we found EBP1 that had two binding sites of miR-4728-5p at the 3’UTR. We next tried to figure out whether HER2-encoded miR-4728-5p could directly regulate EBP1. The luciferase reporter assays verified the direct binding between miR-4728-5p and EBP1 3’UTR. Overexpression of miR-4728-5p significantly repressed EBP1 protein expressions as well as increased HER2 protein levels in BT474 and MCF-7 cells. We also introduced the rescue experiments to confirm the negative regulation of EBP1 by miR-4728-5p. EBP1 overexpression vector was used to specifically restore EBP1 expression inhibited by miR-4728-5p. As a result, restoration of EBP1 in BT474 completely abolished the promoting effect of miR4728-5p on cell proliferation and migration.Based on these results, we draw an intriguing conclusion that HER2-intronic miR-4728-5p could facilitate the expression of its host gene HER2 by directly repressing EBP1. Thus, co-amplification of HER2 and miR-4728-5p plays a dual oncogenic role in the pathogenesis of breast cancer. The feedback loop of HER2-intronic miR-4728-5p/EBP1/HER2 is of great importance in HER2-positive breast cancer and leads to poor prognosis. Our findings may provide a better understanding of HER2 signaling pathway in breast cancer, as well as potential therapeutic targets.S1 Fig(A and B) The Pearson’s correlation analysis between the expression levels of miR-4728-5p and HER2 in LUAD and STAD tissue obtained from the TCGA dataset .(TIF)Click here for additional data file.S2 FigThe RNA expression levels of miR-4728-5p, EBP1 and Erbb2 in MCF-7 and BT474. The expression levels of miR-4728-5p and Erbb2 mRNA are very low. ***p<0.001.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLS)Click here for additional data file.


Caenorhabditis elegans is a well-known model for investigating fundamental cellular processes. In egg-laying, muscle contraction is the relaxation of the vulval muscle to extrude eggs from the vulva. Unlike skeletal muscle, vulval muscle lacks visible striations of the sarcomere. Therefore, vulval muscle must counteract the mechanical stress, caused by egg extrusion and body movement, from inducing cell-shape distortion by maintaining its cytoskeletal integrity. However, the underlying mechanisms that regulate the cellular integrity in vulval muscles remain unclear. Here, we demonstrate that C. elegans egg-laying requires proper vulval muscle 1 (vm1), in which the actin bundle organization of vm1 muscles is regulated by Ras suppressor protein 1 (RSU-1). In the loss of RSU-1, as well as RasLET-60 overactivation, blister-like membrane protrusions and disorganized actin bundles were observed in the vm1 muscles. Moreover, RasLET-60 depletion diminished the defected actin-bundles in rsu-1 mutant. These results reveal the genetic interaction of RSU-1 and RasLET-60in vivo. In addition, our results further demonstrated that the fifth to seventh leucine-rich region of RSU-1 is required to promote actin-bundling protein, α-actinin, for actin bundle stabilization in the vm1 muscles. This expands our understanding of the molecular mechanisms of actin bundle organization in a specialized smooth muscle.Egg-laying behavior in  Caenorhabditis elegans fertilize their oocytes with their own sperms and stores 10 to 15 fertilized eggs in the uterus, after which the eggs are expelled into the environment through the vulva in every 20 min  and ventral cord type C neurons (VCs). Ablation of vm2s eliminates egg-laying , actin filaments, and myosin filaments , smooth e.g., PAT-2 and PAT-3 in C. elegans) consist of α and β subunits, each containing a large extracellular domain, a single transmembrane domain, and a short cytoplasmic tail. Once activated to the extended-open conformation, IntegrinPAT-3 recruits integrin-linked kinase (ILKPAT-4), particularly interesting Cys-His-rich protein (PINCHUNC-97), and parvinPAT-6 to form the IPP (Integrin-PINCH-Parvin) complex  and Unc (uncoordinated) phenotypes -containing domains. Human cell lines lacking RSU-1 are viable, but the proliferation and migration of the cells are markedly compromised  at the neuromuscular junctions (NMJs) of dorsal and ventral SAB motor neurons  acts as a downstream effector in RSU-1-mediated signaling to promote F-actin bundling. Based on our findings, we propose that RSU-1 inhibits RasLET-60 activity to regulate proper actin bundling in C. elegans vulval muscle.Here, we report that Escherichia coli OP50. All strains used in this study are listed in Table S1.All nematode strains were cultured at 22° on NGM (nematode growth medium) plates seeded with let-60(RNAi) clones from Julie Ahringer’s library (rsu-1(RNAi) and atn-1(RNAi) by inserting the corresponding DNA fragments into L4440 vectors. RNAi plasmids were transformed into bacteria HT115. Primers used for DNA amplification are listed in Table S2. RNAi feeding plates were prepared using NGM containing 100 μg/ml ampicillin and 1 mM isopropyl-β-thiogalactoside. HT115 carrying RNAi plasmids were grown in 5 ml of LB containing 100 μg/ml ampicillin at 37° overnight and then seeded on the RNAi plates at room temperature for 10 h. Embryos were hatched on the RNAi plates, and then at the L4 stage, were transferred to new RNAi plates for 12 h and used for confocal imaging.RNAi experiments were performed using the feeding method . We obta library , and we rsu-1(tm6690) strains—were treated with 100 μl of serotonin (3 mg/ml) for 60 min in each condition, and then the eggs expelled from each worm were counted. The developmental stages of the eggs were examined using confocal imaging. Whole-worm images were acquired using a Nikon A1R confocal microscope equipped with a 40×/1.4 numerical aperture (NA) objective. Images were processed using ImageJ.The average numbers of laid eggs were quantified as described  detector, or an Olympus IX83 spinning-disk confocal microscope equipped with a 60×/1.4 NA objective and a charge-coupled device (CCD) camera. Images were processed using ImageJ.Young adult To visualize actin filaments in vulval muscle, young adult worms were stained with iFlour 555-conjugated phalloidin . Worms were fixed with 3% formaldehyde for 3 h at room temperature, washed thrice with phosphate-buffered saline (PBS), and treated with 100% acetone at -20° for 5 min. Next, the worms were washed with PBS and stained with iFlour 555-phalloidin (0.1 μg/ml) overnight at 4° in the dark, and after rinsing with PBS, the stained worms were examined using a Nikon A1R confocal microscope equipped with a 100×/1.4 NA objective.Worms were washed with M9 buffer, fixed overnight with the primary fixative solution , and then incubated with the secondary solution (2% osmium tetroxide) for 4 h at room temperature. After washing with distilled water, the samples were dehydrated using 25%, 50%, 75%, 90%, and 100% acetone, infiltrated with spur resin at 25%, 50%, 75%, and 100%, and then embedded in a plastic capsule at 60° for 2 days. Samples were sectioned at 60-nm thickness, and the thin sections were treated with uranyl acetate and lead citrate before examination by using a Hitachi HT-7700 TEM instrument.RSU-1 and GFP::PINCHUNC-97 were generated by SunyBiotech (China) by using a modified CRISPR/Cas9 method , RSU-1(LRR 1-4), RSU-1(LRR 1-5), RSU-1(LRR 1-6), and RSU-1(LRR 5-7) were constructed by amplifying the corresponding DNA fragments from N2 genomic DNA and cloned into modified pCFJ90. Then, the constructs (80 ng/μl) were injected into young adults gonads together with the original marker pCFJ90 (1.25 ng/μl). The expression of extrachromosomal array were then screened under fluorescent microscope.The extrachromosomal array of truncated To examine actin filaments in vulval muscle cells , Z-stackRSU-1, GFP::PINCHUNC-97, and IntegrinPAT-3::GFP were obtained using a spinning-disk confocal microscope equipped with a 60×/1.4 NA objective (cytoplasmic - Intensitybackground)/. The intensities measured in different groups were compared using two-tailed t-tests in GraphPad Prism.For fluorescence-intensity analysis, images of mCherry::bjective . The intATN-1::GFP patches in vulval muscle  for further analysis in ImageJ. The areas of α-actininATN-1::GFP patches were measured using the “analyze particles tool” in ImageJ. Data from different groups were compared using two-tailed t-tests in GraphPad Prism.To analyze α-actininl muscle , Z-stackhttps://doi.org/10.25387/g3.12319226.All strains used in this work are available upon request or at the Caenorhabditis Genetics Center (CGC). All data to support the conclusions of this article are present in figures and tables. Supplemental material available at figshare: RSU-1 in vulval muscles by examining the egg-laying ability in rsu-1(RNAi) and rsu-1(tm6690) adult worms. The rsu-1(tm6690) mutant allele in the chromosome III. We believe that rsu-1(tm6690) completely eliminates gene function because it removes the 778 bp from the 5ʹ-end of the locus . First, we counted the number of unlaid eggs in control, rsu-1(RNAi), and rsu-1(tm6690) adult worms expressing mCherry::H2B (rsu-1(RNAi) (23% ± 1%) or rsu-1(tm6690) (45% ± 2%) than control (5% ± 1%) worms  worms .rsu-1(RNAi) and rsu-1 mutant worms. Therefore, we stimulated adult worms to lay eggs by applying the monoamine neurotransmitter serotonin. If RSU-1 acts in the egg-laying circuits, similar amount of laid-eggs will be obtained in the control, rsu-1(RNAi), and rsu-1(tm6690) adult worms after treating with serotonin. Otherwise, less embryos will be expelled in rsu-1(RNAi) or rsu-1(tm6690). In control adult worms treated with serotonin, 15 ± 2 eggs were expelled in 1 h. However, this rate decreased to 11 ± 1 egg in rsu-1(RNAi) and it was almost halved in rsu-1(tm6690), only obtained 8 ± 2 eggs per hour (rsu-1(RNAi) and rsu-1(tm6690) adult worms was less than control worms. Collectively, these data suggest that the egg-laying is impaired in rsu-1 null mutant worms.Next, we wonder whether the egg-laying circuits is compromised in per hour . These rRSU-1 contributes to the egg-laying behavior, we further suspected that RSU-1 acts in the vulval muscles, and thereby regulates the vulval opening. First, the subcellular localization of RSU-1 in vulval muscles was examined. We generated a transgenic fluorescent C. elegans strain by using the CRISPR/Cas9 genome-editing technique to insert the mCherry-coding DNA sequence into the 5ʹ-end of endogenous rsu-1 on Chromosome III. Whole-worm imaging revealed that mCherry::RSU-1 was primarily expressed in body-wall muscles and vulval muscles /  . The C/FCHUNC-97 . AltogetRSU-1 expresses in vm1 muscles, we further investigate the function of RSU-1 by examining the morphology of vm1 muscles in control, rsu-1(RNAi), and rsu-1(tm6690) worms. When cytosolic GFP expression is driven by the vm1 muscle-specific egl-15 promoter (Pegl-15::GFP), the morphology of vm1 muscles can be visualized by using confocal imaging. In controls, the 4 vm1 muscles were rod-like in shape and displayed straight and smooth cell boundaries, and cells were oriented toward each other around the vulva and formed a cross shape (rsu-1(RNAi) and rsu-1(tm6690) vm1 muscles were irregular in shape and harbored blister-like protrusions (RSU-1 is required for cell morphology control.Given that ss shape . By conttrusions . These rrsu-1(tm6690) might be caused by aberrant levels of RasLET-60 activity. To address this possibility, we examined the shape of vm1 muscles after downregulating and upregulating RasLET-60 activity. We downregulated RasLET-60 activity by using let-60(RNAi), whereas for upregulation of RasLET-60 activity, we used the strain let-60(n1046) and let-60(n1700), in which these alleles in the chromosome IV encode a RasLET-60 dominant mutant that is constitutively active (let-60(n1046) and let-60(n1700) worms expressing Pegl-15::GFP, ∼48% of vm1 muscles showed blister-like protrusions (rsu-1(tm6690). According to this observation, we then speculate that RasLET-60 in the rsu-1(tm6690) worms might be overactivated. Therefore, knockdown RasLET-60 in rsu-1(tm6690) should suppress the membrane blebbing. To test this, we treated rsu-1(tm6690) or rsu-1(RNAi) worms with let-60(RNAi). The blister-like protrusions were absent in et-60l(RNAi);rsu-1(tm6690) adult worms or decreased to 8% in let-60(RNAi);rsu-1(RNAi) adult worms (let-60(RNAi) was confirmed by evaluating the number of vulva in let-60(n1046);let-60(RNAi), in which the number of vulva in let-60(n1046) were significantly reduced . Collectively, our data indicate that RSU-1 regulates RasLET-60 to maintain the cell shape of vm1 muscles.Cell membrane blebbing was previously reported in MCF10A cells with overactivated R-Ras  vm1 muscles, let-60(n1700) vm1 muscles, and let-60(n1046) vm1 muscles (let-60(RNAi);rsu-1(tm6690) vm1 muscles. We also examined the actin bundles in fixed rsu-1(tm6690) by using transmission electron microscopy (TEM): whereas straight and parallel actin filaments were detected in control vm1 muscles, curved actin filaments with irregular empty spaces between actin filaments were observed in rsu-1(tm6690) vm1 muscles (rsu-1(RNAi) vm1 muscles expressing LifeAct::mKate and Pegl-15::GFP, which were used for (respectively) labeling actin filaments and visualizing the morphology of vm1 muscles , we observed that loss of α-actininATN-1 leads to membrane blebbing (rsu-1(tm6690);atn-1(RNAi) and let-60(n1046);atn-1(RNAi) vm1 muscles  and atn-1(ok84) cells  or let-60(n1046) vm1 muscle cells than control cells, while the patches in let-60(n1046);let-60(RNAi) were similar to control cells  cells , which r muscles . We also muscles . Notablyol cells . These rRSU-1 has seven LRR-containing domains, and they may form an arc or horseshoe shape, with the variable convex face and the parallel β-strands-containing concave face  worms, and then examined the morphology of vm1 muscle cells , whereas LRR1-4::mCherry, LRR1-5::mCherry, and LRR1-6::mCherry did not  . Altogether, these findings suggest that RSU-1 may not function in the focal adhesion or it acts in parallel with an unknown protein in the downstream signaling of the focal adhesion pathway.Body-wall muscles are striated muscles that consist of multiple sarcomeres (rsu-1(RNAi) and rsu-1(tm6690) mutant worms (discussed below) but they were eliminated in the loss of RasLET-60. Furthermore, we observed that RSU-1 was diffusely distributed in the cytoplasm of vm1 muscles. Therefore, based on the results of our genetic studies and subcellular-localization analysis, we propose that the cytosolic RSU-1 performs a cellular function that is independent of the integrin-mediated adhesion pathway in vm1 muscles. In human cells, RSU-1 was reported to regulate p38 activity in a PINCHUNC-97-independent manner  are critical for the inhibition. Therefore, the binding of RSU-1 and RasLET-60 need to investigate further. Nevertheless, the interaction of a LRR protein, FLI-1, with Ras was reported in C. elegans during embryonic cytokinesis and germline development (SOC-2 also directly interact with Ras and Raf to positively modulate Ras pathway in C. elegans (ATN-1 is activated by PIP2 binding and forms an antiparallel dimer (LET-60 activates PI3K to phosphorylate PIP2 into PIP3 (RSU-1 might inhibit RasLET-60-dependent activation of PIP2 phosphorylation, and this, in turn, might promote the activation of α-actininATN-1 for actin-bundle stabilization. This poorly understood mechanism underlying actin organization warrants further investigation.In summary, our results demonstrated that asLET-60 . Althoug


Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential cross-neutralizing epitopes on SARS-like viruses. The ongoing COVID-19 pandemic is caused by SARS-CoV-2. Due to the genetic similarity of SARS-CoV-2 and SARS-CoV, which caused an epidemic in 2003, a few of the SARS-CoV antibodies have now been found to also cross-react with SARS-CoV-2. One such antibody is CR3022, which was isolated from a convalescent SARS patient 14 years ago. However, the 100-fold lower binding to SARS-CoV-2 does not enable neutralization of SARS-CoV-2 compared to SARS-CoV. This study shows that one (P384A) of the four mutational differences in the CR3022 epitope between SARS-CoV and SARS-COV-2 fully accounts for the differences in CR3022 binding affinity and neutralization. These findings advance our understanding of antibody cross-reactivity among SARS-like CoVs with implications for vaccine and therapeutic design. The ongoing COVID-19 pandemic, which is caused by the new coronavirus SARS-CoV-2, continues to escalate. Investigating the immunogenicity and antigenicity of SARS-CoV-2 is germane to vaccine and therapeutic development. The major antigen of coronavirus is the spike (S) glycoprotein, which is expressed as a homotrimer on the virus surface. Since the S protein is essential for virus entry through engaging the host receptor and mediating virus-host membrane fusion, many antibodies to the S protein are neutralizing . The S pCR3022 was previously isolated from a SARS survivor and neutralizes SARS-CoV , CR3022 We therefore aimed to dissect the molecular basis underlying the difference in binding affinity and neutralization potency of CR3022 to SARS-CoV-2 and SARS-CoV. The crystal structure of SARS-CoV RBD in complex with CR3022 was determined to compare with the corresponding SARS-CoV-2 RBD structure . In combD = 66 nM), T430M (KD = 64 nM), and H519N (KD = 60 nM) was comparable to wild type (WT) SARS-CoV-2 RBD (KD = 68 nM), binding of CR3022 to the P384A mutant (KD = 1.4 nM) was greatly increased (D = 1.0 nM) [The epitopes of CR3022 in SARS-CoV-2 and SARS-CoV differ by four residues. We aimed to determine whether amino-acid variants in these four non-conserved residues influence the binding affinity of CR3022 to RBD. Four SARS-CoV-2 RBD mutants, namely A372T, P384A, T430M, and H519N (SARS-CoV-2 numbering), were recombinantly expressed and examined . These mncreased , akin no 1.0 nM) . Thus, t50 of 3.2 μg/ml, which is comparable to its neutralizing activity to SARS-CoV (IC50 of 5.2 μg/ml). This finding validates the CR3022 epitope as a neutralizing epitope in both SARS-CoV-2 and SARS-CoV, provided that the antibody affinity can surpass a threshold for detection of neutralization.While CR3022 can neutralize SARS-CoV ,20, mult50 of 4.4 μg/ml, which is similar to that of CR3022 IgG (3.2 μg/ml)  . This fiWe then examined the sequence conservation of residue 384 in other SARS-related coronaviruses (SARSr-CoV) strains. Most SARSr-CoV strains have Pro at residue 384, as in SARS-CoV-2. Only those strains that are phylogenetically very close to SARS-CoV, such as bat SARSr-CoV WIV1 and bat SARSr-CoV WIV16, have Ala at residue 384 Figs  and S1. We further determined the x-ray structure of SARS-CoV RBD in complex with CR3022 to 2.7 Å resolution Figs . The oveH S96, which is encoded by IGHD3-10 gene segment on CDR H3 [H S96 forms a hydrogen bond (H-bond) with the T385 side chain in both SARS-CoV-2 RBD and SARS-CoV RBD, it can form a second H-bond with the backbone amide of T385 in SARS-CoV RBD . At residue 384, the backbone of SARS-CoV-2 is further from CR3022, as compared to that of SARS-CoV . This din CDR H3 ,20. Whil-CoV RBD , but not-CoV RBD . In addi-CoV RBD , but not-CoV RBD . In summTo understand the binding of CR3022 to the RBD in the context of the homotrimeric S protein, we previously proposed a structural model where CR3022 could only access its epitope on the S protein when at least two RBD are in the “up” conformation and the RBD is rotated relative to its unliganded structure . To furtWhile our results here demonstrate that CR3022 Fab could form a stable complex with SARS-CoV S protein in a prefusion conformation, a recent study reported that prefusion SARS-CoV-2 S protein fell apart upon binding to CR3022 Fab as indicated by cryo-EM . It shouTo address some of these issues, we performed cryo-EM analysis to interrogate the binding of CR3022 to SARS-CoV S protein at higher resolution . FocusedDespite the flexibility of CR3022-bound RBD, bivalent binding of CR3022 to S protein does not seem to occur on the virus surface since an IgG avidity effect was not observed in the neutralization assay  [in vitro [Given the scale of the outbreak, SARS-CoV-2 may persist and circulate in humans for years to come . A numbeo_table) , which o6 tag, and cloned into a customized pFastBac vector [RBD (residues 319–541) of the SARS-CoV-2 spike protein (GenBank: QHD43416.1) and RBD (residues: 306–527) of the SARS-CoV spike (S) protein (GenBank: ABF65836.1) were fused with an N-terminal gp67 signal peptide and a C-terminal Hisc vector . RecombiThe SARS-CoV spike construct (Tor2 strain) for recombinant spike protein expression contains the mammalian-codon-optimized gene encoding residues 1–1190 of the spike followed by a C-terminal T4 fibritin trimerization domain, a HRV3C cleavage site, 8x-His tag and a Twin-strep tags subcloned into the eukaryotic-expression vector pαH. Residues at 968 and 969 were replaced by prolines for generating stable spike proteins as described previously . The spiThe CR3022 Fab heavy (GenBank: DQ168569.1) and light (GenBank: DQ168570.1) chains were cloned into phCMV3. The plasmids were transiently co-transfected into Expi293F cells at a ratio of 2:1 (HC:LC) using ExpiFectamine 293 Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The supernatant was collected at 7 days post-transfection. The Fab was purified with a CaptureSelect CH1-XL Pre-packed Column (Thermo Fisher Scientific) followed by size exclusion chromatography. For crystallization, a VSRRLP variant of the elbow region was used to reduce the conformational flexibility between the Fab constant and variable domains .Purified CR3022 Fab with a VSRRLP modification in the elbow region and SARS-CoV RBD were mixed at a molar ratio of 1:1 and incubated overnight at 4°C. The complex (7.5 mg/ml) was screened for crystallization using the 384 conditions of the JCSG Core Suite (Qiagen) on our custom-designed robotic CrystalMation system (Rigaku) at Scripps Research by the vapor diffusion method in sitting drops containing 0.1 μl of protein and 0.1 μl of reservoir solution. Optimized crystals were then grown in 2 M sodium chloride and 10% PEG 6000 at 4°C. Crystals were grown for 7 days and then flash cooled in liquid nitrogen. Diffraction data were collected at cryogenic temperature (100 K) at Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12–2 with a wavelength of 1.033 Å, and processed with HKL2000 . StructuIndividual mutants for validation experiments were constructed using the QuikChange XL Mutagenesis kit (Stratagene) according to the manufacturer’s instructions.Six molar excess of CR3022 Fab (unmodified) was added to SARS-CoV spike protein 1 hour prior to direct deposition onto carbon-coated 400-mesh copper grids. The grids were stained with 2% (w/v) uranyl-formate for 90 seconds immediately following sample application. Grids were imaged on Tecnai T12 Spirit at 120 keV with a 4k x 4k Eagle CCD. Micrographs were collected using Leginon  and imagSARS-CoV spike protein was incubated with six molar excess of CR3022 Fab for 2 h. 3.5 μL of the complex (0.9 mg/ml) was mixed with 0.5 μL of 0.04 mM lauryl maltose neopentyl glycol (LMNG) solution immediately before sample deposition onto a 1.2/1.3 300-Gold grid (EMS). The grids were plasma cleaned for 7 seconds using a Gatan Solarus 950 Plasma system prior to sample deposition. Following sample application, grids were blotted for 3 seconds before being vitrified in liquid ethane using a Vitrobot Mark IV (Thermo Fisher).-/Å2. Micrographs were collected through Leginon software at a nominal defocus range of -0.4 μm to -1.6 μm and MotionCor2 was used for alignment and dose weighting of the frames [Data collection was performed using a Talos Arctica TEM at 200 kV with a Gatan K2 Summit detector at a magnification of 36,000x, resulting in a 1.15 Å pixel size. Total exposure was split into 250 ms frames with a total cumulative dose of ∼50 ee frames ,56. Micre frames . CTF este frames . Particle frames . Final se frames .θ is the subunit rotation angle, X11, Y22, and Z33 represent the X11, Y22, and Z33 values in the rotation matrix calculated for the superpose.Comparisons of subunit rotation angles among different structures were performed with a software ‘Superpose’ in the CCP4 package ,60. For 6-tagged RBD proteins at 20 to 100 μg/ml in 1x kinetics buffer  were loaded onto Anti-Penta-HIS (HIS1K) biosensors and incubated with the indicated concentrations of CR3022 Fab. The assay consisted of five steps: 1) baseline: 60 s with 1x kinetics buffer; 2) loading: 300 s with His6-tagged S or RBD proteins; 3) baseline: 60 s with 1x kinetics buffer; 4) association: 120 s with samples (Fab or IgG); and 5) dissociation: 120 s with 1x kinetics buffer. For estimating the exact KD, a 1:1 binding model was used.Binding assays were performed by biolayer interferometry (BLI) using an Octet Red instrument (FortéBio) as described previously . Briefly4, 4 mM EGTA, and 1% Triton X-100). Luciferase intensity was then measured using Bright-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions. Percentage of neutralization was calculated using the following equation:Pseudovirus preparation and assay were performed as previously described . BrieflyRBD protein sequences from SARS-CoV-2, SARS-CoV, and SARS-related coronavirus (SARSr-CoV) strains were retrieved from the following accession codes:GenBank QHD43416.1 (SARS-CoV-2)GenBank ABF65836.1 (SARS-CoV)GenBank AID16716.1 (Bat SARSr-CoV Longquan-140)GenBank AAY88866.1 (Bat SARSr-CoV HKU3-1)GenBank AIA62320.1 (Bat SARSr-CoV GX2013)GenBank AIA62310.1 (Bat SARSr-CoV HuB2013)GenBank ACU31032.1 (Bat SARSr-CoV Rs672)GenBank AAZ67052.1 (Bat SARSr-CoV Rp3)GenBank AIA62300.1 (Bat SARSr-CoV SX2013)GenBank ABD75323.1 (Bat SARSr-CoV Rf1)GenBank AVP78031.1 (Bat SARSr-CoV ZC45)GenBank AVP78042.1 (Bat SARSr-CoV ZXC21)GISAID EPI_ISL_410721 (Pangolin BetaCoV Guandong2019)GenBank QHR63300.2 (Bat CoV RaTG13)NCBI Reference Sequence YP_003858584.1 (Bat SARSr-CoV BM48-31)GenBank ALK02457.1 (Bat SARSr-CoV WIV16)GenBank AGZ48828.1 (Bat SARSr-CoV WIV1)http://tree.bio.ed.ac.uk/software/figtree/).Multiple sequence alignment of the RBD sequences was performed by MUSCLE version 3.8.31 . PhylogeS1 FigBranches corresponding to strains that have A384 are colored in red on the phylogenetic tree. Scale bar represents 0.05 amino-acid substitutions per position.(PDF)Click here for additional data file.S2 Fig(A) Final 2Fo-Fc electron density maps for the side chains in the epitope region of SARS-CoV-2 contoured at 1 σ. (B) Final 2Fo-Fc electron density maps for the paratope region of CR3022 contoured at 1 σ. The heavy chain is colored in orange, and light chain in yellow. Epitope and paratope residues are labeled.(PDF)Click here for additional data file.S3 FigStructure of CR3022 in complex with SARS-CoV RBD (this study) is aligned to that with SARS-CoV-2 RBD (PDB 6W41). Structural alignment was performed using CR3022 heavy chain variable domain. Red: CR3022 in complex with SARS-CoV RBD. Blue: CR3022 in complex with SARS-CoV-2 RBD.(PDF)Click here for additional data file.S4 FigThe top panel shows a representative cryo-electron micrograph of the SARS-CoV spike complexed with CR3022 Fab, whereas the bottom panels show the 2D class averages.(PDF)Click here for additional data file.S5 FigFour 3D class averages of complex of the SARS-CoV spike and CR3022 were found during data processing.(PDF)Click here for additional data file.S6 FigThe conformation of CR3022-bound RBD in class 2 and 4 is compared to the conformation of RBD on an unliganded SARS-CoV S protein (PDB 6ACD) .(PDF)Click here for additional data file.S7 FigThe conformation of CR3022-bound RBD in class 2 and 4 is compared to that of dispositions 2 and 3 of ACE2-bound RBD  .(PDF)Click here for additional data file.S8 FigThe angles of approach of CR3022 (blue) and COVA1-16   to RBD a(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Plasmodium falciparum, is poorly characterized, despite being the second most abundant isotype in blood after IgG. Here, we investigated the circulating IgA response in humans to Plasmodium falciparum sporozoites that are injected into the skin by mosquitoes and migrate to the liver via the bloodstream to initiate malaria infection. We found that circulating IgA was induced in three independent sporozoite-exposed cohorts: individuals living in an endemic region in Mali, malaria-naïve individuals immunized intravenously with three large doses of irradiated sporozoites, and malaria-naïve individuals exposed to a single controlled mosquito bite infection. Mechanistically, we found evidence in an animal model that IgA responses were induced by sporozoites at dermal inoculation sites. From malaria-resistant individuals, we isolated several IgA monoclonal antibodies that reduced liver parasite burden in mice. One antibody, MAD2-6, bound to a conserved epitope in the N-terminus of the P. falciparum circumsporozoite protein, the dominant protein on the sporozoite surface. Crystal structures of this antibody revealed a unique mode of binding whereby two Fabs simultaneously bound either side of the target peptide. This study reveals a role for circulating IgA in malaria and identifies the N-terminus of the circumsporozoite protein as a target of functional antibodies.Immunoglobulin (Ig)A antibodies play a critical role in protection against mucosal pathogens. However, the role of serum IgA in immunity to non-mucosal pathogens, such as  Plasmodium falciparum parasite, the deadliest of the five Plasmodium species that infect humans G or IgM isotypes -specific IgA monoclonal antibodies (mAbs) from two Malian individuals and investigated the fine specificity and function of these antibodies. We found that exposure to sporozoites through different routes triggered a specific IgA response. We identified an IgA mAb, MAD2-6, that bound to a conserved epitope on the N-terminus of PfCSP, inhibited sporozoite invasion in vitro and reduced liver parasite burden in vivo. These findings reveal a role for IgA in the immune response to P. falciparum and identify the N-terminus of PfCSP as a target of functional antibodies.Antibodies can play a critical role in mediating protection against all stages of malaria . The vasisotypes –11 basedathogens . Howeverathogens . To inveP. falciparum infection in a cohort study conducted in a region of Mali where the annual malaria season is clearly defined  as a blood-stage antigen comparator. Of the 1.5 x 106 IgA+ (598 wells) and 7.0 x 106 IgG+ (2720 wells) memory B cells screened, we identified 37 positive PfCSP-specific IgA wells (6.2% of wells) and 96 positive PfCSP-specific IgG wells (3.5% of wells)  . In contPlasmodium antigens whose antibodies cross-react with sporozoite surface antigens  prior to controlled human malaria infection (CHMI) with P. falciparum 3D7-infected mosquitoes. The PfSPZ Vaccine induced an IgA response against sporozoites and PfCSP . Six days later, NANP-specific IgA+ plasmablasts were detected in the draining ipsilateral auricular lymph node (LN) and spleen, but not in the contralateral auricular LN, portal/celiac LNs, mesenteric LNs or Peyer’s patches  every time that they were tested as a possible indication of resistance to sporozoite infection. Of the 758 individuals followed by active surveillance for infection, only four met this criterion , we extended the in vivo analysis to compare MAD2-6 with other N-terminal antibodies in a definitive experiment with 9 to 10 mice per group. It has proven difficult to isolate mAbs against the N-terminus, with only a small number of mouse antibodies isolated to date. To our knowledge, mouse antibody 5D5 is the only other mAb targeting the N-terminus that has been tested in vivo, with variable efficacy reported in different studies  not showing stronger antibody binding than malaria-naïve U.S. individuals . In age-HGly96, HVal98, and HThr100 in CDR H3, as well as to the side chain of HThr100 in Fab A  at 2.5 and 2.1 Å resolution, respectively . For simin Fab A  and D. Uainchain . Additioainchain  to E. Adainchain –29. Thesainchain , fig. S9ainchain , 29.H to the CH1 domain, differs from that of the IgG Fab and allows for formation of a hydrophobic core consisting of HLeu11, HPro116, and HTyr203  to the middle of CH1 (HCys127) could help stabilize the constant region  analysis of dried blood spots (DBS) on filter paper . Venous blood samples were collected before and after each malaria season, at the time of diagnosis of the first clinical malaria episode of each malaria season, and 7-10 days later. Venous blood was drawn into sodium citrate-containing cell preparation tubes (BD Biosciences) and peripheral blood mononuclear cells (PBMCs) and plasma were isolated within 2 hours according to the manufacturer’s instructions. Plasma and PBMCs were stored at −80°C and in vapor-phase liquid nitrogen, respectively. For this study, we focused on 758 subjects who had plasma and PBMCs available for analysis. Monoclonal antibodies were isolated from two individuals in the cohort based on clinical and serological data as described in the text, and all IgA monoclonal antibodies that bound to both P. falciparum sporozoites and PfCSP from these two individuals were characterized in this study. For mAb analysis, no randomization or blinding was performed.The cohort study was conducted in the rural village of Kalifabougou, Mali where ng study , and 50.https://clinicaltrials.gov/; trial number NCT01322581). Written informed consent was obtained from participants or parents or guardians of participating children before inclusion in the study.The Kalifabougou cohort study was approved by The Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry at the University of Sciences, Technique and Technology of Bamako, and the Institutional Review Board of the National Institute of Allergy and Infectious Diseases, National Institutes of Health  or 1 μg/mL goat anti-human IgA-Alexa Fluor 647 (Jackson Immunoresearch 109-606-011) and read with the iQue Screener Plus high-throughput flow cytometer. FACS data were analysed on the Forecyt program (Intellicyt).Cryopreserved PBMCs from the Malian cohort were thawed and stained for 20 min at 4˚C in phosphate buffered saline (PBS) + 1% fetal bovine serum (FBS) with the following panel: 1:400 LIVE/DEAD Fixable Aqua, 1:50 CD14-Brilliant Violet (BV)510 (BioLegend 301842), 1:50 CD3-BV510 (BioLegend 317332), 1:50 CD56-BV510 (BioLegend 318340), 1:25 CD19-Electron Coupled Dye (ECD) (Beckman Coulter IM2708U), 3 μg/mL IgA-Alexa Fluor 647 (Jackson Immunoresearch 109-606-011), 1:125 IgD-phycoerythrin-Cyanine 7 (PECy7) (BD Biosciences 561314), and 1:20 IgM-peridinin-chlorophyll-protein (PerCP)-Cy5.5 (BD Biosciences 561285). Sorted IgAH) and light chain variable (VL) genes and percentage of somatic mutations was done using the International Immunogenetics Information System (IMGT) database (H with IgA1 or IgA2 constant region and VL genes were synthesized (Genscript) and used to transfect Expi293 (Thermo Fisher Scientific) cells without a J chain to express only monomeric IgA. Recombinant IgA was purified by fast performance liquid chromatography using a Tricon column  containing the CaptureSelect IgA Affinity Matrix (Thermo Fisher Scientific). The column was attached to an AKTA chromatographic system . After loading of the culture media, the column was washed with endotoxin-free PBS. The bound IgA was then eluted with 0.1 M Glycine-HCl, pH 2.7 and the eluates were immediately neutralized with 1 M Tris-HCl, pH 8. The IgA preparations were then dialyzed extensively against PBS for complete buffer exchange. The antibodies were also expressed with an IgG1 backbone and purified using HiTrap Protein A columns . For select antibodies for which Fabs were expressed, heavy chain plasmids encoding only the VH and CH1 were synthesized and used to transfect Expi293 cells along with light chain plasmids. Fab purification was done with CaptureSelect KappaXP Affinity Matrix (Thermo Fisher Scientific).cDNA was synthesized from B cell clones of interest and antibody heavy and light chains were PCR-amplified using gene-specific primers  and sequdatabase . PlasmidPlasmodium species 18S ribosomal RNA gene. Plasmodium positive samples were identified only as P. falciparum, P. malariae, or both (mixed infections) given the negligible incidence of other Plasmodium species in the study area. Target bands were detected with a DNA 5 K LabChip on the LabChip GX HT  per the manufacturer’s protocol.As previously described , we adaphttps://clinicaltrials.gov/; trial number NCT02015091) after obtaining written informed consent. VRC 314 was a phase 1, open-label, dose-escalation trial that was designed to assess the safety, immunogenicity, and protective efficacy of the Sanaria PfSPZ Vaccine  , and the other group underwent CHMI without prior immunization.Plasma and peripheral blood mononuclear cell (PBMC) samples were obtained from healthy malaria-naive adults (18-45 years of age) enrolled in the VRC 314 clinical trial (ozoites) . SamplesP. falciparum NF54 sporozoites were dissected from the salivary glands of Anopheles stephensi and harvested in PBS containing 10 μM E-64 protease inhibitor (Sigma-Aldrich E3132). 5000 sporozoites were stained with 1:1500 SYBR Green and 1:30 Malian or U.S. plasma for 30 minutes at 4˚C, washed, and then incubated with 1:105 mouse anti-human IgM-BV450 (BD Biosciences 561286), 2.5 μg/mL goat anti-human IgA-Alexa Fluor 594 (Jackson Immunoresearch 109-586-011), and 2.5 μg/mL goat anti-human IgG-Alexa Fluor 647 (Jackson Immunoresearch 109-606-170) for 30 minutes at 4˚C. A mixture of 4 strains of P. falciparum infected erythrocytes was used for the screen with Malian plasma; the laboratory line 3D7, a Cambodian isolate CP806, and two Malian isolates . The blood-stage parasites were cryopreserved in glycerolyte at the late trophozoite stage and thawed with a series of salts according to standard procedure , 2.5 μg/mL goat anti-human IgA-Alexa Fluor 647 (Jackson Immunoresearch 109-606-011), or 2.5 μg/mL goat anti-human IgM-Alexa Fluor 647 (Jackson Immunoresearch 109-606-129). The samples were analyzed with the iQue Screener (Intellicyt).Streptavidin beads with different intensities of PE-channel fluorescence (Spherotech SVFA-2558-6K and SVFB-2558-6K) were incubated with the following biotinylated antigens: 10 μg/mL recombinant PfCSP (aa 21-375 from PlasmoDB ID: PF3D7_0304600.1), 5 μg/mL N-terminus (aa 21-104 from PlasmoDB ID: PF3D7_0304600.1), 5 μg/mL C-terminus (aa 273-375 from PlasmoDB ID: PF3D7_0304600.1), 2 μg/mL NANP (NANPNANPNANPNANPNANPNANPNANPNANPNANP), or 10 μg/mL CD4  or TV1-g+CD14-CD3-CD8-CD56-CD21+CD27+IgA+/IgG+, with CD27++CD38++ cells excluded.Cryopreserved PBMCs from the VRC 314 trial were thawed and stained for 30 minutes at room temperature in the dark with the following panel: 1:500 Blue LIVE/DEAD (Thermo Fisher Scientific L23105), 18 μg/mL IgA-Dylight 405 (Jackson Immunoresearch 109-475-011), 1:50 CD14-BV510 (BioLegend 301842), 1:50 CD3-BV510 (BioLegend 317332), 1:100 CD8-BV510 (301048), 1:50 CD56-BV510 (BioLegend 318340), 1:50 CD27-BV605 (BioLegend 302830), 1:40 CD21-BV711 (BD Biosciences 563163), 1:50 CD19-BV750 (BioLegend 302236), 1:125 IgD-PECy7 (BD Biosciences 561314), 1:20 IgM-Brilliant Ultraviolet (BUV)395 (BD Biosciences 563903), 1:125 CD38-Alexa Fluor 700 (BD Biosciences 560676), 1:40 IgG-allophycocyanin (APC)H7 (BD Biosciences 561297), 1:25 PfCSP-Brilliant Blue (BB)660, and 1:25 PfCSP-BUV737. The PfCSP probes were made by conjugating biotinylated PfCSP to streptavidin tagged with the relevant fluorescent dye. The cells were sorted using a BD FACS Aria II and analyzed with FlowJo software (Tree Star). Cells were gated on live CD19P. berghei sporozoites expressing PfCSP (Pb-PfCSP) within 2 hours. Two days post-challenge, the mice were administered intraperitoneally 150 μl of 30 mg/mL Rediject D-luciferin (Perkin Elmer) and imaged by the IVIS Spectrum in vivo imaging system (Perkin Elmer) to quantify the liver-stage parasite burden. Total flux (photons/second) was measured after choosing an equivalent region of interest around each mouse liver. Liver-stage parasite burden was compared to negative control mice that did not receive a monoclonal antibody and naïve mice that did not receive sporozoites. All mouse research was conducted according to National Institutes of Health (NIH) guidelines and approved by the Vaccine Research Center animal care and use ethics committee .As previously described , female P. falciparum NF54 sporozoites dissected from salivary glands were stained with 1:2000 SYBR Green and four dilutions of VRC 314 U.S. plasma  for 30 minutes at 4˚C, washed, and then incubated with 2.5 μg/mL goat anti-human IgA-Alexa Fluor 647 (Jackson Immunoresearch 109-606-011) or goat anti-human IgG-Alexa Fluor 647 (Jackson Immunoresearch 109-606-170) for 30 minutes at 4˚C. The binding data with the sporozoites were acquired with the iQue Screener Plus high-throughput flow cytometer and analyzed with FlowJo software (Tree Star).4 chimeric Pb-PfCSP sporozoites. Control mice were injected with a pool of matched salivary grand extract from uninfected mosquitoes. Six days later, cells were isolated from left  and right  auricular lymph nodes, portal and celiac lymph nodes, Peyer’s patches, mesenteric lymph nodes, and spleen for flow cytometry. Live cells were pre-incubated for 10 minutes at 4˚C with 1:100 anti-CD16/CD32 mAb to block Fcγ receptors II/III and 1:1000 LIVE/DEAD Fixable aqua (Thermo Fisher Scientific L34966), and then stained for 15 minutes at 4˚C with 1:250 anti-mouse CD19-APCeF780 (Invitrogen 47-0193-82), 1:250 GL7-Alexa Fluor 647 (BD Biosciences 561529), 1:250 CD95-BV421 (BD Biosciences 562633), 1:100 CD138-BV786 (BD Biosciences 740880), 1:100 IgA-PE (Invitrogen 12-4204-82), 1:100 IgG1-BUV805 (BD Biosciences 741942), 1:100 IgG2a-BUV805 (BD Biosciences 749396), 1:100 IgG2b-BUV805 (BD Biosciences 749136), 1:100 IgG3-BUV805 (BD Biosciences 749007), 1:100 NANP tetramer-FITC, and 1:100 NANP tetramer -BV605 in BD brilliant stain buffer (BD Biosciences 563794), and fixed with BD Cytofix/Cytoperm (BD Biosciences 51-2090KZ) per manufacturer’s instructions. The NANP probes were made by conjugating biotinylated NANP to streptavidin tagged with the relevant fluorescent dye. After fixation, intracellular IgA and IgG were further stained with IgA-PE, IgG1-BUV805, IgG2a-BUV805, IgG2b-BUV805, IgG3-BUV805, NANP tetramer-FITC, and NANP tetramer-BV605 in BD brilliant stain buffer as above. Data were acquired using an BD LSRFortessa X-20 flow cytometer with FACSDiva software (BD Biosciences) and analyzed using FlowJo software (Tree Star). All mouse research was conducted according to National Institutes of Health (NIH) guidelines and approved by the Vaccine Research Center (VRC) animal care and use ethics committee .Four-to six-week-old female BALB/c mice were purchased from Jackson Laboratories and challenged by a single intradermal injection on the left ear with 3-3.6 × 10P. falciparum NF54 sporozoites dissected from salivary glands or midguts were stained with 1:2000 SYBR Green and dilutions of IgA or IgG monoclonal antibodies for 30 minutes at 4˚C, washed, and then incubated with 2.5 μg/mL goat anti-human kappa-Alexa Fluor 647 (Southern Biotech 2062-31) or 2.5 μg/mL goat anti-human lambda-Alexa Fluor 647 (Southern Biotech 2072-31) for 30 minutes at 4˚C. To compare the binding of MAD2-6 IgA to live and dead sporozoites, the sporozoites were stained with a range of dilutions of the antibody for 30 minutes at 4˚C, followed by staining with 2.5 μg/mL IgA-Alexa Fluor 647 (Jackson Immunoresearch 109-606-011) for 30 minutes at 4˚C. Sporozoites were labeled with 1 μg/mL mAb10-Dylight 405 and stained with 5 μg/mL of propidium iodide prior to loading onto the flow cytometer. Data were acquired with the iQue Screener Plus high-throughput flow cytometer and analyzed with FlowJo software (Tree Star).96-well half-area plates were coated with 100 μg/mL heparin or MAD2-6 IgA, incubated at 37°C for 1 hour, and blocked with 1% BSA in PBS. Serially diluted P14-P19 PfCSP peptides and recombinant PfCSP  at a range of concentrations and 0.5 μg/mL of biotinylated P17 was added to the plates. Next, streptavidin-alkaline phosphatase (Southern Biotech 7100-04) was added to the plate, and the plates were developed and read after 30 minutes as described above.Mapping of MAD2-6, 5D5, and BL18 mAbs was performed using PfCSP overlapping peptides (peptides 13-22) that were 15 amino acids in length (GenScript) and overlapped by 11 residues spanning the N-terminal region of PfCSP. The assay was performed using the MSD U-Plex Assay platform  according to the manufacturer’s instructions.Kinetics of antibody binding to PfCSP peptides were measured using streptavidin-capture biosensors in the Octet Red384 system (fortéBio). Assays were performed with agitation at 30°C. Monoclonal antibodies were plated in solid black tilt-well 384-well plates (Geiger Bio-One). Loading of the biotinylated P17 peptide was done for 300 seconds, followed by immersing the biosensors into buffer (PBS + 1% BSA) for 60 seconds to assess baseline assay drift. Association with whole IgG  was performed for 300 seconds, followed by dissociation in buffer for 600 seconds. Data analysis and curve fitting were performed using Octet Data Analysis software. Experimental data were fitted with the binding equations describing a 1:1 heterologous ligand interaction. Global analyses of the complete data sets, assuming binding was reversible (full dissociation), were carried out using nonlinear least-squares fitting allowing a single set of binding parameters to be obtained simultaneously for all concentrations of a given monoclonal antibody dilution series.MAD2-6 IgG Fab was concentrated to 10 mg/mL and mixed with the EKLRKPKHKKLKQ peptide, which overlaps with P17 (KLRKPKHKKLKQPAD) recognized by MAD2-6 mAb in the initial screening, in a 5:1 molar ratio of peptide to Fab. Upon identifying the minimal epitope of MAD2-6 IgG Fab, MAD2-6 IgA Fab was crystallized with a shorter peptide, KPKHKKLKQ, at the same concentration and molar ratio. The peptides used for co-crystallization were ordered from Innopep Inc. with a purity of >98% and contained chlorine counter ions. Crystal screening was carried out using a high-throughput, robotic CrystalMation system (Rigaku) at The Scripps Research Institute, which was based on the sitting drop vapor diffusion method with 35 μL reservoir solution and each drop consisting of 0.1 μL protein + 0.1 μL precipitant. High-quality crystals of the MAD2-6 IgG Fab-peptide co-complex diffracted to 2.14 Å and were grown in a reservoir solution containing 2.4 M ammonium sulfate and 0.1 M MES pH 6.0. Crystals of MAD2-6 IgA Fab-peptide co-complex diffracted to 2.50 Å and were grown in a well solution containing 20% polyethylene glycol 3350 and 0.2 M potassium citrate pH 8.3. Crystals for both co-complexes were grown at 20˚C and appeared after 3 days. MAD2-6 IgG and IgA Fab co-complex crystals were cryoprotected by soaking in a well solution supplemented with 20% glycerol and 20% ethylene glycol, respectively, before being flash-cooled in liquid nitrogen. X-ray diffraction data were collected at the Advanced Proton Source (APS) beamline 23ID-B. The datasets were indexed, integrated, and scaled using the HKL-2000 package . The strAssociations between plasma IgA binding to sporozoites, plasma IgG binding to sporozoites, plasma IgA binding to PfCSP and donor age were evaluated using a two-tailed Pearson’s correlation. Segmented regression of plasma IgA versus age was performed using the “segmented” package library in R and the median age was used as the initial value of the breakpoint for the two segments. The association between P17-specific IgA and IgG (n = 525 subjects) and the number of febrile malaria episodes during the ensuing transmission season was determined using age-adjusted Poisson regression analysis, with Bonferroni correction. In the in vivo experiment comparing the efficacy of N-terminus antibodies, differences in parasite liver burden between each antibody and the no mAb control were determined using the Kruskal-Wallis with Dunn’s multiple comparison test for panels with multiple antibodies and the two-tailed Mann-Whitney test for the panel with MAD2-302 and MAD3-23. Total fluorescence intensity of CSP-stained sporozoites and trails between the MAD2-6, 2A10 and buffer control were compared using one-way ANOVA with Dunnett’s multiple comparisons. In the figure legends, N refers to the number of independent experiments unless stated otherwise.Supplementary MaterialsData File S1


Pex2-knockout mouse. Peroxisome deficiency in the conditional Pex2-knockout adult mouse brain induces the upregulated expression of BDNF and its inactive receptor TrkB-T1 in hippocampus, which notably results in memory disturbance. Our results suggest that peroxisome deficiency gives rise to the dysfunction of hippocampal circuit via the impaired BDNF signaling.Peroxisome is an intracellular organelle that functions in essential metabolic pathways including β-oxidation of very-long-chain fatty acids and biosynthesis of plasmalogens. Peroxisome biogenesis disorders (PBDs) manifest severe dysfunction in multiple organs including central nervous system (CNS), whilst the pathogenic mechanisms are largely unknown. We recently reported that peroxisome-deficient neural cells secrete an increased level of brain-derived neurotrophic factor (BDNF), resulting in the cerebellar malformation. Peroxisomal functions in adulthood brain have been little investigated. To induce the peroxisome deficiency in adulthood brain, we here established tamoxifen-inducible conditional  PEX genes encoding peroxisome biogenesis factors, termed peroxins (Pex). Molecular mechanisms of peroxisome biogenesis have been investigated by several approaches including functional analyses of Pex proteins. Pex2 is a peroxisomal membrane protein containing RING  zinc finger at the C-terminal part , and α-oxidation of branched-chain fatty acids . Peroxisnal part . Pex2 annal part  functionnal part . Mono-uband Pex6 . Deficieand Pex6 .Pex gene-defective ZS model mice have been generated, showing abnormal development of CNS as observed in ZS patients  in the inferior olivary nucleus and elevates an inactive truncated form of its receptor, TrkB-T1, in cerebellum , the most severe PBDs, display malformation of central nervous system (CNS) such as disturbance of cortical laminar structure, dysmorphology of Purkinje cells, and dysplasia of inferior olivary nucleus . To datepatients . Using trebellum . A combije cells . We alsoje cells . Taken tenerated . CNS-speelopment . On the elopment , suggestPex14C/ΔCΔ mouse at early stages after birth . Briefly, genomic DNA corresponding to Pex2 locus was isolated from bacterial artificial clone (ID: RP23-184F21) containing C57BL/6 genomic DNA. FRT-Neo-FRT-loxP (FNFL) cassette and a loxP site were engineered into flanking exon 7 of Pex2 gene. The gene targeting vector was as follows: a 2.8-kb short homology arm , FNFL cassette, floxed sequence containing exon 7 , loxP, and 5.6 kb long homology arm  into a vector pBS-DTA carrying the diphtheria toxin A chain (DTA) (Unitech). The replacement vector was linearized by SacII site and electroporated into C57BL/6J embryonic stem (ES) cells that were cultured on G418-resistant mouse embryonic fibroblasts. The resistant colonies were picked up and screened for homologous recombination by Southern blot analysis. Targeted ES cells were injected into C57BL/6 blastocysts to produce chimeric mice. The Neo cassette was then removed in vivo by using flp/FRT recombination by crossing Neo-positive offspring from chimeric breeding with transgenic flp deleter mice. To generate conditional Pex2-knockout mice, Pex2flox/flox mice were crossed with Cre-ER transgenic mice [B6.Cg-Tg(CAG-cre/Esr1∗)5Amc/J] (Pex2 offspring with Cre-ER (Pex2flox/flox/+/–Cre-ER) were backcrossed to Pex2flox/flox mice. Homozygous floxed Pex2 littermates with Cre-ER (Pex2flox/flox/+/–Cre-ER) and homozygous floxed Pex2 littermates lacking Cre-ER (Pex2flox/flox) were used as conditional Pex2-knockout mice and control mice, respectively. PCR analysis of tail-biopsy genomic DNA was performed using primers 2F (5′-GACTTCTGAGGCACTCATACCTAAC-3′), 2R (5′-ATTGTGAGTTTCAGCATTTCTATGG-3′) to amplify 277 and 435 bp fragments specific for the wild-type allele and targeted allele, respectively. Primers 2F and 2R2 (5′-GACTTCTGAGGCACTCATACCTAAC-3′) were used to amplify 620 bp fragment specific for the disrupted Pex2 allele. CreF (5′-CGCGATTATCTTCTATATCTTCAGG-3′) and CreR (5′-AGGTAGTTATTCGGATCATCAGCTA-3′) were for 500 bp fragment specific for the Cre gene.)5Amc/J]  obtainedPex2flox/flox/–/–Cre-ER, and Pex2flox/+/Cre-ER+/– mice were used as control mice.Tamoxifen (Sigma) dissolved at 20 mg/ml in corn oil (Sigma) was orally administrated (10 mg/40 g of body weight) for 5 consecutive days . Two weeApparatus for conditioning and context test consisted of an acrylic square chamber (33 cm × 25 cm × 28 cm) with metal grids  covered by white acrylic lid . Grids wPex14C/ΔCΔ mouse on a C57BL/6 background was previously described (Pex14C+/Δ) were intercrossed to produce homozygous mutant animals. PCR analysis of tail biopsy genomic DNA was undertaken using primers P14F (5′-GTATAAATGTGGGAGTTTCCCTGG-3′) and P14R (5′-GTACTTGTGAACTCTGCTGGTAC-3′) to amplify a 599-bp fragment specific for the wild-type allele and primers P14F and KN52-2 (5′-GTGTTGGGTCGTTTGTTCGG-3′) to amplify a 169 bp fragment specific for the disrupted Pex14 gene.escribed . HeterozMouse monoclonal antibodies to α-tubulin and nestin (Rat-401) were purchased from BD Biosciences  and Invitrogen , respectively. Mouse monoclonal antibody to 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase) was from Sigma . Rabbit antibodies to BDNF (N-20) and TrkB (H-181) were from Santa Cruz Biotechnology . We used rabbit antisera to PTS1 peptide , rat catsn-glycero-3-phosphocholine , 1, 2-didodecanoyl-sn-glycero-3-phosphocholine , and 1, 2-didodecanoyl-sn-glycero-3-phosphoethanolamine  were added as internal standards. After incubation for 5 min at room temperature, 1 ml each of water and chloroform was added and the samples were then centrifuged at 2,000 rpm for 5 min in Himac CF-16RX  to collect the lower organic phase. To re-extract lipids from the water phase, 1 ml chloroform was added. The combined organic phase was evaporated under a nitrogen stream and the extracted lipids were dissolved in methanol.Total cellular lipids were extracted by the Bligh and Dyer method . BrieflyLC-MS/MS analysis of phospholipids was performed as described  using a Total RNA was extracted from the tissues using TRIzol reagent (Invitrogen) and first-strand cDNA was synthesized with a PrimeScript RT reagent kit . Quantitative real-time RT-PCR was performed with SYBR Premix Ex Taq II (Takara Bio) using an Mx3000P QPCR system . Several sets of primers used are listed in Mice were deeply anesthetized with isoflurane and then decapitated. The mouse brains were fixed in 4% paraformaldehyde (PFA) in PBS, pH 7.4, for overnight at 4°C and were transferred to 30% sucrose in PBS for 2 days. The brains were embedded in the Tissue-Tek OCT compound  and subsequently frozen at −80°C. Cryosections were cut at a thickness of 20 μm using a cryostat Microm HM550-OMP (Thermo Fisher Scientific) and were then mounted on MAS-coated glass slides . The sections were permeabilized by ice-cold methanol, blocked by blocking buffer  and were then incubated for overnight at 4°C with primary antibody diluted in blocking buffer. After washing with PBS, the sections were incubated with appropriate secondary antibody conjugated to Alexa 488 or 567; for marker staining of nuclei, the sections were incubated with Hoechst 33,242 in PBS for 2 min at room temperature, and then mounted with PermaFluor. For the staining of nestin, anti-nestin antibody was biotinylated by Biotin Labeling Kit  according to the manufacturer’s instructions and visualized by FITC-streptavidin . Images were obtained under a LSM710 with Axio Observer.Z1  or AF 6000LX . Quantitative analysis was performed by Image J software . Quantification of colocalization in randomly selected cells (40∼50 cells) was performed by calculating Pearson’s correlation coefficient using ZEN 2012 software (Carl Zeiss).Immunoblotting was performed as described . Precisi1. All Student’s t-tests used were one-tailed. A P-value less than 0.05 was considered statistically significant.Statistical analysis was performed using R softwareThe animal ethics committee of Kyushu University approved all animal experiments.Pex2-knockout mouse using tamoxifen-inducible Cre/loxP recombination system  mouse. To assess the peroxisomal matrix protein import, we analyzed the proteolytic processing/conversion of peroxisomal matrix proteins including ADAPS, 3-ketoacyl-CoA thiolase, AOx, and SCPx. The precursors of these proteins including AOx-A chain were elevated in Pex2-KD mouse . Immunoblotting showed that hippocampal BDNF expression was not altered in neonatal Pex14C/ΔCΔ mouse  mouse. –/–CMV-Tx-Pex5 mouse showed motor dysfunction, demyelination, axonal impairment, and microgliosis at 5 or 8 months after tamoxifen administration. In this study, we performed the behavioral experiment with Pex2-KD mouse at 2∼3 weeks after the tamoxifen administration. At such early time-points, memory impairment was emerged in Pex2-KD mouse (Pex5 knockout (–/–Nes-Pex5) mouse that manifests the cognitive impairment (–/–Nes-Pex5 mouse, the defect of peroxisome biogenesis in CNS emerges during embryonic day (–/–Nes-Pex5 mouse appears to be owing to the abnormal brain development (Pex2-KD mouse is intact before the administration of tamoxifen. Thus, the defect of peroxisome biogenesis in Pex2-KD mouse induced by administration of tamoxifen apparently impairs the maintenance of neural functions. Taken together, these results suggested that peroxisomal integrity is essential for maintaining the hippocampal memory functions.KD mouse , while tKD mouse . The invpairment . In Nes-onic day . Therefoelopment . On the Pex-knockout mouse. Liver-specific restoration of peroxisome biogenesis in –/–Pex5 mouse improved the brain morphology (Pex5-knockout mouse showed the neuronal migration defect in cortex and malformation of cerebellum (Pex2-KD mouse.It has been reported that inter-organ interaction caused the abnormal brain development in rphology . Likewisrebellum . TherefoPex14C/ΔCΔ mouse (Pex2-KD mouse also showed the elevated levels of BDNF and TrkB-T1 in hippocampus (Pex14C/ΔCΔ mouse (We recently reported that peroxisomal deficiency causes the upregulation of BDNF and TrkB-T1, giving rise to an abnormal morphology of Purkinje cells in the cerebellum of ΔC mouse . In thispocampus . Our recpocampus . HoweverΔC mouse . The BDNΔC mouse , 7. The Pex2-KD mouse shows the upregulation of BDNF and TrkB-T1 in hippocampus. Taken together, peroxisome deficiency in hippocampus likely induces the attenuation of BDNF-TrkB signaling, resulting in the memory disturbance of Pex2-KD mouse. Therefore, peroxisome biogenesis is essential for not only morphogenesis of CNS, but also maintaining the neuronal functions.BDNF plays an important role in activity-dependent synaptic plasticity such as long-term potentiation in hippocampus . The impPex2-KD mouse hippocampus was mosaically, not completely, impaired as assessed by catalase staining (Pex2-KD mouse manifests the increased level of BDNF and TrkB-T1 (Peroxisome biogenesis in staining . Nonethe TrkB-T1 , 7 and t TrkB-T1 . These f TrkB-T1 . CollectThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by the Animal Ethics Committee of Kyushu University.YA, ST, MH, TY, and YF conceived and designed the study. YN, KN, ST, and HU generated conditional knockout mice and performed behavioral experiments. YA and YN performed the biochemical and immunohistological analyses of brains. YA, YN, ST, MH, and YF analyzed and interpreted data and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.