However, this method typically requires 300 μg of total RNA, which limits its application to patient tumors. In this study, we present a refined m6A MeRIP-seq protocol and analysis pipeline that can be applied to profile low-input RNA samples from patient tumors. We optimized the key parameters of m6A MeRIP-seq, including the starting amount of RNA, RNA fragmentation, antibody selection, MeRIP washing/elution conditions, methods for RNA library construction, and the bioinformatics analysis pipeline. With the optimized immunoprecipitation (IP) conditions and a postamplification rRNA depletion strategy, we were able to profile the m6A epitranscriptome using 500 ng of total RNA. We identified approximately 12,000 m6A peaks with a high signal-to-noise (S/N) ratio from 2 lung adenocarcinoma (ADC) patient tumors. Through integrative analysis of the transcriptome, m6A epitranscriptome, and proteome data in the same patient tumors, we identified dynamics at the m6A level that account for the discordance between mRNA and protein levels in these tumors. The refined m6A MeRIP-seq method is suitable for m6A epitranscriptome profiling in a limited amount of patient tumors, setting the ground for unraveling the dynamics of the m6A epitranscriptome and the underlying mechanisms in clinical settings.N6-Methyladenosine (m 6A) is one of the most abundant and conserved mRNA modifications. It has been reported to influence multiple steps of RNA life cycle and play an important role in the initiation and progression of human cancers. m6A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq) is a powerful technique to map the m6A location transcriptome-wide. However, this method typically requires 300 μg of total RNA, which limits its application to patient tumors. In this study, we presented an optimized MeRIP-seq protocol that allows us to profile m6A epitranscriptiome using as low as 500 ng of total RNA. By applying our refined protocol to 2 lung cancer patient tumors and integrating with proteomic data, we identified dynamics at the m6A level that account for the discordance between mRNA and protein levels in these tumors.N6-Methyladenosine (m A secon Input]) . Next, w49 cells . With 1 100-fold . Consistnd of IP , althoug6A profiling, 2 analysis approaches have been applied for m6A peak calling. The first one employed and tuned the most popular peak calling software, MACS  > 1) and protein (Tumorlight/Stable Isotope Labeling by Amino acids in Cell culture [SILAC]heavy > 0.5) levels, and 18 of these 45 genes have discordant fold changes between the 2 tumors at RNA and protein levels (SLC2A1), while the mRNA levels were similar between the 2 tumors, the protein level was 1.9-fold higher in tumor1 compared with tumor2  in the A549 cell line. The knockdown efficiency was confirmed by both RT-qPCR and western blot  has been reported to be able to drive Epidermal Growth Factor Receptor (EGFR) protein translation ) > 1  > 1 , and thoS1 Fig(A) Different RNA samples were chemically fragmented for the specified time points, ethanol-precipitated, and separated on the High Sensitivity RNA ScreenTape. After 6 minutes, RNA fragments centered around approximately 200 nt.  Representative electropherogram of fragmented RNA using 5 μg total RNA from A549 cells after 6- and 7-minute incubation.(TIFF)Click here for additional data file.S2 Fig6A motifs detected from top 5,000 m6A summit centered around 200 nt regions. (D) The location and frequency of the top motif to the summit; ***p < 1 × 10−4. (E) The distribution characteristic of the peaks detected by MACS based on 2 published m6A datasets. (F) Motif discovered by MACS based on 2 published m6A datasets. Data related to this figure can be found in 6A, N6-methyladenosine; MeRIP-seq, m6A RNA immunoprecipitation followed by high-throughput sequencing.(A) Peaks detected by MACS and MeTPeak with and without the duplication reads. (B) Changes in peak number with increasing depth of uniquely mapped reads for IP/Input. The dashed line means the overlapped peaks called by both MeTpeak and MACS. (C) Consistent m(TIFF)Click here for additional data file.S3 Fig6A peaks from different libraries. Right: the motif called based on Millipore or Synaptic 32 μg unique peaks. (B) Analysis of the percentage of m6A peaks in each of the 5 nonoverlapping transcript segments. (C) Relative enrichment of m6A peaks around the TSS and stop codon region. (D) Sequence logo representing the deduced top motifs for 12 μg and 6 μg libraries. (E) Density curves of the motif flanking the peak summit.  Comparison between the Millipore 2 μg and the control dataset. Data related to this figure can be found in 6A RNA immunoprecipitation followed by high-throughput sequencing; TSS, transcription start site.(A) Comparison between the control dataset (300 μg) and the Millipore or Synaptic 32 μg library data. Left: Venn diagrams show the overlap between m(TIFF)Click here for additional data file.S4 FigSETD7/GAPDH with different starting amount of total RNA. (B) Correlation between 2 replicates for both Input (top) and IP (bottom) of the 0.5 μg MeRIP-seq data. (C) The S/N ratio of SETD7/GAPDH (top) and the SETD7 IP yield (percentage of the input) (bottom) using different amounts of Millipore antibody  with fixed amount of total RNA (2 μg). Data related to this figure can be found in 6A RNA immunoprecipitation followed by high-throughput sequencing; S/N, signal-to-noise.(A) The S/N ratio of (TIFF)Click here for additional data file.S5 FigE. coli K-12 total RNA before and after DNase treatment on the High Sensitivity RNA ScreenTape. (B) Representative electropherogram of E. coli K-12 total RNA before and after purification and DNase treatment.(A) The gel image separation profile of (TIFF)Click here for additional data file.S6 Fig6A peaks before normalization (bottom). (C) The correlation between IP fold change and Input fold change (top). MA plots for different m6A peaks after normalization were shown in the bottom. (D) Number of differential peaks distributed along the log-transformed fold change. Data related to this figure can be found in (A) Venn diagram of peaks detected in 2 ADC tumors (tumor1 and tumor2). (B) The correlation between IP fold change and Input fold change before the ERCC spike-in normalization (top). MA plots for different m(TIFF)Click here for additional data file.S7 FigSETD7/GAPDH. Bottom: SETD7 IP yield (percentage of the input). (B) MeRIP-seq library of different amplification cycles using SMARTer Stranded Total RNA-Seq Kit version 2  kit. One out of 50 μl of PCR product was used for gel electrophoresis by DNA tape station. The smear centered at 300 bp is the library DNA. IP, immunoprecipitation; MeRIP-seq, m6A RNA immunoprecipitation followed by high-throughput sequencing; S/N, signal-to-noise.(A) Comparison of low-salt, high-salt, and low/high salt combination washing conditions. Top: pulldown efficiency as measured by S/N ratio of (TIFF)Click here for additional data file.S1 Table6A RNA immunoprecipitation followed by high-throughput sequencing.MeRIP-seq, m(XLSX)Click here for additional data file.S2 Table6A RNA immunoprecipitation followed by high-throughput sequencing.MeRIP-seq, m(XLSX)Click here for additional data file.S3 Table6A, N6-Methyladenosine.lincRNA, long intergenic noncoding RNA; m(XLSX)Click here for additional data file.S4 Table6A, N6-Methyladenosine; MeRIP-seq, m6A RNA immunoprecipitation followed by high-throughput sequencing.m(XLSX)Click here for additional data file.S5 Table6A RNA immunoprecipitation followed by high-throughput sequencing.MeRIP-seq, m(XLSX)Click here for additional data file.S1 Text6A RNA immunoprecipitation followed by high-throughput sequencing.MeRIP-seq, m(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


N-biose  have been determined. The resolution of P[8] VP8* crystal structures in complex with H1 antigen and LNB and site-directed mutagenesis experiments revealed that both glycans bind to the P[8] VP8* protein through a binding pocket shared with other members of the P[II] genogroup . Our results show that the L-fucose moiety from H1 only displays indirect contacts with P[8] VP8*. However, the induced conformational changes in the LNB moiety increase the ligand affinity by two-fold, as measured by surface plasmon resonance (SPR), providing a molecular explanation for the different susceptibility to rotavirus infection between secretor and non-secretor individuals. The unexpected interaction of P[8] VP8* with LNB, a building block of type-1 human milk oligosaccharides, resulted in inhibition of rotavirus infection, highlighting the role and possible application of this disaccharide as an antiviral. While key amino acids in the H1/LNB binding pocket were highly conserved in members of the P[II] genogroup, differences were found in ligand affinities among distinct P[8] genetic lineages. The variation in affinities were explained by subtle structural differences induced by amino acid changes in the vicinity of the binding pocket, providing a fine-tuning mechanism for glycan binding in P[8] rotavirus.Rotavirus is the leading agent causing acute gastroenteritis in young children, with the P[8] genotype accounting for more than 80% of infections in humans. The molecular bases for binding of the VP8* domain from P[8] VP4 spike protein to its cellular receptor, the secretory H type-1 antigen , and to its precursor lacto- N-biose  is also bound to this pocket with diminished affinity. The P[8] VP8* structures show a marginal role for the L-fucose moiety from H1 in protein interaction. However, its presence provides conformational changes in the LNB moiety that increase the affinity of VP8* for the H1 ligand and would account for a stronger RV binding to mucosa in individuals expressing H1 (secretors). We thus offer a mechanistic explanation for the different incidence of P[8] RV infection in different secretor phenotypes.The interaction of viruses with host glycans has become an important topic in the study of enteric virus infectivity. This interaction modulates several aspects of the viral cycle, including viral attachment, which in most cases depends on the host glycobiology dictated by the secretor and Lewis genotypes. The capacity to synthesize secretory type-I antigen H  at the mucosae, dictated by the presence of one or two functional copies of the fucosyl-transferase FUT2 gene (secretor status), has been clearly linked to infectivity in important enteric viruses such as the noroviruses. However, a big controversy existed about the contribution of H1 antigen to infection in the leading cause of viral gastroenteritis in young children (rotavirus). It has not been until recently that epidemiological data evidenced a diminished incidence of rotavirus in non-secretor individuals unable to produce H1. In the present manuscript we offer the evidence that P[8] RV bind H1 via a binding site common for the P[II] RV genogroup and that the H1 precursor lacto- This binding pocket was first suggested by protein sequence analyses in other members belonging to the P[II] genogroup of rotaviruses   genotypes. We provide the molecular bases for the role of secretor antigen in rotavirus binding to its receptor. Our results show two-fold increase in the affinity for the H1 antigen compared to LNB. This increase is explained by reduced contacts of the L-fucose with solvent molecules and the structural stabilization of LNB moiety in the competent conformation for binding. Furthermore, we show how subtle differences at the H1/LNB binding pocket in different P[8] lineages influence antigen affinities, giving clues for the relevance of the host glycobiology in P[8] rotavirus impact in humans. The partial anti-adhesin effect of LNB against rotavirus reported in the present article and the acquired knowledge on rotavirus-host cells interaction during virus attachment might open new avenues for the treatment and prevention of rotavirus infections.Diverse interactions between histo-blood group antigens (HBGAs) and rotavirus have been described and it is believed that HBGAs expressed on the surface of target cells serve as viral receptors. The distal VP8* portion  of the rotavirus spike protein VP4 from P[8], P[4], P6] and P19] genotypes recognize the secretor HBGAs. P[8] and P[4] are closely related genetically and both genotypes were reported to bind the Lewis and P genotype additionally displayed low binding to this trisaccharide. However, and contrarily to previous reports [7], in our assays genotypes P[4] and P[8] exhibited very low or absence of binding to Lewisb . Remarkably, VP8* from P[8] genotype recognize the H1 antigen precursor lacto-N-biose  and the Rotarix vaccine (P[8]Rotarix) strains (both lineage I) gave lower signals in the ELISA tests with H1 and LNB than the VP8* from the lineage IV strain (P[8]LIV) and from a clinical isolate belonging to lineage III (P[8]c) (c) and the cultivable P[8]Wa (a) for each interacting pair by SPR  suggesting that the H1 L-fucose moiety contributes actively to the binding. Surprisingly, the affinity constant for the interaction of VP8* from P[8]Wa with H1 was three times higher (Kda = 80.2 ± 2.21 μM) than that of P[8]c  from P[8] genotype were produced . In orde (P[8]c) . The newe P[8]Wa . The resr by SPR . The Kdaof P[8]c . FurtherTo further characterize the role of VP8* interaction with the H1 precursor LNB, complete virions of the Wa strain  and double-layered particles  were assayed by an ELISA-like binding assay. The Wa TLP, but not the Wa DLP, were able to bind H1 antigen  and its c genotype to LNB we settled up a binding blocking assay where LNB  and its structurally-related disaccharide galacto-N-biose , were tested as potential inhibitors of binding. The results showed a moderate but significant (p < 0.05) reduction in the binding to the H1 antigen by both disaccharides  and L-fucose were also tested (p = 0.032). Interestingly, soluble L-fucose significantly increased the binding of VP8* to the H1 antigen  while the second form, VP8*-Apo2, diffracts to 1.5 Å and presents two copies in the ASU  to the VP8* apo forms of the linage I P[8]Wa and P[8]Rotarix   .c VP8* binds H1 and LNB in a pocket formed by one of the β-sheets and the C-terminal α helix , R209A  and E212A  were obtained , defining the Lewis status , resulting in different secretor status and by the FUT3 gene  in the P[8]c VP8* structure taking H1 antigen as a reference showed that the α1,4-linked L-fucose points towards the inside of the glycan binding pocket, clashing with different residues (mainly T185 and E212) that generate this cavity  added to the non-reducing end of the H1 galactose is pointing towards the solvent without showing any interaction with the protein  differed from other members of the same lineage, including the Wa strain, by the replacement of leucine 167 by phenylalanine. Since L167 is placed at the bottom of the sugar-binding pocket , emulating P[8]Wa. This replacement resulted in a VP8* with a diminished interaction with the H1 antigen, with an affinity constant . The VP8I173V variant retained the binding ability to LNB with an apparent affinity constant . These results support that subtle amino acid changes at the loop close to the binding pocket may have contributed to modulate the glycan affinity between P[8] VP8* from lineages I and III. Differences at this site are also evident in the structures of VP8* from the P[II] genogroup. While conservation between P[8] and P[4] is high, the sequence divergence between P[4] and P[6]  individuals. Furthermore, a weaker interaction to LNB may also explain why infection of P[8] rotaviruses can occur, at a lower level, in non-secretor individuals  lineages, suggest that simple qualitative ELISA tests do not always provide reliable results and that other techniques need to be implemented in order to assess VP8* affinities for HBGAs. However, structural data from P[4] and P[6] genotypes predict interaction with A- and B-types HBGAs, as the N-acetyl-galactosamine (A-type) and galactose (B-type) located at the non-reducing ends in these glycans do not make any steric hindrance [N-acetyl-galactosamine in H1 bound to P[8]c VP8* , although sharing identical key binding residues to lineage III, do not bind H1 [We showed that variations in the binding domain in the P[8] lineages exist that justify the differences in the affinity for H1 and LNB as measured by SPR. Additionally, even if the architecture of the binding site is similar for most P[8] lineages and other genotypes belonging to the P[II] group, other protein residues outside this site may possess epistatic effects over the capacity of the binding pocket to accommodate H1 and LNB, explaining the diverse affinities among P[II] genotypes and in the different P[8] lineages. This is exemplified by the fact that we were able to associate a residue that does not participate in direct protein-ligand contacts  in P[8] bind H1 . It is p bind H1 , 24–26,  bind H1 . It is w bind H1 . Many st bind H1 , 29, but bind H1 . Studies bind H1 , but ourin vitro. The anti-adhesin potential of this simple disaccharide (LNB) is susceptible for being exploited in antiviral strategies. LNB is present in human milk in its free form [Despite the need for more exhaustive research on the relevance of HBGA and host specificity/infectivity in P[8] rotavirus, surface glycans possess a clear application in the development of antiviral strategies. It is established that human milk, in addition to other antiviral components, carry a set of oligosaccharides  that share structural similarities to HBGA  and coulree form  but mainree form . Thus, Lree form . LNB wouree form , makes tree form , 21 and,Detailed determination of the interactions between viruses and their host is crucial to develop appropriate antiviral strategies. We have defined the molecular interactions of P[8] VP8* from human rotavirus with its ligand HBGA giving a physical explanation as to why the secretor status influences rotaviral infectivity. Notwithstanding, extra structural elements beyond the identified binding site in VP8* are probably responsible for modulating HBGA interactions within P[8] lineages. Dissection of additional VP8* structural features affecting ligand binding is under way.The VP8* (amino acids 64–224 from the VP4 protein of rotavirus) belonging to the P[4], P[6], P[8], P[9], P[11], P[14] and P[25] genotypes were cloned into the, pGEX-2T, expression vector  in order to express N-terminal GST fusions.E. coli and its codon usage was optimized. P[11] VP8* and P[25] VP8* were not available as clinical samples. The recombinants GST::VP8* proteins were expressed in E. coli BL21 (DE3) (Novagen) and purified by affinity chromatography using GSTrap columns coupled to an ÄKTA prime FPLC system . All sequences are included as fasta files in the supplementary data.To amplify P[4], P[6], P[8], P[9] and P[14] VP8*s coding region, RNA was extracted from human stool samples  containing rotavirus of known P genotype using the Trizol reagent following the standard procedure (Invitrogen). Viral RNA was retro-transcribed using the SuperScript Reverse Transcriptase (Invitrogen) and random-primers, and the cDNA was amplified by PCR using Pfu polymerase (Stratagene) with primers detailed in Selected residues in the GST::VP8* P[8] were replaced for alanine or valine according to the structural data of the LNB binding site. Four mutants (M1–M4) were constructed using a Quick-Change site-directed mutagenesis kit (Stratagene) and appropriate oligonucleotides , and thea, Leb, Lec , H type-1, H type-2 and blood group A and B trisaccharides were purchased from Glyconz  and Wa (P[8]Wa) genotypes to the H type-1 and to its precursor  was determined incubating two fold serial dilutions of the VP8* proteins, ranging from 100 μg/ml to 1.5 μg/ml. The binding assays were performed in triplicate using the protocol described above.The ECN-biose  produced and purified in our laboratory as previously described [To confirm the binding of the VP8* from the P[8] genotype to the H type-1 precursor LNB, a blocking assay was performed using soluble LNB and its related disaccharide galacto-escribed .Wa VP8* protein at their EC50 for each ligand and 20 mM of each of the soluble disaccharides (LNB and GNB) and monosaccharides . A positive binding control without sugar was also included. The tubes containing the mixes of VP8* with sugars were maintained 1 hour at 37°C, followed by an overnight incubation at 4°C. The next day the coated streptavidin plates were washed with PBS-T, the VP8*-sugar solutions were added to the plates and incubated during 4 hours at 4°C. The plates were washed three times with PBS-T and detection of the interactions was performed as described above. The results are presented as the percentage (%) of binding of each condition compared to the binding of the positive binding control (without blocking sugar). All experiments were performed in triplicate.Streptavidin microtiter plates were coated with biotinylated H type-1 antigen or LNB at 2 μg/ml with water and incubated for 1 hour at 37°C, followed by an overnight incubation at 4°C. Blocking assays were performed in parallel, using glass tubes containing the P[8]c and P[8]2 flasks (approximately 1.5 ×107 cells/flask) were infected with Wa strain at a multiplicity of infection (MOI) of ≤ 0.1 and processed as previously described . Briefly, ten MA104 cells confluent 150-cm2, 0.05% Tween 20, pH 7,4) buffer and the binding and washing steps were always carried out in this solution. DLPs assays were carried out in Tris-buffered saline buffer with Tween 20 . TLP and DLP were incubated in the plate overnight at 4°C. After binding, the plates were washed three times in TNC-T (for TLP) or TBS-T (for DLP) with 0.05% Tween 20 (TNC-T and TBS-T), and a mouse anti-VP6 antibody was added at 1:100 in TNC-T or TBS-T and incubated 1 h at 37°C. The plates were then washed three times with TNC-T or TBS-T, and a HRP-conjugated anti-mouse IgG was added at 1:10.000 and incubated at 37°C for 1 h. After three final washes the binding was revealed by QuantaBlue reagent (ThermoFisher) following the manufacturer recommendations. Fluorescence units were recorded by a MultiScan microplate reader.An ELISA-like binding assay was employed to determine the binding ability of rotavirus TLP and DLP to the H type-1 antigen and to its precursor LNB. Streptavidin plates were coated with the biotinylated oligosaccharides as described above. After washing with PBS-T, two fold serial dilutions of TLPs and DLP were added to the plate (ranging from 10 μg/ml to 0.078 μg/ml). The TLP were always maintained in TNC-T , a flow rate of 5 μl/min with 2700 seconds of contact time and a dissociation time of 1800 seconds. The regeneration step consisted in a wash step with 10 mM Glycine-HCl pH 2 for 20 seconds at the same flow rate. The assays were performed with purified VP8* at different concentrations . Each run included three blanks without sample. The affinity data were obtained after analysis of sensorgrams performed with the BIAevaluation 2.0 software . Since multivalent oligosaccharides are immobilized on a sensor chip surface, avidity and rebinding effects can take place and apparent affinity constants (Kda) are calculated with this experimental setup. Kda values were obtained from the steady-state kinetics experiment as the ligand concentration needed to achieve a half-maximum binding at equilibrium. The experiments were made in triplicate. Graphical representation of signal/concentration curves were plotted using GraphPad Prism 6 for MacOsX.The affinity assays were based on SPR and performed in a Biacore T100 instrument . H1 PAA-biotin and LNB PAA-biotin were diluted to a concentration of 1 mg/ml in water and captured with streptavidin present in a SA sensor chip . H1 was immobilized in channel 2 (630 RU) of the sensor chip and LNB was immobilized in channel 4 (624 RU). The channels 1 and 3 were used as the reference surfaces for channels 2 and 4, respectively. The immobilization process was performed by conditioning the sensor chip surface with three consecutive 1-minute injections of 1 M NaCl 50 mM NaOH before biotinylated ligands were immobilized at a flow rate of 15 μl/min. The affinity assays of VP8* polypeptides to biotinylated sugars were performed at 10°C using 1X HBS-EPc VP8* protein solution and the corresponding reservoir solution, and were equilibrated against 100 lμl reservoir solution. Both P[8]c VP8* Apo structures were crystallized at 10 mg/ml. VP8* Apo1 was crystallized in a reservoir solution of 1.2 M (NH4)2SO4, 3% iso-propanol and 0.1 sodium citrate pH 4.6, whereas VP8* Apo2 was crystallized in 1.5 M Li2SO4 and 0.1 M Tris-HCl pH 6.5. In both cases 2 M Li2SO4 was used to cryoprotect the crystal when freezing in liquid nitrogen. For the crystallization in presence of glycans, the ligands were mixed with the protein at 10 mM final concentration of ligand and 10 mg/ml of protein final concentration. P[8]c VP8* LNB was crystallized in a reservoir solution consisting in 25% PEG 3,350 0.1 M Bis-Tris pH 5.5. The cryosolution used for crystal freezing was its reservoir solution increased up to 35% PEG 3,350. P[8]c VP8* H1 was crystallized against a a reservoir solution consisting in 25% PEG 6,000, 0.1 M Na-HEPES pH 7.5, 0.1 M LiCl, and PEG 6,000 was increased up to 35% for cryoprotection. X-ray diffraction was carried out at 100K at Alba  and DLS  synchrotrons and the best data sets used to solve the structures were collected at the indicated beamlines and wavelengths  and JCSG+ (Molecular Dimensions) in 96-well plates. Crystallization drops were generated by mixing equal volumes (0.3 μl) of P[8]elengths . DiffracC VP8* Apo1 structure was solved by molecular replacement carried out with the program Phaser [c VP8* Apo1 structure was then used as a model for molecular replacement to solve the P[8]c VP8* Apo2, P[8]c VP8*LNB and P[8]c VP8*H1 structures. All the final models were generated by iterative cycles of refinement using the Refmac c VP8* Apo2, P[8]c VP8*LNB and P[8]c VP8*H1, respectively. Structure Superposition and RMSD calculations were carried out with Superpose [P[8]m Phaser  and usinm Phaser ) as a mom Phaser . P[8]c Ve Refmac  and manuuperpose  from CCPa values obtained by SPR an unpaired t-test was applied. All statistical analyses were performed with GraphPad Prism version 6.0 for MacOsx (GraphPad Software). p values <0.05 were considered to be statistically significant.To assess statistical differences in the ELISA-like binding experiments where many groups were compared an ANOVA test was performed. To analyze significative differences in the KdThis study was conducted with the approval of the Ethics Committee of the University of Valencia (code H1544010468380).The human stool samples from Hospital Clínico Universitario de Valencia were anonymized previously to their inclusion in the present study.S1 FigWa corresponds to the lineage I and the P[8]c corresponds to the lineage III strain RVA/Human-wt/VLC/3455/2015/[G1P8]. Lineage IV correspond to strain RVA/Human-tc/BGD/MMC71/2005/G1P[8].The genotype of each one of the proteins is indicated as well as the molecular weight marker (Mw). The molecular weights (in kDa) of the marker are indicated at the right of the gel. The P[8](PPTX)Click here for additional data file.S2 Figpanel a to panel j.The interaction pairs are indicated in each panel from (PPTX)Click here for additional data file.S3 Figc) and from the cultivable Wa strain (P[8]Wa) to the H1 antigen and to its precursor lacto-N-biose (LNB).The graph shows the concentration-dependent binding of VP8* from the clinical isolate (P[8](PPTX)Click here for additional data file.S4 Figa The backbone structures of P[8]c VP8* in its apo form , and in complex H1 (green) and LNB (blue) are superimposed. The glycans bound to P[8]c VP8* are shown in stick representation with carbon atoms colored according to the corresponding structure. b Superposition of the backbone structures of P[8]c VP8* H1 (green) with VP8* apo forms of the linage I P[8]Wa , P[19] , P[14] VP8*  and P ( PDB 5VKS) and Pc VP8* H1 structures shows that the Lewisb Fuc (in sticks with carbon atoms colored in cyan) is projected from the H1 glycan (in sticks with carbon atoms in green) towards the VP8* protein  clashing with different sugar recognizing residues as T185 and E212. In the case of the A-type I antigen, the presence of an additional GalNAc moiety (in sticks with carbon atoms colored in magenta) has not steric problems since it is project towards the solvent.Modeling of the Lewis(PPTX)Click here for additional data file.S7 FigWa  in its apo forms are superimposed with P[8]C in complex with H1 (green) and the backbone of the structural elements conforming the glycan binding site are shown. The bound H1 antigen is represented in sticks with carbon atoms in yellow (GlcNAc), green  and slate blue (Fuc). The residues interacting with the sugars are shown in stick representation, with carbon atoms colored according to the structural element to which they correspond. Oxygen and nitrogen atoms are colored in red and dark-blue, respectively, in all the structure. A subtle displacement of the loop connecting β9-β10 strands between the glycan free and glycan-bound forms is observed and indicated by and around. Similar displacement is observed in the ligang-binding residue Y169. The loop displacement is facilitated by the presence of two Gly (G170 and G171) residues represented as spheres. The hydrophobic residue  in the backside of the glycan binding pocket at position 173 is shown as stick.The structures of P[8] PDN 2DWR), P[8]Ro PDB 5JDB) and P(PPTX)Click here for additional data file.S1 TableAll biotinylated oligosaccharides were purchased from Glyconz.(PPTX)Click here for additional data file.S2 Table(PPTX)Click here for additional data file.S3 Table(PPTX)Click here for additional data file.S4 Tablec VP8* (lineage III). The residues that were mutated to alanine  are shown in bold. The underlined residue (position 173) does not make ligand contacts but differs between lineage I and lineages II, III and IV.Residues critical for H1 and LNB interaction were determined in P[8](PPTX)Click here for additional data file.S5 TableBamHI restriction site is underlined.(PPTX)Click here for additional data file.S6 Table(PPTX)Click here for additional data file.


Multiple regulatory mechanisms tailor BK current properties across tissues, such as alternative splicing, posttranslational modifications, and auxiliary subunits. Another potential mechanism for modulating BK channel activity is genetic variation due to single nucleotide polymorphisms (SNPs). The gene encoding the human BK α subunit, KCNMA1, contains hundreds of SNPs. However, the variation in BK channel activity due to SNPs is not well studied. Here, we screened the effects of four SNPs  on BK currents in HEK293T cells, selected based on predicted protein pathogenicity or disease linkage. We found that the SNPs C495G and R800W had the largest effects on BK currents, affecting the conductance–voltage relationship across multiple Ca2+ conditions in the context of two BK channel splice variants. In symmetrical K+, C495G shifted the V1/2 to more hyperpolarized potentials (by −15 to −20 mV) and accelerated activation, indicating C495G confers some gain-of-function properties. R800W shifted the V1/2 to more depolarized potentials (+15 to +35 mV) and slowed activation, conferring loss-of-function properties. Moreover, the C495G and R800W effects on current properties were found to persist with posttranslational modifications. In contrast, A138V and N599D had smaller and more variable effects on current properties. Neither application of alkaline phosphatase to patches, which results in increased BK channel activity attributed to channel dephosphorylation, nor bidirectional redox modulations completely abrogated SNP effects on BK currents. Lastly, in physiological K+, C495G increased the amplitude of action potential (AP)-evoked BK currents, while R800W had a more limited effect. However, the introduction of R800W in parallel with the epilepsy-linked mutation D434G (D434G/R800W) decreased the amplitude of AP-evoked BK currents compared with D434G alone. These results suggest that in a physiological context, C495G could increase BK activation, while the effects of the loss-of-function SNP R800W could oppose the gain-of-function effects of an epilepsy-linked mutation. Together, these results implicate naturally occurring human genetic variation as a potential modifier of BK channel activity across a variety of conditions.BK Ca The human BK channel gating ring structure  with the human residues . The hBKQEERL variant was generated by deleting the “SRKR” exon from splice site 1 and the Ca2+ bowl exon from site 3 within a PciI fragment . The human “QEERL” exon at site 4 was introduced by subcloning the PciI fragment into a PciI-digested mouse BKQEERL clone (GenBank KF530043), resulting in the final construct called hBKQEERL (GenBank MG279688). hBKVYR and hBKQEERL were sequenced in entirety and verified to have no extraneous mutations. SNP sequences or mutations were introduced into hBKQEERL and hBKVYR backgrounds by site-directed mutagenesis  in the pcDNA3.1+ mammalian expression vector and verified by sequencing. All channel constructs contain an N-terminal Myc tag and an EYFP tag (241 amino acids) inserted after residue 742 in the RCK2 domain.The human BK channel (hBK) complementary DNA was generated by replacing eight amino acids in the mouse clone  were cultured in a 37°C incubator with 5% carbon dioxide in 35- and 60-mm tissue culture dishes in media containing Dulbecco’s modified Eagle medium , 10% fetal bovine serum , 1% penicillin/streptomycin , and 1% L-glutamine . Cells were transfected at 50–70% confluency with wild-type (WT) or SNP-containing BK expression constructs using either Lipofectamine 2000  or Trans-IT LT1  at 1/5 and 1/2 ratios of DNA/transfection reagent (μg/μl), respectively, according to manufacturer protocols. After 6–12 h, cells were re-plated onto glass coverslips pre-coated with poly-L-lysine  and recorded from 20 h to 48 h post-transfection. BK-expressing cells were identified by the fluorescence signal from the EYFP tag in the BK α subunit. No differences in expression were observed between WT and any of the SNP-containing channels.+ experiments, the external (pipette) solution contained (mM): 140 KMeSO3, 2 KCl, 2 MgCl2, and 20 HEPES. Internal (bath) solution contained (in mM): 140 KMeSO3, 2 KCl, and 20 HEPES. pH was adjusted to 7.2 with KOH. Appropriate amounts of CaCl2, calculated in WebMaxC2+. Solutions were buffered with either 5-mM EGTA (0-μM Ca2+) or 5-mM HEDTA (1- and 10-μM Ca2+). No Ca2+ buffer was used for 100-μM Ca2+ solutions.Inside out patch clamp recordings of macroscopic BK currents were conducted at room temperature using thin-walled borosilicate glass pipettes with resistances of 1–4 MΩ . Data were acquired at 50 kHz and online filtered at 10 kHz with the MultiClamp 700B amplifier . For symmetrical K+, macroscopic BK currents were elicited using a voltage protocol stepping from holding potentials of −100 or −150 to +350 mV (in +10 mV increments) for 20 ms and back to −80 mV for 10 ms to generate tail currents. Conductance–voltage (G–V) curves were obtained by measuring the instantaneous tail current amplitudes 200 μs after the start of the −80-mV step, dividing the current amplitude by the K+ driving force, and normalizing to the maximum conductance (Gmax) and plotting against the activating voltage step. Driving force was calculated from the voltage of the tail step (Vm) subtracted by equilibrium potential for K+ (Veq). In symmetrical K+ conditions, Veq for K+ is 0 mV, and Veq is −80 mV for physiological K+ conditions. The half maximal voltage of activation (V1/2) was determined by fitting G–V curves to a Boltzmann function: In symmetrical K2+), and currents were recorded from inside out patches in control bath solution, or bath solution containing Alk P, 1 min after, patches were excised. For redox experiments, the reducing agent dithiothreitol  and oxidizing agent hydrogen peroxide  were diluted to working concentrations in the internal bath solution (with 10-μM Ca2+). Baseline currents were recorded 1 min after patches were excised. After recording baseline currents, vehicle control-, DTT-, or H2O2-containing solution was perfused into the bath, and posttreatment currents were recorded 10 min later. For Mg2+ experiments, currents were recorded in 1- or 10-μM Ca2+ internal bath solutions that contained either 0-, 1-, or 3-mM Mg2+. Reagents were stored at −20°C  or 4°C (H2O2) as per the manufacturers’ instructions, and all reagents were freshly diluted in the internal solution on the same day experiments were performed.For dephosphorylation experiments, calf intestinal alkaline phosphatase  was warmed to room temperature and diluted to 10 U/ml in the internal bath solution  solution contained (in mM): 134 NaCl, 6 KCl, 1 MgCl2, 10 glucose, and 10 HEPES with pH adjusted to 7.4 with NaOH. The internal (bath) solution contained: 110 K-aspartate, 10 NaCl, 30 KCl, 10 HEPES, 1 MgCl2, 5 HEDTA, and 10-μM free-Ca2+, with pH adjusted to 7.2 with KOH. To generate G–V relationships, currents were evoked from a holding potential of −100 mV, followed by 20-ms voltage steps from −150 to +150 mV (in +10-mV increments), followed by a 10-ms tail step to −150 mV. G–V analysis was performed on the tail currents, and all current kinetics were obtained as described previously.For physiological KFollowing square waveform current recordings, BK currents were recorded in response to AP waveforms. Three types of representative AP commands were used to evoke BK current: neuronal . Graphs were generated in Prism 8.0 , and statistical analysis was performed using Prism 8.0 or Origin 8.5 . One-way ANOVA with Bonferroni KCNMA1 coding sequence is comprised of 29 constitutive and eight alternative exons spanning 768 kilobases on human chromosome 10 [Zemen et al., KCNMA1 gene encoding the BK channel α subunit (gene ID: 3778) were identified from publicly available National Center for Biotechnology Information dbSNP datasets . The mutation R272Q produced a high MutPred2 score of 0.780. This mutation has been shown to enhance voltage-dependent gating and left-shifts the G–V relationship to more hyperpolarized potentials  Ca2+ concentrations compared with WT channels  to −100 mV, compared with the −200 mV leftward shift observed for WT currents. The R800W SNP, located within RCK2, eliminates a positive charge and introduces a larger, hydrophobic residue. R800W is next to G798 and N801, two residues regulating the flexible interface that governs interactions between RCK1 and RCK2  Ca2+ from BK channel constructs transfected into HEK293T cells. Since BK channels acquire tissue-specific functional diversity through alternative splicing, two different human BK splice variant backgrounds were used, hBKQEERL and hBKVYR  were most apparent in 1- and 10-μM Ca2+, where hBKQEERL values were left-shifted by −27 mV (P < 0.0001) and −18 mV (P = 0.002), respectively (QEERL currents had faster activation time constants (τ Activation), while deactivation time constants (τ Deactivation) were mostly unchanged  relationships produced by hBKch other . In symmcurrents . The difectively . In addinchanged . Thus, tQEERL variant background. BK currents from each channel variant exhibited robust activation upon membrane depolarization or elevation of intracellular Ca2+  in the G–V relationship (P = 0.03), slower activation (P = 0.002), and faster deactivation (P = 0.001) compared with WT hBKQEERL currents  and slower activation (P < 0.0001) compared with WT hBKQEERL. This suggests R800W hinders the activation of BK channels.Next, SNP substitutions A138V, C495G, N599D, and R800W were initially introduced into the hBKlar Ca2+ . The larCa2+ , suggesting C495G facilitates the opening of BK channels. C495G currents exhibited slightly faster activation (P = 0.002) and slower deactivation (P = 0.0005) at 10-μM Ca2+ compared with WT hBKQEERL currents  along with faster activation (P = 0.03) compared with WT hBKQEERL currents  compared with WT hBKQEERL currents, as well as slower activation (P = 0.0001), suggesting that N599D does not facilitate channel opening at 1-μM Ca2+. The G–V relationship and current kinetics for A138V currents at 1- and 10-μM Ca2+ were not significantly different from WT hBKQEERL currents.Unlike C495G and R800W, A138V and N599D had less consistent effects on BK currents at 1- and 10-μM Ca2+ . N599D c2+ concentrations (100 μM), the G–V relationships for A138V, C495G, N599D, and R800W currents were all significantly right-shifted to more depolarized potentials compared with WT hBKQEERL currents , C495G produced mostly increases in channel activity via left-shifts in the G–Vs at 1- and 10-μM Ca2+. Conversely, R800W could be summarized as decreasing channel activity via right-shifts of the G–Vs and slowed activation kinetics. N599D did not produce effects that could be themed as readily, with G–V shifts in both directions (1- and 100-μM Ca2+) or not at all (0- and 10-μM Ca2+). Unlike C495G, N599D, and R800W, SNP A138V produced the fewest changes in activation and kinetics across Ca2+ conditions, correlating with the lower MutPred2 score.Taken together, these results show that candidate SNPs could affect multiple aspects of BK current properties. We found that C495G and R800W had the most consistent effects on BK currents. As predicted by high MutPred2 scores, C495G, N599D, and R800W affected current activation and kinetics to varying degrees across CaVYR . Similarly, the effect of alkaline phosphatase was not precluded by R800W and still resulted in a left-shift of the G–V compared with baseline R800W currents  due to alkaline phosphatase was reduced for C495G and increased for R800W compared with WT hBKVYR currents . This suggests that at an intermediate Ca2+ concentration, the effects of two alterations within the RCK1 domain that enhance channel activation can be additive, postulating C495G could have a small potential to exacerbate the gain-of-function properties of D434G under some conditions. Similarly, R800W altered D434G activation when introduced in parallel , 1- (P < 0.0001), and 10-μM Ca2+ (P = 0.004). Interestingly, the V1/2 for D434G/R800W currents were no longer significantly different from WT hBKVYR at 0-, 1-, and 100-μM Ca2+ conditions. Thus, the effects of R800W on current properties appear to be more additive, with the net effect of abrogating the increased activation produced by D434G. These data show that SNP variation, in principle, could affect the properties of a disease-linked mutation.Next, we tested whether the C495G or R800W SNPs could influence the properties of BK channels containing the D434G mutation. C495G and R800W SNPs were each introduced in parallel with the D434G mutation within the same BK α subunit, and the resulting currents were first recorded in symmetrical K+ . D434G/Cparallel . The V1/VYR channels were recorded in physiological K+ solutions containing physiological concentrations of Na+  and K+ , while R800W currents exhibited slower activation (P < 0.0001) and faster deactivation (P = 0.0006) compared with WT currents , and deactivation was accelerated  compared with 1-min baselines, indicating both WT and C495G currents undergo a right G–V shift over time under these conditions  for WT currents, while DTT produced a decrease  and a decreased V1/2  in DTT  in DTT . These d2+  compared with WT currents at 1-μM Ca2+ . This leftward shift in the V1/2 value in the presence of 1-mM Mg2+ was larger for A138V currents than for WT currents, which eliminated the net difference between WT and A138V in 1-μM Ca2+/1-mM Mg2+ conditions , and posttranslational conditions. These data first show that a predictive approach was able to identify some functionally consequential SNP residues in the human BK channel, which would not have been possible by evaluating the limited allele frequency data currently available for these residues , which had a relatively small effect on the G–V relationship compared with mutations at E600 (mouse E535A), a neighboring residue critical for CaR800W is located near a proposed flexible interface between RCK1 and RCK2 . Mutatio2+ , suggesting introduction of SNPs produces the changes in BK current properties beyond what might be expected for YFP insertion alone. In addition, the effect of the D434G mutation has been assessed in studies employing both tagged and untagged channels. The data in our study with this mutation are consistent in direction and magnitude with previous studies using untagged BK channels, including the leftward G–V shifts of 17–48 mV  by which each SNP residue alters gating.SNPs A138V and N599D showed the most variable effects across conditions, correlated with lower MutPred scores  and loca+ with standard voltage protocols, R800W did not significantly reduce the AP-evoked current compared with WT currents. A potential explanation for this result is that the experiments were performed on the hBKVYR variant  suggesting the loss-of-function effects of R800W become more apparent on a left-shifted channel background. Consistent with this, R800W significantly reduced the increased AP-evoked currents caused by the left-shifted D434G mutation, even in the context of the hBKVYR variant . Bethesda (MD): National Center for Biotechnology Information, National Library of Medicine. dbSNP accession: rs144215383, rs201243440, rs140520584, rs199681253 and rs137853333. Available at: ML, AP and AM designed the experiments and wrote the manuscript. ML and AP performed the experiments. ML, AP, JL and AM analyzed the data.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.


Toxoplasma gondii is a ubiquitous intracellular apicomplexan parasite that can cause zoonotic toxoplasmosis. Effective vaccines against T. gondii infection are necessary to prevent and control the spread of toxoplasmosis. The present study analyzed the B-linear epitopes of T. gondii DOC2 (TgDOC2) protein and then cloned the C-terminus of the TgDOC2 gene (TgDOC2C) to construct the pVAX-TgDOC2C eukaryotic vector. After intramuscular injection of pVAX-TgDOC2C, immune responses were monitored. Two weeks after the last immunization, the protective effects of pVAX-TgDOC2C against acute and chronic toxoplasmosis were evaluated by challenges with T. gondii RH tachyzoites (genotype I) and PRU cysts (genotype II). The DNA vaccine elicited strong humoral and cellular immune responses with high levels of IgG antibody, IL-2 and IFN-γ production compared to those of the controls. The percentage of CD4+ and CD8+ T cells in mice immunized with pVAX-TgDOC2C was significantly increased compared to that of mice injected with empty pVAX I or PBS. After acute infection with 103 lethal tachyzoites, mice immunized with pVAX-TgDOC2C survived longer (12.5 days) than mice treated with pVAX I (8 days) and PBS (7.5 days). Mice immunized with pVAX-TgDOC2C had significantly less brain cysts (1600.83 ± 284.61) compared to mice immunized with pVAX I (3016.67 ± 153.84) or PBS (3100 ± 246.98). Together, these results demonstrated that TgDOC2C confers protective immunity against T. gondii infection and may be a promising candidate antigen for further development of an effective multicomponent vaccine for veterinary use against toxoplasmosis in livestock animals. Toxoplasma gondii are common in all warm-blooded animals, including birds and humans  female Kunming mice aged 6–8 weeks were obtained from the Center of Laboratory Animals, Lanzhou Institute of Biological Products . The present study was approved by the Animal Administration and Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences . All mice used for the experiments were raised and handled in strict accordance with the Good Animal Practice requirements of the Animal Ethics Procedures and Guidelines of the People’s Republic of China.T. gondii RH strain (genotype I) were maintained in the State Key Laboratory of Veterinary Etiological Biology through serial passage in Kunming mice via intraperitoneal injection. Tachyzoites were harvested from the peritoneal exudates. The obtained tachyzoites were also used for the preparation of Toxoplasma lysate antigen (TLA). The PRU cysts (genotype II) were maintained monthly through oral passage in Kunming mice, and they were purified from infected brains.Tachyzoites of the T. gondii was extracted from the tachyzoites of the RH strain using the Trizol reagent (Invitrogen) according to the manufacturer’s protocol. The TgDOC2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using the following pair of primers: C2F (5′-ATGATGAAACTAAAGGAAATGG-3′) and C2R (5′-TCAGGTGCGCCCAGCCAGATCCAGTTG-3′). The PCR product was sequenced by Sangon Biotech Co., Ltd. (China). The potential epitopes of TgDOC2 were predicted using DNAStar 8.0  software with the Jameson-Wolf index.Total RNA of GGTACCACGGAGAGAGAACGGAGAC-3′) and C2CR (5′-CCGGAATTCTCAGGTGCGCCCAGCCAGATCCAGT-3′), in which Kpn I and EcoR I restriction sites were introduced and underlined. PCR products were ligated into the pVAX I eukaryotic vector  through Kpn I/EcoR I sites to construct the recombinant pVAX-TgDOC2C plasmid. The concentration of extracted recombinant plasmids was determined by spectrophotometry at OD260 and OD280. The pVAX-TgDOC2C vector was diluted in sterile phosphate buffered saline (PBS) at a concentration of 1 μg/μl.The C-terminus of the TgDOC2 gene (TgDOC2C) was amplified by PCR from the TgDOC2 gene using a pair of primers, namely, C2CF  following the manufacturer’s instructions. Forty-eight hours posttransfection, the expression of pVAX-TgDOC2C in vitro was assayed by indirect immunofluorescence assay (IFA). In brief, pVAX-TgDOC2C-transfected cells were fixed with absolute acetone and washed with PBSTr (0.1% Triton-X-100 in PBS). Cells were then incubated with goat anti-T. gondii tachyzoites polyclonal antibody (in PBSTr at 1:50) for 1 h. After washing with PBSTr, cells were stained by fluorescein isothiocyanate (FITC)-labeled rabbit anti-goat IgG antibody  for 1 h. HEK293 cells transfected with the empty pVAX I vector were treated as the negative control.The pVAX-TgDOC2C recombinant plasmid was transfected into HEK293 cells using Lipofectamine6) were lysed by freezing and thawing five times, and the lysates were then subjected to SDS-PAGE. After electrotransfer, the nitrocellulose (NC) membrane  was incubated with 5% bovine serum albumin (BSA) in PBST (PBS with 0.05% Tween-20) at room temperature (RT) for 1 h to block the non-specific binding sites. The membrane was washed 3 times with PBST and then was cultured with pVAX-TgDOC2C-vaccinated mouse sera  at RT for 1 h. After washing 3 times with PBST, the membrane was incubated with goat anti-mouse IgG-HRP antibody  for 1 h at RT. The specific band was visualized with ClarityTM Western ECL Blotting Substrates  according to the manufacturer’s protocol. Mouse sera from PBS-treated mice were used as the negative control.The expression of pVAX-TgDOC2C in HEK293 cells was detected by Western blot analysis. Briefly, transfected HEK293 cells . The reaction was stopped after the addition of 1 M H2SO4, and the absorbance was measured at 450 nm. All samples were performed in triplicate.The levels of IgG antibodies in sera from mice in each group were determined by ELISA using the SBA Clonotyping System-HRP Kit  following the manufacturer’s protocol. In brief, 96-well microtiter plates were coated with 5 μg/ml TLA at 4°C overnight. After washing with PBST, plates were then treated with 1% skim milk in PBST for 1 h while shaking at RT to block non-specific binding sites. Serum  from each mouse was pipetted into a well and incubated for 1 h at 37°C. Each well was then incubated with 100 μl of anti-mouse IgG  for 1 h at 37°C. Peroxidase activity was revealed by incubating with 100 μl of substrate solution . Harvested splenocytes were resuspended in DMEM medium supplemented with penicillin/streptomycin (p/s) and 10% fetal bovine serum (FBS).5/well in 96-well microtiter plates. Cells were cultured in DMEM medium supplemented with 10% FBS and 100 U/ml p/s. Splenocytes were then incubated with 10 μg/ml TLA or 5 μg/ml concanavalin A  at 37°C in 5% CO2. Splenocytes cultured with medium alone (M) served as the blank control. The levels of proliferative responses in vitro were estimated using the MTS assay  after 92 h. The stimulation index (SI) was calculated using the following equation: OD570TLA/OD570M or OD570ConA/OD570M.The density of purified splenocytes was adjusted into 2 × 106 cells/ml in PBS containing 2% FBS. Staining with phycoerythrin (PE)-labeled anti-CD3 , allophycocyanin (APC)-labeled anti-CD4 (eBioscience) and fluorescein isothiocyanate (FITC)-labeled anti-CD8 (eBioscience) was performed at 4°C for 30 min in the dark. After rinsing twice with PBS, cells were fixed in FACScan solution (1% BSA and 0.1% sodium azide diluted in PBS) and 2% paraformaldehyde. The fluorescence profiles of stained samples were analyzed using a FACScan flow cytometer (BD Biosciences) using SYSTEM II software (Coulter).CD4+ and CD8+ T lymphocytes were examined according to previous methods . SplenocPurified splenocytes from mice in each group were stimulated with TLA (10 μg/ml) in 96-well flat-bottom microtiter plates. Supernatants in each well were harvested to determinate IL-4 and IL-2 levels at 24 h and IFN-γ levels at 96 h using ELISA kits according to the manufacturer’s instructions . Briefly, the supernatants and assay buffer A were pipetted into the same well, and plates were then incubated while shaking at 200 rpm for 2 h at RT. After washing the plates four times with 1× wash buffer, 100 μl of IL-4, IL-2 or IFN-γ detection solution was added into each well and incubated at RT. After 1 h, the contents of the plates were discarded, and the avidin-HRP solution was added after washing four times and incubated while shaking for 30 min at RT. The substrate solution E was then added and incubated for 20 min in the dark. To stop the reaction, 100 μl of stop solution was added. Absorbance was read by a Model 680 microplate reader  at 450 nm. Sensitivity limits for the assays were 50 pg/ml for IL-2, 20 pg/ml for IFN-γ and 10 pg/ml for IL-4. Three independent experiments were performed for analysis of the data.t-test between two groups. The difference in survival time was calculated by the Chi-square test. All figures regarding the statistical analysis were prepared by GraphPad Prism version 5.0 . A value of P < 0.05 was considered significant.The differences in IgG levels, CD4+ T cells and CD8+ T cells as well as the reduction in brain cysts were calculated by one-way ANOVA among at least three groups and by the http://toxodb.org/website. The cloned TgDOC2 gene showed 100% identity to the reference sequence (Gene ID: TGGT1_240910).The amplified TgDOC2 gene was identified through alignment with the corresponding sequence from the The potential B-linear epitopes of the TgDOC2 protein was predicted using DNAStar 8.0 software. The results showed that potential B-linear epitopes of the TgDOC2 protein were mainly gathered in the C-terminal end Figure . Thus, tin vitro was first examined by IFA. Green fluorescence was identified in HEK293 cells transfected with pVAX-TgDOC2C plasmids, but no fluorescence was observed in cells transfected with pVAX I alone  = 66.21, P < 0.0001]. However, no differences were detected in serum samples of mice from pVAX I  and PBS  groups following immunization. After the third immunization, a significantly higher level of IgG antibody was examined in the sera of mice inoculated with pVAX-TgDOC2C compared to those receiving pVAX I  and PBS   = 23.95, P < 0.0001] and ConA  compared to those from the pVAX I and PBS groups.As shown in Table F = 57.53, P < 0.0001]  = 9.3, P = 0.002]  = 52.66, P < 0.0001] and IL-2  than those in mice in the pVAX I and PBS groups (Table P > 0.05).Splenocytes from mice injected with pVAX-TgDOC2C had significantly higher levels of IFN-γ  and PBS (3100 ± 246.98) . Immunization of mice with pVAX-TgDOC2C resulted in a cyst reduction rate of 48.36% compared to that of PBS-injected mice.The protective effect of the pVAX-TgDOC2C vaccine against P = 0.0004) during acute infection and a decrease in brain cysts  during chronic infection. Significant progress has been made in recent years in the investigation of immune effects of various T. gondii antigens, such as ASP3 . In the present study, the increasing humoral immune response was associated with the enhanced protection against T. gondii infection, which was similar to the results from DNA immunization with TgROM5, TgeIF4A, TgROP18, and other antigens (The IgG antibodies against toxoplasmosis control antigens .The present study demonstrated that immunization of mice with pVAX-TgDOC2C induces a strong humoral and cellular Th1-type immune response, resulting in reduced brain cyst numbers and the extent of survival time. The vaccine potential of pVAX-TgDOC2C indicates the possibility of inclusion in further development of a multicomponent or an epitope-based vaccine against toxoplasmosis in food-producing animals.The datasets supporting the conclusions of this article are included within the article.X-QZ and N-ZZ conceived and designed the experiments. QG, MW, F-KZ, and L-YH performed the experiments. N-ZZ and J-LH analyzed the data and wrote the paper. N-ZZ, QG, and X-QZ critically revised the manuscript. All authors read and approved the final version of the manuscript.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.


There are no FDA licensed vaccines or therapeutics for Venezuelan equine encephalitis virus (VEEV) which causes a debilitating acute febrile illness in humans that can progress to encephalitis. Previous studies demonstrated that murine and macaque monoclonal antibodies (mAbs) provide prophylactic and therapeutic efficacy against VEEV peripheral and aerosol challenge in mice. Additionally, humanized versions of two neutralizing mAbs specific for the E2 glycoprotein, 1A3B-7 and 1A4A-1, administered singly protected mice against aerosolized VEEV. However, no studies have demonstrated protection in nonhuman primate (NHP) models of VEEV infection. Here, we evaluated a chimeric antibody 1A3B-7 (c1A3B-7) containing mouse variable regions on a human IgG framework and a humanized antibody 1A4A-1 containing a serum half-life extension modification (Hu-1A4A-1-YTE) for their post-exposure efficacy in NHPs exposed to aerosolized VEEV. Approximately 24 hours after exposure, NHPs were administered a single bolus intravenous mAb. Control NHPs had typical biomarkers of VEEV infection including measurable viremia, fever, and lymphopenia. In contrast, c1A3B-7 treated NHPs had significant reductions in viremia and lymphopenia and on average approximately 50% reduction in fever. Although not statistically significant, Hu-1A4A-1-YTE administration did result in reductions in viremia and fever duration. Delay of treatment with c1A3B-7 to 48 hours post-exposure still provided NHPs protection from severe VEE disease through reductions in viremia and fever. These results demonstrate that post-exposure administration of c1A3B-7 protected macaques from development of severe VEE disease even when administered 48 hours following aerosol exposure and describe the first evaluations of VEEV-specific mAbs for post-exposure prophylactic use in NHPs. Viral mutations were identified in one NHP after c1A3B-7 treatment administered 24 hrs after virus exposure. This suggests that a cocktail-based therapy, or an alternative mAb against an epitope that cannot mutate without resulting in loss of viral fitness may be necessary for a highly effective therapeutic. in vitro virus neutralization by a given antibody did not predict efficacy in NHPs. Importantly, we identified viral escape mutations in one NHP after treatment, highlighting the need for development of novel antibodies for inclusion in cocktail-based therapy against VEEV.Endemic in the Americas, Venezuelan equine encephalitis virus (VEEV) can be transmitted to humans, horses, and other animals through the bite of a mosquito. Beyond its natural prevalence, VEEV was previously developed as a biological weapon making the development of vaccines and therapeutics of the upmost importance. Despite over 60 years of research to identify effective therapeutics for VEEV disease, to-date no anti-VEEV therapeutics have progressed beyond pre-clinical testing in a mouse model. Here, we present the first evaluation of an anti-VEEV therapeutic in a nonhuman primate (NHP). We found that a monoclonal antibody given either one or two days after an aerosol exposure to VEEV protected from severe VEE disease. We also found the level of  Togaviridae family, Venezuelan equine encephalitis virus (VEEV), is one of the most extensively studied alphaviruses due to its historical production as a biological agent by multiple State actors , 24N. betry BLI; . Howevertry BLI; . Compariin vitro . Since tThe half-life of chimeric and human(ized) mAbs administered to NHP by the intravenous (IV) route is varied in the literature, but is generally between 7–11 days . In a ph5 PFU VEEV TrD in Experiment 1  and a target inhaled dose of 1.0x106 PFU VEEV TrD in Experiment 2  by the aerosol route. The target inhaled exposure dose was intentionally increased by a log in Experiment 2 based on the results from Experiment 1. Twenty-four hours (±1 hr) after exposure, cohorts were treated IV with 25 mg/kg c1A3B-7 , Hu-1A4A-1-YTE  or equal volume by weight vehicle control . Blood was collected daily through day 7 and weekly thereafter until day 28. Based on markers of VEEV disease defined in NHP vaccine studies  * MV * Run Time, where aerosol concentration is the amount of virus (PFU/mL * volume in AGI sampler /sampler flow rate (mL/min) * sampling duration (min); minute volume (mL/min) and run time (min) is the total time of exposure and air wash.Nicotiana benthamiana plants and purified over a 3-column  process. The Hu-1A4A-1-N was modified to include half-life extension substitutions, specifically YTE /Baseline. Presence of lymphopenia was designated as 1 when percent change for lymphocytes was less than or equal to -30% and as 0 when not present. Neutropenia was calculated in a similar fashion. NHP level summary measures  were calS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 FigBALB/c mice were exposed to 100 PFU (A & C) or 10,000 PFU (B & D) of VEEV TrD by the subcutaneous route and then administered PBS daily by intraperitoneal route for either 5 or 9 days, as indicated, or left untreated. Average weight (A & B) and survival (C & D) were monitored.(TIF)Click here for additional data file.


Findings from the community-based Canadian Sentinel Practitioner Surveillance Network (SPSN) suggest children were more affected by the 2018/19 influenza A(H1N1)pdm09 epidemic.To compare the age distribution of A(H1N1)pdm09 cases in 2018/19 to prior seasonal influenza epidemics in Canada.The age distribution of unvaccinated influenza A(H1N1)pdm09 cases and test-negative controls were compared across A(H1N1)pdm09-dominant epidemics in 2018/19, 2015/16 and 2013/14 and with the general population of SPSN provinces. Similar comparisons were undertaken for influenza A(H3N2)-dominant epidemics.In 2018/19, more influenza A(H1N1)pdm09 cases were under 10 years old than controls . In particular, children aged 5–9 years comprised 14% of cases, greater than their contribution to controls (4%) or the general population (5%) and at least twice their contribution in 2015/16  or 2013/14 . Conversely, children aged 10–19 years (11% of the population) were under-represented among A(H1N1)pdm09 cases versus controls in 2018/19 , 2015/16  and 2013/14 .Children under 10 years old contributed more to outpatient A(H1N1)pdm09 medical visits in 2018/19 than prior seasonal epidemics in Canada. In 2018/19, all children under 10 years old were born after the 2009 A(H1N1)pdm09 pandemic and therefore lacked pandemic-induced immunity. In addition, more than half those born after 2009 now attend school (i.e. 5–9-year-olds), a socio-behavioural context that may enhance transmission and did not apply during prior A(H1N1)pdm09 epidemics. The 2018/19 seasonal influenza epidemic in the northern hemisphere has primarily been due to influenza A viruses with fewer influenza B detections than usual . In CanaThe 2018/19 influenza A(H1N1)pdm09 epidemic in Canada began early in week 43, with paediatric hospitalisations above the seasonal norm as early as week 45 and national surveillance indicators showing the epidemic peak during week 52 2018 . SubstanThe Canadian SPSN interim report, which captured cases as at week 2 2019, suggested that children aged less than 10 years were disproportionately affected by the 2018/19 influenza A(H1N1)pdm09 epidemic [As described in prior SPSN publications -10, nasaSupplementary Figure S1). Cases tested positive for the dominant influenza A subtype of a given season; controls tested negative for any influenza virus. Patients without available influenza A subtype information were excluded. To assess age distribution in the absence of vaccine effects, analyses were restricted to unvaccinated patients aged 1 year or older. Unvaccinated patients were those self-reporting no influenza vaccine receipt (or as reported by their parent/guardian) in the season during which their specimen was collected.The current study is restricted to specimens collected from patients presenting within 7 days of ILI onset between 1 November and 30 April each seasonal influenza epidemic, except 2018/19 for which collection dates spanned from 1 November to specimens available as at 18 March 2019 -10 . During the study period the protocol was approved by the following ethics review committees in participating provinces: University of Calgary, Calgary, Alberta; University of British Columbia, Vancouver, British Columbia; University of Toronto (2013/14–2016/17), University Health Network (2013/14–2016/17), and Public Health Ontario (2017/18–2018/19), Toronto, Ontario; and Comité d’éthique de santé publique, Québec.The age distribution of unvaccinated influenza A(H1N1)pdm09 cases and unvaccinated controls in 2018/19 was primarily compared with prior influenza A(H1N1)pdm09-dominant seasonal epidemics (i.e. 2013/14 and 2015/16) -9. Age-r(i) The percentage of ILI specimens testing positive for the specified influenza A subtype, by age group. Age groups included: children aged 1–4 years; 5–9 years and 10–19 years; and adults aged 20–49 years; 50–64 years; and 65 years and older (elderly adults). Percentage positivity estimates were accompanied by 95% confidence intervals (CIs) and compared by chi-squared analysis.(ii) The percentage distribution of unvaccinated cases and controls by single year of age. Percentage histograms displayed the percentage of all cases (of the specified influenza A subtype) that belonged to a given age in years such that percentages sum 100% across the age range each season. The percentage distribution of influenza test-negative controls was superimposed upon the same season-specific plots to standardise for potential age-related sampling variation. The median age of cases and controls was compared within and across seasons by the Wilcoxon rank-sum test.Supplementary Table S1) [(iii) The percentage distribution of unvaccinated cases and controls by age group. Proportions were accompanied by 95% CIs and compared by chi-squared analysis. Case/control distributions by age group were also compared with the general population of SPSN provinces (2018 data) (able S1) .In Higher influenza A(H1N1)pdm09 test-positivity was also observed among children aged 1-4 years in 2018/19 compared to 2015/16 and 2013/14  and among adults 20-49 years old , but these differences were less striking. Other age groups showed no significant differences across these seasons.The next highest percentage influenza A positivity (54%) also involved children aged 5–9 years but during the influenza A(H3N2) epidemic in 2016/17 (p = 0.002 compared with 2018/19). Children aged 10–19 years also had higher influenza A(H3N2) positivity rates in 2016/17 (47%), greater than for any other season for that age group (36% in 2014/15 (p = 0.03); p < 0.005 any other seasons’ comparisons). Among those aged 65 years and older, influenza test-positivity was also generally higher during A(H3N2) vs A(H1N1)pdm09 epidemics, marginally significant in comparing positivity across combined A(H3N2) vs A(H1N1)pdm09 epidemics  and between 2016/17 vs 2013/14  .As shown in Comparing the age distributions of cases and controls across seasons (not shown) the median age of A(H1N1)pdm09 cases in 2018/19 (31 years) was lower than for cases in 2015/16 and 2013/14 ; whereas, the median age of controls did not significantly differ (or was higher) in 2018/19 (34 years) compared to controls in 2015/16 and 2013/14 . Comparing the age distributions of cases versus controls within the same seasons (as shown in Influenza A(H3N2) cases were more evenly distributed across the age range . In 2017The median age of A(H3N2) cases did not significantly differ from controls in any season  [In 2018/19, children less than 10 years old comprised a greater proportion of influenza A(H1N1)pdm09 cases than controls , greater also than their share of the general population of SPSN provinces (10%) or their contribution to cases in 2015/16  or 2013/14 . (Supplementary Table S1) [The proportion of A(H1N1)pdm09 cases that were children aged 1–4 years increased from 7% in 2013/14 and 10% in 2015/16 to 15% in 2018/19  , Table 2Supplementary Table S1) [The proportion of A(H1N1)pdm09 cases that were children aged 5–9 years also increased from 5% in 2013/14 and 7% in 2015/16 to 14% in 2018/19 (p < 0.001 comparing both prior epidemics to 2018/19) , Table 2Supplementary Table S1) [Supplementary Table S1) [The case contribution by other age groups in 2018/19 was more comparable to, or below that of, their contribution to controls or to cases and controls during prior seasons . In partable S1) . Childreable S1) .Findings across successive seasons of influenza monitoring by the Canadian SPSN show greater involvement of children aged less than 10 years among outpatient medical visits during the 2018/19 influenza A(H1N1)pdm09 epidemic. In particular, children aged 5–9 years were disproportionately affected, comprising 14% of all cases in 2018/19 which is at least twice that of their contribution during prior A(H1N1)pdm09 epidemics in 2013/14 (5%) and 2015/16 (7%). Over-sampling of children 5–9 years old during the 2018/19 season is unlikely to explain these observations given their contribution to influenza test-negative controls (4%), commensurate with the general population of SPSN provinces (5%) [Moving cohort effects can be followed in relation to major immunological priming events, such as pandemics, to anticipate relative age-related susceptibility during subsequent epidemics. In that regard, our observations in 2018/19 may have an immuno-epidemiological explanation. Infection rates during the 2009 pandemic were very high, especially in children (ca 47% among those aged 5–19 years), resulting in high levels of infection-induced immunity ,16. SubsFurthermore, in 2018/19, more than half those born after the 2009 pandemic now attend school (i.e. aged 5–9 years)—a context that did not apply in previous A(H1N1)pdm09 seasonal epidemics and may have enhanced transmission. School children are efficient propagators of infections transmitted by the respiratory or close contact route [Most contacts of children are of a similar age, but also include siblings and parents and these contacts tend to be of long duration ,19. The Immunological cohort effects may also be expressed in more complex ways across the life span. Priming to the particular influenza viruses of first childhood exposure may leave lasting immunological imprints that can negatively or positively affect responses during subsequent influenza virus encounters -23. SuchThe SPSN places no age restrictions on influenza testing and includes patients meeting the same ILI testing indication. No relevant system changes were introduced during the study period to account for the age-related shift in influenza A(H1N1)pdm09 cases we observed. Although particular diagnostic assays varied, all test sites are accredited clinical and public health diagnostic and reference laboratories subject to regular proficiency testing programs, conducted at least thrice annually. Either way, assay changes were not applied differentially by age to SPSN specimens. Although children may shed more virus and/or for longer periods ,27, thisSupplementary Figure S2 and Supplementary Figure S3). This is not unexpected since analyses overall remain driven by the majority of SPSN participants (about two-thirds) [To assess age distribution in the absence of vaccine effects, which vary by age, subtype and season -10,28, w-thirds) -10, who -thirds) . InclusiLess than 25% of SPSN participants aged 1–9 years received influenza vaccine each season , includiOur findings only include patients seeking outpatient care; our system cannot assess patterns among inpatients or those not seeking medical attention. Differences in healthcare seeking or testing behaviours by age and/or epidemic may skew surveillance trends based upon case detection alone. To account for possible age-related sampling variation, we assessed trends among influenza test-negative controls identified according to the same study protocol as for cases, recognising, however, that non-influenza causes of ILI may separately vary by age and season. School-aged children 5–19 years old may have higher influenza attack rates ,17, but Limitations of our study include that we used data available season-to-date for 2018/19 (cases spanning to mid-March 2019) but for other years spanning to end-of-season (late April). The timing, peak and mix of influenza and non-influenza causes of ILI may vary each season and their distribution by age may differ toward seasons’ end. This may be especially relevant to the current study given that the involvement of children can be most pronounced during the initial phases of an influenza epidemic [Children less than 10 years old, notably children aged 5–9 years, contributed more to medically-attended cases of influenza A(H1N1)pdm09 illness in Canada in 2018/19 compared to prior seasonal influenza epidemics. Age-related differences likely reflect a combination of immuno-epidemiologic and socio-behavioural factors. Over the next decade, children born after 2009 and lacking pandemic-induced immunity to A(H1N1)pdm09 viruses will be entering the pre-teen and teenage period (10-19 years) associated with the highest social contact rates. The implication for greater transmission during subsequent influenza A(H1N1)pdm09 epidemics warrants ongoing monitoring.