E2 Residues Research Articles

Characterization of 17β-estradiol-degrading enzyme from Microbacterium sp. MZT7 and its function on E2 biodegradation in wastewater

Background17β-estradiol (E2) residues exhibit harmful effects both for human and animals and have got global attention of the scientific community. Microbial enzymes are considered as one of the effective strategies having great potential for removal E2 residues from the environment. However, limited literature is available on the removal of E2 from wastewater using short-chain dehydrogenase.ResultsIn this study, 17β-estradiol degrading enzyme (17β-HSD-0095) was expressed and purified from Microbacterium sp. MZT7. The optimal pH and temperature for reaction was 7 and 40 °C, respectively. Molecular docking studies have shown that the ARG215 residue form a hydrogen bond with oxygen atom of the substrate E2. Likewise, the point mutation results have revealed that the ARG215 residue play an important role in the E2 degradation by 17β-HSD-0095. In addition, 17β-HSD-0095 could remediate E2 contamination in synthetic livestock wastewater.ConclusionsThese findings offer some fresh perspectives on the molecular process of E2 degradation and the creation of enzyme preparations that can degrade E2.Graphical

Classical Swine Fever Virus Structural Glycoprotein E2 Interacts with Host Protein ACADM during the Virus Infectious Cycle.

The E2 glycoprotein is one of the four structural proteins of the classical swine fever virus (CSFV) particle. E2 has been shown to be involved in many virus functions, including adsorption to host cells, virus virulence and interaction with several host proteins. Using a yeast two-hybrid screen, we have previously shown that the CSFV E2 specifically interacts with swine host protein medium-chain-specific acyl-Coenzyme A dehydrogenase (ACADM), an enzyme that catalyzes the initial step of the mitochondrial fatty acid beta-oxidation pathway. Here, we show that interaction between ACADM and E2 also happens in swine cells infected with CSFV using two different procedures: coimmunoprecipitation and a proximity ligation assay (PLA). In addition, the amino acid residues in E2 critically mediating the interaction with ACADM, M49 and P130 were identified via a reverse yeast two-hybrid screen using an expression library composed of randomly mutated versions of E2. A recombinant CSFV, E2ΔACADMv, harboring substitutions at residues M49I and P130Q in E2, was developed via reverse genomics from the highly virulent Brescia isolate. E2ΔACADMv was shown to have the same kinetics growth in swine primary macrophages and SK6 cell cultures as the parental Brescia strain. Similarly, E2ΔACADMv demonstrated a similar level of virulence when inoculated to domestic pigs as the parental Brescia. Animals intranasally inoculated with 105 TCID50 developed a lethal form of clinical disease with virological and hematological kinetics changes undistinguishable from those produced by the parental strain. Therefore, interaction between CSFV E2 and host ACADM is not critically involved in the processes of virus replication and disease production.

Role of the conserved E2 residue G259 in classical swine fever virus production and replication

The E2 glycoprotein of classical swine fever virus (CSFV) plays multiple roles in the viral life cycle. The chimeric live attenuated C strain with the E2 substitution of bovine viral diarrhea virus (BVDV) is a promising marker vaccine candidate. In this study, the recombinant chimeric CSFV/bE2 cDNA clone harboring heterologous E2 (bE2) of BVDV was constructed by genetic approaches. Recombinant infectious virus rCSFV/bE2 (P11) was recovered by 11 serial passages of transfected PK15 cells. Viral genome sequencing showed that a glutamic acid to glycine mutation (E260G) at position 260 of the bE2 was observed in rCSFV/bE2 P11. Alignment of amino acid sequences displayed that the glycine was one of three conserved residues in pestivirus E2. When the glutamic acid to glycine substitution (E260G) was introduced into chimeric CSFV/bE2 cDNA clone, the high-titer infectious rCSFV/bE2E260G was rescued. The glycine to glutamic acid substitution at corresponding position in CSFV E2 resulted in significantly decreased rCSFV/E2G259E production. We further identified that the conserved E2 residue G259 played a critical role in the release and binding activity of CSFV and that the E2 residues G259 and V111 modulated synergistically infectious virus production and replication.

Antigenic and immunogenic evaluation of permutations of soluble hepatitis C virus envelope protein E2 and E1 antigens.

Yearly, about 1.5 million people become chronically infected with hepatitis C virus (HCV) and for the 71 million with chronic HCV infection about 400,000 die from related morbidities, including liver cirrhosis and cancer. Effective treatments exist, but challenges including cost-of-treatment and wide-spread undiagnosed infection, necessitates the development of vaccines. Vaccines should induce neutralizing antibodies (NAbs) against the HCV envelope (E) transmembrane glycoprotein 2, E2, which partly depends on its interaction partner, E1, for folding. Here, we generated three soluble HCV envelope protein antigens with the transmembrane regions deleted (i.e., fused peptide backbones), termed sE1E2 (E1 followed by E2), sE2E1 (E2 followed by E1), and sE21E (E2 followed by inverted E1). The E1 inversion for sE21E positions C-terminal residues of E1 near C-terminal residues of E2, which is in analogy to how they likely interact in native E1/E2 complexes. Probing conformational E2 epitope binding using HCV patient-derived human monoclonal antibodies, we show that sE21E was superior to sE2E1, which was consistently superior to sE1E2. This correlated with improved induction of NAbs by sE21E compared with sE2E1 and especially compared with sE1E2 in female BALB/c mouse immunizations. The deletion of the 27 N-terminal amino acids of E2, termed hypervariable region 1 (HVR1), conferred slight increases in antigenicity for sE2E1 and sE21E, but severely impaired induction of antibodies able to neutralize in vitro viruses retaining HVR1. Finally, comparing sE21E with sE2 in mouse immunizations, we show similar induction of heterologous NAbs. In summary, we find that C-terminal E2 fusion of E1 or 1E is superior to N-terminal fusion, both in terms of antigenicity and the induction of heterologous NAbs. This has relevance when designing HCV E1E2 vaccine antigens.

A β-Hairpin Motif in the Envelope Protein E2 Mediates Receptor Binding of Bovine Viral Diarrhea Virus.

Pestivirus envelope protein E2 is crucial to virus infection and accomplishes virus-receptor interaction during entry. However, mapping of E2 residues mediating these interactions has remained unexplored. In this study, to investigate the structure-function relationship for a β-hairpin motif exposed to the solvent in the crystal structure of bovine viral diarrhea virus (BVDV) E2, we designed two amino acidic substitutions that result in a change of electrostatic potential. First, using wild type and mutant E2 expressed as soluble recombinant proteins, we found that the mutant protein had reduced binding to susceptible cells compared to wild type and diminished ability to inhibit BVDV infection, suggesting a lower affinity for BVDV receptors. We then analyzed the effect of β-hairpin mutations in the context of recombinant viral particles. Mutant viruses recovered from cell culture supernatant after transfection of recombinant RNA had almost completely inhibited ability to re-infect susceptible cells, indicating an impact of mutations on BVDV infectivity. Finally, sequential passaging of the mutant virus resulted in the selection of a viral population in which β-hairpin mutations reverted to the wild type sequence to restore infectivity. Taken together, our results show that this conserved region of the E2 protein is critical for the interaction with host cell receptors.

Cryo-EM structure of the mature and infective Mayaro virus at 4.4\u2009\xc5 resolution reveals features of arthritogenic alphaviruses

Mayaro virus (MAYV) is an emerging arbovirus of the Americas that may cause a debilitating arthritogenic disease. The biology of MAYV is not fully understood and largely inferred from related arthritogenic alphaviruses. Here, we present the structure of MAYV at 4.4 Å resolution, obtained from a preparation of mature, infective virions. MAYV presents typical alphavirus features and organization. Interactions between viral proteins that lead to particle formation are described together with a hydrophobic pocket formed between E1 and E2 spike proteins and conformational epitopes specific of MAYV. We also describe MAYV glycosylation residues in E1 and E2 that may affect MXRA8 host receptor binding, and a molecular “handshake” between MAYV spikes formed by N262 glycosylation in adjacent E2 proteins. The structure of MAYV is suggestive of structural and functional complexity among alphaviruses, which may be targeted for specificity or antiviral activity.

Amelioration of Beta Interferon Inhibition by NS4B Contributes to Attenuating Tembusu Virus Virulence in Ducks.

Our previous studies reported that duck Tembusu virus nonstructural protein 2A (NS2A) is a major inhibitor of the IFNβ signaling pathway through competitively binding to STING with TBK1, leading to a reduction in TBK1 phosphorylation. Duck TMUV NS2B3 could cleave and bind STING to subvert the IFNβ signaling pathway. Here, we found that overexpression of duck TMUV NS4B could compete with TBK1 in binding to STING, reducing TBK1 phosphorylation and inhibiting the IFNβ signaling pathway by using the Dual-Glo® Luciferase Assay System and the NanoBiT protein-protein interaction (PPI) assay. We further identified the E2, M3, G4, W5, K10 and D34 residues in NS4B that were important for its interaction with STING and its inhibition of IFNβ induction, which were subsequently introduced into a duck TMUV replicon and an infectious cDNA clone. We found that the NS4B M3A mutant enhanced RNA replication and exhibited significantly higher titer levels than WT at 48-72 hpi but significantly decreased mortality (80%) in duck embryos compared to WT (100%); the NS4B G4A and R36A mutants slightly reduced RNA replication but exhibited the same titer levels as WT. However, the NS4B R36A mutant did not attenuate the virulence in duck embryos, whereas the G4A mutant significantly decreased the mortality (70%) of duck embryos. In addition, the NS4B W5A mutant did not affect viral replication, whereas the D34A mutant slightly reduced RNA replication, and both mutants exhibited significantly lower titer levels than the WT and significantly decreased mortality (90% and 70%, respectively) in duck embryos. Hence, our findings provide new insight into the development of attenuated flaviviruses by targeting the disabling viral strategies used to evade the innate defense mechanisms.

Development of a structural epitope mimic: an idiotypic approach to HCV vaccine design

HCV vaccine development is stymied by the high genetic diversity of the virus and the variability of the envelope glycoproteins. One strategy to overcome this is to identify conserved, functionally important regions—such as the epitopes of broadly neutralizing antibodies (bNAbs)—and use these as a basis for structure-based vaccine design. Here, we report an anti-idiotype approach that has generated an antibody that mimics a highly conserved neutralizing epitope on HCV E2. Crucially, a mutagenesis screen was used to identify the antibody, designated B2.1 A, whose binding characteristics to the bNAb AP33 closely resemble those of the original antigen. Protein crystallography confirmed that B2.1 A is a structural mimic of the AP33 epitope. When used as an immunogen B2.1 A induced antibodies that recognized the same epitope and E2 residues as AP33 and most importantly protected against HCV challenge in a mouse model.

The molecular basis of OH-PCB estrogen receptor activation

Polychlorinated bisphenols (PCBs) continue to contaminate food chains globally where they concentrate in tissues and disrupt the endocrine systems of species throughout the ecosphere. Hydroxylated PCBs (OH-PCBs) are major PCB metabolites and high-affinity inhibitors of human estrogen sulfotransferase (SULT1E1), which sulfonates estrogens and thus prevents them from binding to and activating their receptors. OH-PCB inhibition of SULT1E1 is believed to contribute significantly to PCB-based endocrine disruption. Here, for the first time, the molecular basis of OH-PCB inhibition of SULT1E1 is revealed in a structure of SULT1E1 in complex with OH-PCB1 (4ʹ-OH-2,6-dichlorobiphenol) and its substrates, estradiol (E2), and PAP (3’-phosphoadenosine-5-phosphosulfate). OH-PCB1 prevents catalysis by intercalating between E2 and catalytic residues and establishes a new E2-binding site whose E2 affinity and positioning are greater than and competitive with those of the reactive-binding pocket. Such complexes have not been observed previously and offer a novel template for the design of high-affinity inhibitors. Mutating residues in direct contact with OH-PCB weaken its affinity without compromising the enzyme’s catalytic parameters. These OH-PCB resistant mutants were used in stable transfectant studies to demonstrate that OH-PCBs regulate estrogen receptors in cultured human cell lines by binding the OH-PCB binding pocket of SULT1E1.

Enhanced dissipation of trace level organic contaminants by floating treatment wetlands established with two macrophyte species: A mesocosm study

This study evaluated removal efficiencies of six contaminants of emerging concern (CECs) in floating treatment wetland (FTW) mesocosms established with either Japanese Sweetflag (Acorus gramineus Sol. ex Aiton) or canna lilies (Canna Hybrida L. ‘Orange King Humbert’). The CECs included: acetaminophen (APAP), atrazine (ATZ), carbamazepine (CBZ), perfluorooctanoic acid (PFOA), sulfamethoxazole (SMX), and 17β-estradiol (E2). Each treatment was planted with different numbers of plants (i.e., 0, 10, 15, and 20), and the experiments lasted for 17 weeks. Dissipation of CECs was greater in planted treatments than in non-planted controls, and the planting number had little effect on dissipation of CECs. All residues of APAP and E2 dissipated rapidly within 2 weeks in all planted treatments. At the end of the experiment, residues of ATZ and SMX completely dissipated in the canna treatments, but not in the sweetflag treatments (75.8–87.6% and 96.3–97.1%, respectively). During the 17 week study, moderate dissipation of CBZ was observed in treatments including cannas (79.5–82.6%) and sweetflag (69.4–82.3%), while less dissipation was observed for PFOA (9.0–15.0% with sweetflag and 58.4–62.3% with cannas). Principal component analysis indicates that aqueous persistency of CECs and species of plants used influenced the dissipation of CECs in FTWs. Of the two species evaluated, canna was the most promising plant species for FTW systems designed to remove these CECs from surface water.

Identification of specific amino acid residues in the border disease virus glycoprotein E2 that modify virus growth in pig cells but not in sheep cells.

Border disease virus (BDV) envelope glycoprotein E2 is required for entry into cells and is a determinant of host tropism for sheep and pig cells. Here, we describe adaptive changes in the BDV E2 protein that modify virus replication in pig cells. To achieve this, two BDV isolates, initially collected from a pig and a sheep on the same farm, were passaged in primary sheep and pig cells in parallel with a rescued variant of the pig virus derived from a cloned full-length BDV cDNA. The pig isolate and the rescued virus shared the same amino acid sequence, but the sheep isolate differed at ten residues, including two substitutions in E2 (K771E and Y925H). During serial passage in cells, the viruses displayed clear selectivity for growth in sheep cells; only the cDNA-derived virus adapted to grow in pig cells. Sequencing revealed an amino acid substitution (Q739R) in the E2 domain DA of this rescued virus. Adaptation at the same residue (Q739K/Q739R) was also observed after passaging of the pig isolate in sheep cells. Use of reverse genetics confirmed that changing residue Q739 to R or K (each positively charged) was sufficient to achieve adaptation to pig cells. Furthermore, this change in host tropism was suppressed if Q739R was combined with K771E. Another substitution (Q728R), conferring an additional positive charge, acquired during passaging, restored the growth of the Q739R/K771E variant. Overall, this study provided evidence that specific, positively charged, residues in the E2 domain DA are crucial for pig-cell tropism of BDV.

Development and validation of an analytical method for the determination of 17β-estradiol residues in muscle of tambaqui (Colossoma macropomum Cuvier, 1818) by LC-MS/MS and its application in samples from a fish sexual reversion study

Tambaqui (Colossoma macropomum Cuvier, 1818) is the main native fish species farmed in Brazil, and 17β-estradiol (E2) is a natural steroid hormone commonly used for the production of female fish monosex population, which, in tambaqui, shows a higher growth rate than the male. Thus, to assess whether the fish meat of treated tambaqui contains hormonal residue levels, a high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS) method for the determination of E2 residues in fish muscle was developed and validated. A QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) dispersive solid phase extraction method was used for the sample preparation. The chromatographic separation was performed in a Poroshel EC-18 reverse phase column. The mobile phase was a mixture of acetonitrile with 0.01% ammonium hydroxide (A) and water with 0.01% ammonium hydroxide (B). The ratio of A:B phases was 60:40 (v/v) used in an isocratic mode. The method validation was performed according to Commission Decision 2002/657/EC and Veterinary International Conference Harmonization (VICH GL49). Since matrix effects were observed, matrix-matched analytical curves are recommended for quantitation. The linearity, selectivity, intraday and interday precision, accuracy, decision limit, detection capability, and detection and quantitation limits of the method are reported. The limits of detection and quantitation were 0.3 ng/g and 1.0 ng/g, respectively. At these limits and slaughtering fish 7 months after the end of the treatment, the muscle of tambaqui did not show detectable hormone residue level. Thus, consumption of tambaqui edible tissue from fish treated with E2 for the purpose of sexual reversion is unlikely to represent a risk associated with the exposure of human subjects as residue levels of this hormone are not detected in the fish muscle.

Effect of Biochar on 17β-Estradiol Degradation in Composted Poultry Manure: Residue and Bioassay Analysis

While composting is normally a valid approach to decrease 17β-estradiol (E2) contents of manure prior to land application, E2 residues in compost pose a risk to the environment. Biochar is a useful material in composting due to its microstructure. The study aims to compare the effect of amending poultry manure (PM) with two different biochars at three levels on E2 degradation. The seven treatments consisted of compost with no biochar (control) and compost with 5%, 10%, or 15% wheat stalk biochar (WSB) or rice husk biochar (RHB). The results indicated composting with WSB and RHB reduced E2 extractable contents by 71.0–78.5% and 74.2–88.1%, versus 67.8% without biochar. The abundance of two bacteria capable of E2 degradation: Bacillus licheniformis and Bacillus subtilis, increased significantly (p < 0.05) over control in biochar-amended treatments. Moreover, the estrogenic activity (assessed by proliferative assay of MCF-7 cells) of the compost was diminished by the addition of biochar. Our results suggest that E2 degradation was accelerated by adding RHB, possibly by RHB’s provision of a better microenvironment due to larger surface area and pore volume than WSB. Thus, the addition of 15% RHB is recommended for the treatment of PM compost to enhance E2 degradation.

Occurrence, seasonal variation and risk evaluation of selected endocrine disrupting compounds and their transformation products in Jiulong river and estuary, China

In the present study, 13 endocrine disrupting compounds (EDCs) and their transformation products (TPs) were monitored in Jiulong River and its estuary, China in different seasons. The analytes included antimicrobials [triclosan (TCS) and triclocarbon (TCC)]; estrogens [estrone (E1), estradiol (E2), estriol (E3) and ethinylestradiol (EE2)]; alkylphenol ethoxylates [4-n-nonylphenol (NP) and 4-n-octylphenol (OP)] and the TPs [methyl triclosan (MeTCS), carbanilide (NCC), dichlorocarbanilide, 4-hydroxy estrone (4-OH E1) and 4-hydroxy estradiol (4-OH E2)]. A significant seasonal variation was observed for most EDCs. Approximately, 79% of the total E2 residues were detected in the normal season in comparison with the wet season to indicate recalcitrant behavior of E2 during the normal season. Risk assessment revealed that E2 was most potent among the EDCs to cause highest risk to both vertebrate and invertebrate aquatic species, whereas, E1, EE2, MeTCS and TCS also showed relatively high risk for some surface water aquatic species.

Differential interaction strategies of hepatitis c virus genotypes during entry – An in silico investigation of envelope glycoprotein E2 - CD81 interaction

Hepatitis C Virus is a blood borne pathogen responsible for chronic hepatitis in more than 71 million people. Wide variations across strains and genotypes are one of the major hurdles in therapeutic development. While genotype 1 remains the most extensively studied and abundant strain, genotype 3 is more virulent and second most prevalent. This study aimed to compare differences in the glycoprotein E2 across HCV genotypes at nucleotide, protein and structural levels. Nucleotide sequences of E2 from 29 strains across genotypes 1a, 1b, 3a and 3b revealed a stark preference for C-richness which was attributed to a distinct bias for C-rich codons in genotype 1. Genotype 3 exhibited a similar preference to a lesser extent. Amino acid level comparison revealed majority of the changes at the C-terminal half of the proteins leaving the N-terminal region conspicuously conserved apart from the two hyper variable regions. Amino acid changes across genotypes were mostly polar-nonpolar alterations. In silico models of E2 glycoproteins and docking analysis with the energy minimized PDB-CD81 model revealed unique interacting residues in both E2 and CD81. While several CD81 binding residues were common for all four genotypes, number and composition of interacting residues varied. The interacting residues of E2 were however unique for each genotype. E2 of genotype 3a and CD81 had the strongest interaction. In conclusion this is the first comprehensive study comparing E2 sequences across genotypes 1a, 1b, 3a and 3b revealing stark genotype-specific differences which requires more extensive investigation.

Identification of structural glycoprotein E2 domain critical to mediate replication of Classical Swine Fever Virus in SK6 cells

Envelope glycoprotein E2 of Classical Swine Fever Virus (CSFV) is involved in several critical virus functions. To analyze the role of E2 in virus replication, a series of recombinant CSFVs harboring chimeric forms of E2 CSFV and Bovine viral diarrhea virus (BVDV) were created and tested for their ability to infect swine or bovine cell lines. Substitution of native CSFV E2 by BVDV E2 abrogates virus replication in both cell lines. Substitution of individual domains in CSFV Brescia E2 by the homologous from BVDV produces chimeras that efficiently replicate in SK6 cells with the exception of a chimera harboring BVDV E2 residues 93–168. Further mapping revealed a critical area in E2 required for CSFV replication in SK6 cells between protein residues 136–156. This is the first report categorically defining a discrete portion of E2 as essential to pestivirus infection in susceptible cells.

17β-Estradiol residues and estrogenic activities in the Hawkesbury River, Australia

Two highly sensitive ELISAs for the specific detection of 17β-estradiol (E2) residues were developed, showing the limits of detection (LOD, a concentration at 15% inhibition of color development) of 0.04 ± 0.02 μg/L and 0.05 ± 0.03 μg/L. The average recovery rate of the river water samples spiked with E2 at 1–50 ng/L range was 111.5% (68.6–252%) with the % relative standard deviation (RSD) of 0.5–86.3%. The ELISA demonstrated a good correlation with the GC-MS analyses of the spiked river water samples (r = 0.909). Applying the developed E2 ELISA assay to the monitoring of E2 residues in Hawkesbury River (New South Wales, Australia) found that all the tested creek samples contained E2 residues less than the biologically significant level of 10 ng/L. However, 25% of the water samples tested demonstrated the estrogen activity (determined by the yeast estrogen screening (YES) assay) above the levels that have been linked to the adverse effects in fish and other aquatic organisms (> 20 E2 Eq ng/L). It was apparent that the E2 residues together with the EE2 residues (reported in our previous study) contributed to most of the observed estrogenic activity in Hawkesbury River.

Structure-based drug design for envelope protein E2 uncovers a new class of bovine viral diarrhea inhibitors that block virus entry

Antiviral targeting of virus envelope proteins is an effective strategy for therapeutic intervention of viral infections. Here, we took a computer-guided approach with the aim of identifying new antivirals against the envelope protein E2 of bovine viral diarrhea virus (BVDV). BVDV is an enveloped virus with an RNA genome responsible for major economic losses of the cattle industry worldwide. Based on the crystal structure of the envelope protein E2, we defined a binding site at the interface of the two most distal domains from the virus membrane and pursued a hierarchical docking-based virtual screening search to identify small-molecule ligands of E2. Phenyl thiophene carboxamide derivative 12 (PTC12) emerged as a specific inhibitor of BVDV replication from in vitro antiviral activity screening of candidate molecules, displaying an IC50 of 0.30 μM against the reference NADL strain of the virus. Using reverse genetics we constructed a recombinant BVDV expressing GFP that served as a sensitive reporter for the study of the mechanism of action of antiviral compounds. Time of drug addition assays showed that PTC12 inhibited an early step of infection. The mechanism of action was further dissected to find that the compound specifically acted at the internalization step of virus entry. Interestingly, we demonstrated that similar to PTC12, the benzimidazole derivative 03 (BI03) selected in the virtual screen also inhibited internalization of BVDV. Furthermore, docking analysis of PTC12 and BI03 into the binding site revealed common interactions with amino acid residues in E2 suggesting that both compounds could share the same molecular target. In conclusion, starting from a targeted design strategy of antivirals against E2 we identified PTC12 as a potent inhibitor of BVDV entry. The compound can be valuable in the design of antiviral strategies in combination with already well-characterized polymerase inhibitors of BVDV.

Production of antibodies and development of an enzyme-linked immunosorbent assay for 17β-estradiol in milk

ABSTRACTThe residue of 17β-estradiol (E2) in milk could potentially lead to the occurrence of various reproductive diseases; therefore, a rapid and sensitive method for monitoring E2 residues in milk was highly necessary. In this study, we produced new polyclonal and monoclonal antibodies using E2-3-O-carboxymethyl ether as a hapten and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the detection of E2 in milk. The results showed that the sensitivity of polyclonal antibody was higher than that of monoclonal antibody, providing a half maximum inhibition concentration (IC50) against E2 of 0.17 ng/mL, high cross-reactivity (CR) to E2 benzoate (150%) and oestriol (18.02%), and negligible CR with other oestrogen compounds. Under optimized conditions, the developed icELISA based on the polyclonal antibody had a limit of detection values of 0.093 μg/L, which was enough sensitive to detect E2 in milk. In spiked samples (0.5, 1, and 2 μg/L), the recoveries ranged from 83.12% to 94.58% with coefficients of variation <12.8%. These results indicated that the icELISA method we developed was suitable for screening of E2 residue in milk.

Global mapping of antibody recognition of the hepatitis C virus E2 glycoprotein: Implications for vaccine design

The E2 envelope glycoprotein is the primary target of human neutralizing antibody response against hepatitis C virus (HCV), and is thus a major focus of vaccine and immunotherapeutics efforts. There is emerging evidence that E2 is a highly complex, dynamic protein with residues across the protein that are modulating antibody recognition, local and global E2 stability, and viral escape. To comprehensively map these determinants, we performed global E2 alanine scanning with a panel of 16 human monoclonal antibodies (hmAbs), resulting in an unprecedented dataset of the effects of individual alanine substitutions across the E2 protein (355 positions) on antibody recognition. Analysis of shared energetic effects across the antibody panel identified networks of E2 residues involved in antibody recognition and local and global E2 stability, as well as predicted contacts between residues across the entire E2 protein. Further analysis of antibody binding hotspot residues defined groups of residues essential for E2 conformation and recognition for all 14 conformationally dependent E2 antibodies and subsets thereof, as well as residues that enhance antibody recognition when mutated to alanine, providing a potential route to engineer E2 vaccine immunogens. By incorporating E2 sequence variability, we found a number of E2 polymorphic sites that are responsible for loss of neutralizing antibody binding. These data and analyses provide fundamental insights into antibody recognition of E2, highlighting the dynamic and complex nature of this viral envelope glycoprotein, and can serve as a reference for development and rational design of E2-targeting vaccines and immunotherapeutics.