Low Cross-reactivity Research Articles

Low Immune Cross-Reactivity between West Nile Virus and a Zika Virus Vaccine Based on Modified Vaccinia Virus Ankara.

Zika virus (ZIKV) is a mosquito-borne flavivirus whose infection in pregnant women is associated with a spectrum of birth defects, which are together referred as Congenital Zika Syndrome. In addition, ZIKV can also induce Guillain–Barré syndrome, which is an autoimmune disease with neurological symptoms. The recent description of the first local infections of ZIKV in the European continent together with the expansion of one of its potential vectors, the Asian tiger mosquito (Aedes albopictus), invite us to be prepared for future outbreaks of ZIKV in this geographical region. However, the antigenic similarities of ZIKV with other flaviviruses can lead to an immune cross-reactivity with other circulating flaviviruses inducing, in some cases, flavivirus-disease exacerbation by antibody-dependent enhancement (ADE) of infection, which is a major concern for ZIKV vaccine development. Until now, West Nile virus (WNV) is the main medically relevant flavivirus circulating in the Mediterranean Basin. Therefore, anticipating the potential scenario of emergency vaccination against ZIKV in areas of Europe where WNV is endemic, in this investigation, we have evaluated the cross-reactivity between WNV and our previously developed ZIKV vaccine candidate based on modified vaccinia virus Ankara (MVA) vector expressing ZIKV structural proteins (MVA-ZIKV). To this end, mice were first immunized with MVA-ZIKV, subsequently challenged with WNV, and then, the ZIKV- and WNV-specific immune responses and protection against WNV were evaluated. Our results indicate low cross-reactivity between the MVA-ZIKV vaccine candidate and WNV and absence of ADE, supporting the safety of this ZIKV vaccine candidate in areas where the circulation of WNV is endemic.

Performance of Chimeric Trypanosoma cruzi Antigens in Serological Screening for Chagas Disease in Blood Banks.

Chagas disease (CD) is among the top 10 causes of inability to blood donation. Blood donation centers screen for anti-Trypanosoma cruzi antibodies using highly sensitive immunoenzymatic (ELISA) or chemiluminescent methods, which can lead to false positive results. Since positive samples cannot be used, to avoid the loss of valuable blood donations, it is necessary to improve specificity without reducing the sensitivity of the tests used for blood screening. For this purpose, our group has developed four chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) that have been evaluated in phase I and II studies with high performance and low cross-reactivity rates. The study included a panel of 5,014 serum samples collected from volunteer blood donors at the Hematology and Hemotherapy Foundation of the State of Bahia (Brazil). They were subjected to the detection of anti-T. cruzi antibodies, using all four IBMP antigens individually and latent class analysis (LCA) as a reference test, since there is no gold standard test for this purpose. Considering the sample size analyzed, LCA classified 4,993 (99.6%) samples as T. cruzi-negative and 21 (0.42%) as T. cruzi-positive. Sensitivity values ranged from 85.71% for IBMP-8.1 and 90.48% for IBMP-8.2–95.24% for IBMP-8.3 and 100% for IBMP-8.4, while specificity ranged from 99.98% for IBMP-8.3 and IBMP-8.4–100% for IBMP-8.1 and IBMP-8.2. Accuracy values ranged from 99.4 to 99.98%. The pretest probability for the molecules was 0.42, whereas the positive posttest probability ranged from 95.24 to 99.95% and the negative posttest probability ranged from 0.00001 to 0.0006% for all antigens. The higher odds ratio diagnosis was found for IBMP-8.4, which has been shown to be a safe single antigen for serological screening of CD in blood samples. The use of chimeric IBMP antigens is an alternative to reduce the number of bags discarded due to false-positive results. These molecules have high diagnostic performance and were shown to be suitable for use in screening CD in blood banks, isolated (IBMP-8.4) or in combination; and their use in blood banks could significantly reduce unnecessary disposal of blood bags or the risk of T. cruzi transmission.

Alternative Hapten Design for Zearalenone Immunoreagent Generation.

Appropriate hapten design and synthesis have been identified as critical steps to generate high-performance immunoreagents and to develop sensitive and selective immunoanalytical methods. Antibodies and immunoassays for the major mycotoxin zearalenone have been reported and marketed. However, zearalenone haptens have mostly been prepared by the oxime active ester technique, and hapten characterization has generally been poor or non-existent. In the present study, novel haptens of zearalenone with longer linkers and with alternative tethering sites have been designed for immunizing and assay conjugate preparation. All of these molecules were purified and spectroscopically verified, and a structure-activity relationship evaluation was carried out. This approach revealed that the hapten with the linker at the carbonyl group generated antibodies with a higher affinity than the hapten functionalized at the phenyl moiety. Antibodies produced with the latter hapten, on the other hand, showed lower cross-reactivity values to the major zearalenone metabolites. Finally, similar immunoassay sensitivity was achieved with all of the antibodies when heterologous haptens were employed. Furthermore, by altering the structure of the competing antigen, the immunoassay selectivity was modified. These results demonstrate that immunochemical methods for zearalenone rapid analysis can still be improved in terms of sensitivity and selectivity.

A Novel Peptide as a Specific and Selective Probe for Klebsiella pneumoniae Detection.

Klebsiella pneumoniae is infamous for generating hospital-acquired infections, many of which are difficult to treat due to the bacterium’s multidrug resistance. A sensitive and robust detection method of K. pneumoniae can help prevent a disease outbreak. Herein, we used K. pneumoniae cells as bait to screen a commercially available phage-displayed random peptide library for peptides that could be used to detect K. pneumoniae. The biopanning-derived peptide TSATKFMMNLSP, named KP peptide, displayed a high selectivity for the K. pneumoniae with low cross-reactivity to related Gram-negative bacteria. The specific interaction between KP peptide and K. pneumoniae lipopolysaccharide resulted in the peptide’s selectivity against K. pneumoniae. Quantitative analysis of this interaction by enzyme-linked immunosorbent assay revealed that the KP peptide possessed higher specificity and sensitivity toward K. pneumoniae than commercially available anti-Klebsiella spp. antibodies and could detect K. pneumoniae at a detection limit of 104 CFU/mL. These results suggest that KP peptide can be a promising alternative to antibodies in developing a biosensor system for K. pneumoniae detection.

Comprehensive mapping of SARS-CoV-2 peptide epitopes for development of a highly sensitive serological test for total and neutralizing antibodies.

Quantification of the anti-SARS-CoV-2 antibody response has proven to be a prominent diagnostic tool during the COVID-19 pandemic. Antibody measurements have aided in the determination of humoral protection following infection or vaccination and will likely be essential for predicting the prevalence of population level immunity over the next several years. Despite widespread use, current tests remain limited in part, because antibody capture is accomplished through the use of complete spike and nucleocapsid proteins that contain significant regions of overlap with common circulating coronaviruses. To address this limitation, a unique epitope display platform utilizing monovalent display and protease-driven capture of peptide epitopes was used to select high affinity peptides. A single round of selection using this strategy with COVID-19 positive patient plasma samples revealed surprising differences and specific patterns in the antigenicity of SARS-CoV-2 proteins, especially the spike protein. Putative epitopes were assayed for specificity with convalescent and control samples, and the individual binding kinetics of peptides were also determined. A subset of prioritized peptides was used to develop an antibody diagnostic assay that showed low cross reactivity while detecting 37% more positive antibody cases than a gold standard FDA EUA test. Finally, a subset of peptides were compared with serum neutralization activity to establish a 2 peptide assay that strongly correlates with neutralization. Together, these data demonstrate a novel phage display method that is capable of comprehensively and rapidly mapping patient viral antibody responses and selecting high affinity public epitopes for the diagnosis of humoral immunity.

Aflatoxin B1 in the egg chain: monitoring with specific indirect competitive ELISA in northern Paraná, Brazil

An indirect competitive immunoassay (ic-ELISA) was developed using monoclonal antibody produced by hybridoma AF4, which showed high specificity and reactivity with aflatoxin B1 (AFB1) and aflatoxicol, but low cross-reactivity to other analogs. This low cost reliable method was applied for AFB1 monitoring in the poultry chain of a high agribusiness potential region (northern Paraná state, Brazil). Maize, laying hens feed and egg samples were collected from two poultry farms (with production above 200,000 eggs/day) and evaluated by intralaboratory validated ic-ELISA. The sensitivity of such a validated assay, detecting picogram levels of aflatoxins, demonstrated to be proper for surveying daily ingested cumulative toxins and estimating risks. Additionally, more than 61.00% of positive egg samples ranged between the limit of quantification (LOQ – 0.035 ng/g) and 1.00 ng/g, values commonly not covered by commercial kits. Positive data (>LOQ) occurred in 22 maize (56.40%), 34 feed (85.00%) and 192 (48.00%) egg samples. Mean contamination in maize was 1.51±0.94 ng/g (range 0.11-3.91 ng/g), 1.26±0.96 ng/g in feed (0.10-3.58 ng/g), and 1.01±0.77 ng/g in egg (0.05-3.85 ng/g). No statistical difference was observed between farms (P>0.05) for any of the matrices analysed. However, the difference between median values in maize (0.98 ng/g – Farm A; 1.76 ng/g – Farm B) indicated a higher contamination trend in farm B, possibly due to inadequate local storage. Although there is no limit stipulated for AFB1 contamination in eggs, the levels detected in samples were low and do not represent an immediate risk to animal production or human consumption. Nevertheless, the high frequency of positive maize and feed samples in this field of agribusiness should be highlighted. Sensitive aflatoxin monitoring procedures must be strategically carried out from raw materials to animal derived products, aiming harmless production, which also assures human health.

A Novel Humanized Anti-Interleukin-6 Antibody HZ0408b With Anti-Rheumatoid Arthritis Therapeutic Potential.

Interleukin-6 (IL-6), a pleiotropic cytokine that regulates immune responses and inflammatory reactions, plays a pivotal role in the development of rheumatoid arthritis (RA). Blockade of IL-6 signaling with the monoclonal antibody (mAb) represents an important advancement in RA treatment. Although two IL-6 receptor antibodies are already available in the clinic, there is no mAb specifically targeting the human IL-6 to block IL-6 signaling for RA treatment. In this study, we have developed a novel humanized anti-IL-6 mAb HZ-0408b with potent binding and neutralizing activity to human IL-6. We demonstrated that HZ-0408b has a high species specificity and low cross-reactivity. Moreover, HZ-0408b showed a more potent inhibitory effect on IL-6 signaling than Siltuximab, an FDA-approved anti-IL-6 chimeric mAb. HZ-0408b is comparable to Olokizumab, a humanized mAb against IL-6 that is already in phase III studies. We observed that HZ-0408b is well tolerated at doses that can achieve therapeutic serum levels in cynomolgus monkey. Most importantly, we proved that HZ-0408b treatment significantly ameliorated joint swelling after the onset of arthritis and dramatically reduced plasma C-reactive protein (CRP) levels in a monkey collagen-induced arthritis (CIA) model. Collectively, our findings using non-human primates indicate that humanized anti-IL-6 mAb HZ-0408b has excellent safety and efficacy profiles for RA therapy.

Saliva-based COVID-19 detection: A rapid antigen test of SARS-CoV-2 nucleocapsid protein using an electrical-double-layer gated field-effect transistor-based biosensing system

Facing the unstopped surges of COVID-19, an insufficient capacity of diagnostic testing jeopardizes the control of disease spread. Due to a centralized setting and a long turnaround, real-time reverse transcription polymerase chain reaction (real-time RT-PCR), the gold standard of viral detection, has fallen short in timely reflecting the epidemic status quo during an urgent outbreak. As such, a rapid screening tool is necessitated to help contain the spread of COVID-19 amid the countries where the vaccine implementations have not been widely deployed. In this work, we propose a saliva-based COVID-19 antigen test using the electrical double layer (EDL)-gated field-effect transistor-based biosensor (BioFET). The detection of SARS-CoV-2 nucleocapsid (N) protein is validated with limits of detection (LoDs) of 0.34 ng/mL (7.44 pM) and 0.14 ng/mL (2.96 pM) in 1× PBS and artificial saliva, respectively. The specificity is inspected with types of antigens, exhibiting low cross-reactivity among MERS-CoV, Influenza A virus, and Influenza B virus. This portable system is embedded with Bluetooth communication and user-friendly interfaces that are fully compatible with digital health, feasibly leading to an on-site turnaround, an effective management, and a proactive response taken by medical providers and frontline health workers.

Development and Validation of a Novel ELISA for the Specific Detection of Antibodies against Mycobacterium avium Subspecies paratuberculosis Based on a Chimeric Polyprotein.

Bovine paratuberculosis (PTB) is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The optimization of detection tests specific for MAP is crucial to improve PTB control. In this work, we aimed to develop and validate a diagnostic tool based on an ELISA to specifically detect anti-MAP antibodies from bovine serum samples. For that purpose, we designed a recombinant polyprotein containing four specific antigens from MAP and optimized the ELISA. The validation consisted of the assessment of 10 sera from PTB-infected and healthy bovines with different OD values. The diagnostic performance of the polyprotein-ELISA was evaluated by testing 130 bovine serum samples (47 healthy, 48 MAP-infected, and 35 M. bovis-infected bovines). The ELISA using the polyprotein yielded an area under the ROC curve (AUC) of 0.9912 (95% CI, 0.9758–1.007; P < 0.0001). Moreover, for this ELISA, the cut-off selected from the ROC curve based on the point with a sensitivity of 95.56% (95% CI, 0.8485–0.9946) and specificity of 97.92 (95% CI, 0.8893–0.9995) was 0.3328. Similar results were obtained with an ELISA using the commercial Paratuberculosis Protoplasmatic Antigen (PPA). However, the ELISA with the polyprotein antigen showed a better performance against sera from animals infected with Mycobacterium bovis compared to the ELISA with PPA: lower cross-reactivity (2.85% versus 25.71%). These results demonstrate a very low cross-reactivity of the polyprotein with antibodies present in serum samples from animals infected with M. bovis. The designed polyprotein and the validated ELISA could be very useful for the specific identification of MAP-infected animals in herds.

Development of a sensitive chicken IgY-based enzyme-linked immunosorbent assay for detection of mebendazole in pork and mutton

Chicken egg yolk IgY has proven to be qualified for analysis of targets in immunoassays. In order to explore the feasibility of chicken IgY-based ELISA for detection of mebendazole (MEB), the chicken IgY against MEB was generated in the laying hens. An enzyme-linked immunosorbent assay (ELISA) based on chicken IgY was developed for detection of MEB with a half-maximum signal inhibition concentration (IC50) of 3.65 ng mL−1 and a limit of detection of 0.25 ng mL−1. The assay showed a lower cross reactivity (less than 1%) with other structures analogues (except amino-MEB with the values of 70.7%). The average recoveries of MEB spiked in pork and mutton muscle samples ranged from 93.6% to 106.3% with relative standard deviation less than 8.78% and 10.85% for intra-assay and inter-assay, respectively, and agreed well with those of high-performance liquid chromatography. Our results indicate that generated IgY could be used as a robust reagent for routine screening analysis of small molecular compounds residues in food samples.

65. Impact of an Antibiotic Side-Chain-Based Cross-Reactivity Chart on Antibiotic Use in Patients With β-lactam Allergies and Pneumonia

Backgroundβ-lactam antibiotics with dissimilar R-group side chains are associated with low cross-reactivity. Despite this, patients with β-lactam allergies are too often treated with alternative antibiotic therapy. An institutional β-lactam side-chain-based cross-reactivity chart was developed and implemented to guide in antibiotic selection for β-lactam allergies patients. MethodsThis single center, retrospective, cohort study analyzed the impact of the implementation of the cross-reactivity chart for patients with documented β-lactam allergies with pneumonia. Study time periods were defined as January 2013 to October 2014 prior to implementation of the chart (historical cohort) and January 2017 to October 2018 (intervention cohort) following institutional implementation and adoption. The primary outcome was the incidence of β-lactam utilization between time periods. Propensity-weighted scoring and interrupted time-series analyses compared outcomes across time periods. ResultsA total of 341 and 623 patient encounters were included in the historical and intervention cohorts, respectively. There was a significant increase in the use of β-lactams for treatment of pneumonia (70.4% vs 89.3%; p < 0.001) and the use of any alternative therapy decreased between cohorts (58.1% vs. 36%; p < 0.001) (Figure 1). β-lactam use per patient significantly improved between cohorts in patients with mild, Type 1 IgE-mediated hypersensitivity reactions (HSRs) and in patients with unknown reactions. There was no difference in overall HSRs between cohorts (2.4% vs. 1.45; p = 0.628), or in patients who received β-lactam antibiotics (1.3% historical group vs 1.1% intervention group; p = 0.467). Median alternative antibiotic days of therapy (3 vs. 2; p = 0.027) and duration of therapy per patient (3 days vs. 2 days; p = 0.023) decreased between cohorts. There was a significant increase in mortality while healthcare facility-onset Clostridioides difficile infections decreased between cohorts.β-Lactam vs. Alternative Therapy Use per Patients by Calendar Quarter ConclusionImplementation of a β-lactam side-chain-based cross-reactivity chart significantly increased the utilization of β-lactams in patients with pneumonia without increasing HSRs. Disclosures All Authors: No reported disclosures

Development of a colorimetric aptasensor for aflatoxin B1 detection based on silver nanoparticle aggregation induced by positively charged perylene diimide

A sensitive colorimetric aptasensor for rapid quantification of aflatoxin B1 (AfB1) has been successfully developed. The sensing strategy is based on the aggregation and localized surface plasmon resonance (LSPR) of silver nanoparticles (AgNPs) that involves interactions of a specific aptamer, positively charged perylene diimide (PCPD), and AgNPs nanoprobes. The aptamer can explicitly bind to AfB1, resulting in a conformational change of the aptamer, leading to a dissociation of the PCPD-aptamer complex. Eventually, the released PCPD induces aggregation of AgNPs, producing a new color tone derived from the surface plasmon wavelength shift of AgNPs. The absorbance ratio between 520 nm and 393 nm (A520/393 nm) of AgNPs aggregation has been applied to calculate AfB1 concentration in the sample. A high concentration of AfB1 displays the increasing A520/393 nm. Inversely, a low concentration of AfB1 exhibits the decreasing A520/393 nm. A calibration graph of the aptasensor exhibits excellent linear response to the AfB1 amount in the range of 0.2–6.0 ng/mL. A detection limit for AfB1 determination is 0.09 ng/mL with very low cross-reactivity from other mycotoxins, signifying high selectivity. Furthermore, the proposed method has been successfully applied to determine AfB1 in peanut, corn, and chicken feed samples. Obtained recoveries are more significant than 80%, with RSD lower than 10%. Hence, an innovative aptasensor can be used as an alternative method for quantifying AfB1 in real samples.

Development of single- and multiplex immunoassays for rapid detection and quantitation of amodiaquine in ACT drugs and rat serum.

Amodiaquine (AQ) is a commonly used antimalarial drug, and N-desethyl-AQ (N-DEAQ) is an active metabolite of AQ. Given the significance of drug quality in the management of malaria cases, this study aims to develop antibody-based assays for the detection and quantitation of AQ without the need for sophisticated equipment. Two monoclonal antibodies (mAbs) against AQ, designated as JUN7 and TE7, were selected, which showed 72.7% and 9.5% cross-reactivity to N-DEAQ, respectively. These mAbs showed <0.1% cross-reactivity to other commonly used antimalarial drugs. An indirect competitive enzyme-linked immunosorbent assay (icELISA) based on JUN7 showed a 50% inhibitory concentration (IC50) of 0.16ng/mL and a working range of 0.06-0.46ng/mL. A lateral flow immunoassay (LFIA) based on JUN7 was also developed with a working range of 2.58-30.86ng/mL. The icELISA and LFIA were applied for the quantification of AQ in commercial drugs, and the results were comparable to those determined using high-performance liquid chromatography. In addition, a combination dipstick for simultaneous, qualitative analysis of AQ and artesunate was developed. All immunoassays based on JUN7 can be applied for quality control of AQ-containing artemisinin-based combination therapies. As TE7 showed low cross-reactivity to N-DEAQ, an icELISA based on TE7 was developed with an IC50 of 0.38ng/mL and a working range of 0.14-1.67ng/mL. The TE7 icELISA was applied for the study of pharmacokinetics of AQ in rat serum after intragastric administration, and the results were consistent with those of previous studies.

Antibiotic Use in Patients With β-Lactam Allergies and Pneumonia: Impact of an Antibiotic Side Chain-Based Cross-Reactivity Chart Combined With Enhanced Allergy Assessment.

Backgroundβ-lactam antibiotics with dissimilar R-group side chains are associated with low cross-reactivity. Despite this, patients with β-lactam allergies are often treated with non-β-lactam alternative antibiotics. An institutional β-lactam side chain–based cross-reactivity chart was developed and implemented to guide in antibiotic selection for patients with β-lactam allergies.MethodsThis single-center, retrospective cohort study analyzed the impact of the implementation of the cross-reactivity chart for patients with pneumonia. Study time periods were defined as January 2013 to October 2014 prior to implementation of the chart (historical cohort) and January 2017 to October 2018 (intervention cohort) following implementation. The primary outcome was the incidence of β-lactam utilization between time periods. Propensity-weighted scoring and interrupted time-series analyses compared outcomes.ResultsA total of 341 and 623 patient encounters were included in the historical and intervention cohorts, respectively. There was a significantly greater use of β-lactams in the intervention cohort (70.4% vs 89.3%; P < .001) and decreased use of alternative therapy (58.1% vs 36%; P < .001). There was no difference in overall allergic reactions between cohorts (2.4% vs 1.6%; P = .738) or in reactions caused by β-lactams (1.3% vs 0.9%; P = .703). Inpatient mortality increased (0% vs 6.4%; P < .001); however, no deaths were due to allergic reactions. Healthcare facility–onset Clostridioides difficile infections decreased between cohorts (1.2% vs 0.2%; P = .032).ConclusionsImplementation of a β-lactam side chain–based cross-reactivity chart and enhanced allergy assessment was associated with increased use of β-lactams in patients with pneumonia without increasing allergic reactions.

Recombinant Porcine Factor VIII in Patients with Congenital Hemophilia a with Inhibitors Undergoing Surgery: Phase 3, Multicenter, Single Arm, Open-Label Study

Recombinant Porcine Factor VIII in Patients with Congenital Hemophilia a with Inhibitors Undergoing Surgery: Phase 3, Multicenter, Single Arm, Open-Label Study

Antibacterial Profile of a Microbicidal Agent Targeting Tyrosine Phosphatases and Redox Thiols, Novel Drug Targets.

The activity profile of a protein tyrosine phosphatase (PTP) inhibitor and redox thiol oxidant, nitropropenyl benzodioxole (NPBD), was investigated across a broad range of bacterial species. In vitro assays assessed inhibitory and lethal activity patterns, the induction of drug variants on long term exposure, the inhibitory interactions of NPBD with antibiotics, and the effect of plasma proteins and redox thiols on activity. A literature review indicates the complexity of PTP and redox signaling and suggests likely metabolic targets. NPBD was broadly bactericidal to pathogens of the skin, respiratory, urogenital and intestinal tracts. It was effective against antibiotic resistant strains and slowly replicating and dormant cells. NPBD did not induce resistant or drug-tolerant phenotypes and showed low cross reactivity with antibiotics in synergy assays. Binding to plasma proteins indicated lowered in-vitro bioavailability and reduction of bactericidal activity in the presence of thiols confirmed the contribution of thiol oxidation and oxidative stress to lethality. This report presents a broad evaluation of the antibacterial effect of PTP inhibition and redox thiol oxidation, illustrates the functional diversity of bacterial PTPs and redox thiols, and supports their consideration as novel targets for antimicrobial drug development. NPBD is a dual mechanism agent with an activity profile which supports consideration of tyrosine phosphatases and bacterial antioxidant systems as promising targets for drug development.

Novel DNA Aptameric Sensors to Detect the Toxic Insecticide Fenitrothion.

Fenitrothion is an insecticide belonging to the organophosphate family of pesticides that is widely used around the world in agriculture and living environments. Today, it is one of the most hazardous chemicals that causes severe environmental pollution. However, detection of fenitrothion residues in the environment is considered a significant challenge due to the small molecule nature of the insecticide and lack of molecular recognition elements that can detect it with high specificity. We performed in vitro selection experiments using the SELEX process to isolate the DNA aptamers that can bind to fenitrothion. We found that newly discovered DNA aptamers have a strong ability to distinguish fenitrothion from other organophosphate insecticides (non-specific targets). Furthermore, we identified a fenitrothion-specific aptamer; FenA2, that can interact with Thioflavin T (ThT) to produce a label-free detection mode with a Kd of 33.57 nM (9.30 ppb) and LOD of 14 nM (3.88 ppb). Additionally, the FenA2 aptamer exhibited very low cross-reactivity with non-specific targets. This is the first report showing an aptamer sensor with a G4-quadruplex-like structure to detect fenitrothion. Moreover, these aptamers have the potential to be further developed into analytical tools for real-time detection of fenitrothion from a wide range of samples.

Upconversion-Linked Immunoassay for the Diagnosis of Honeybee Disease American Foulbrood

American foulbrood (AFB) caused by the bacterium Paenibacillus larvae is the most destructive disease of the honeybee brood. Therefore, rapid and sensitive detection methods are required to limit spreading of this pathogen, which has a major impact on agriculture and biodiversity. While P. larvae is typically detected by microbial cultivation or polymerase chain reaction, antibody-based detection represents a viable alternative. Here, we prepared an antibody specific for P. larvae and used it for the development of an upconversion-linked immunosorbent assay (ULISA). Photon-upconversion nanoparticles (UCNP) were conjugated to streptavidin via a PEG-linker using copper-catalyzed click chemistry to replace the enzyme label in conventional enzyme-linked immunosorbent assay (ELISA). The ULISA showed low cross-reactivity and provided a limit of detection of 2.9 × 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sup> CFU/mL, representing a 22-fold improvement compared to the ELISA. This level is within the bacterial loads present in honeybee larvae during an AFB infection. The assay was successfully applied to the analysis of spiked samples of bees, larvae, and hive debris.

Human reference gut microbiome catalog including newly assembled genomes from under-represented Asian metagenomes

BackgroundMetagenome sampling bias for geographical location and lifestyle is partially responsible for the incomplete catalog of reference genomes of gut microbial species. Thus, genome assembly from currently under-represented populations may effectively expand the reference gut microbiome and improve taxonomic and functional profiling.MethodsWe assembled genomes using public whole-metagenomic shotgun sequencing (WMS) data for 110 and 645 fecal samples from India and Japan, respectively. In addition, we assembled genomes from newly generated WMS data for 90 fecal samples collected from Korea. Expecting genome assembly for low-abundance species may require a much deeper sequencing than that usually employed, so we performed ultra-deep WMS (> 30 Gbp or > 100 million read pairs) for the fecal samples from Korea. We consequently assembled 29,082 prokaryotic genomes from 845 fecal metagenomes for the three under-represented Asian countries and combined them with the Unified Human Gastrointestinal Genome (UHGG) to generate an expanded catalog, the Human Reference Gut Microbiome (HRGM).ResultsHRGM contains 232,098 non-redundant genomes for 5414 representative prokaryotic species including 780 that are novel, > 103 million unique proteins, and > 274 million single-nucleotide variants. This is an over 10% increase from the UHGG. The new 780 species were enriched for the Bacteroidaceae family, including species associated with high-fiber and seaweed-rich diets. Single-nucleotide variant density was positively associated with the speciation rate of gut commensals. We found that ultra-deep sequencing facilitated the assembly of genomes for low-abundance taxa, and deep sequencing (e.g., > 20 million read pairs) may be needed for the profiling of low-abundance taxa. Importantly, the HRGM significantly improved the taxonomic and functional classification of sequencing reads from fecal samples. Finally, analysis of human self-antigen homologs on the HRGM species genomes suggested that bacterial taxa with high cross-reactivity potential may contribute more to the pathogenesis of gut microbiome-associated diseases than those with low cross-reactivity potential by promoting inflammatory condition.ConclusionsBy including gut metagenomes from previously under-represented Asian countries, Korea, India, and Japan, we developed a substantially expanded microbiome catalog, HRGM. Information of the microbial genomes and coding genes is publicly available (www.mbiomenet.org/HRGM/). HRGM will facilitate the identification and functional analysis of disease-associated gut microbiota.

Expansion of Unique Hepatitis C Virus-Specific Public CD8+ T Cell Clonotypes during Acute Infection and Reinfection.

Hepatitis C virus (HCV) infection resolves spontaneously in ∼25% of acutely infected humans where viral clearance is mediated primarily by virus-specific CD8+ T cells. Previous cross-sectional analysis of the CD8+ TCR repertoire targeting two immunodominant HCV epitopes reported widespread use of public TCRs shared by different subjects, irrespective of infection outcome. However, little is known about the evolution of the public TCR repertoire during acute HCV and whether cross-reactivity to other Ags can influence infectious outcome. In this article, we analyzed the CD8+ TCR repertoire specific to the immunodominant and cross-reactive HLA-A2-restricted nonstructural 3-1073 epitope during acute HCV in humans progressing to either spontaneous resolution or chronic infection and at ∼1 y after viral clearance. TCR repertoire diversity was comparable among all groups with preferential usage of the TCR-β V04 and V06 gene families. We identified a set of 13 public clonotypes in HCV-infected humans independent of infection outcome. Six public clonotypes used the V04 gene family. Several public clonotypes were long-lived in resolvers and expanded on reinfection. By mining publicly available data, we identified several low-frequency CDR3 sequences in the HCV-specific repertoire matching human TCRs specific for other HLA-A2-restricted epitopes from melanoma, CMV, influenza A, EBV, and yellow fever viruses, but they were of low frequency and limited cross-reactivity. In conclusion, we identified 13 new public human CD8+ TCR clonotypes unique to HCV that expanded during acute infection and reinfection. The low frequency of cross-reactive TCRs suggests that they are not major determinants of infectious outcome.