Albuquerque, NM, United States
Albuquerque, NM, United States

Time filter

Source Type

We studied eight subjects in the United States with previous or recent ZIKV infection (Extended Data Table 2). The studies were approved by the Institutional Review Board of Vanderbilt University Medical Center; samples were obtained after informed consent was obtained by the Vanderbilt Clinical Trials Center. Two subjects (972 and 973) were infected with an African lineage strain in 2008 (one subject while working in Senegal, the second acquired the infection by sexual transmission from the first, as previously reported24). The other six subjects were infected during the current outbreak of an Asian lineage strain, following exposure in Brazil, Mexico or Haiti. Peripheral blood mononuclear cells (PBMCs) from heparinized blood were isolated with Ficoll-Histopaque by density gradient centrifugation. The cells were used immediately or cryopreserved in the vapour phase of liquid nitrogen until use. Ten million PBMCs were cultured in 384-well plates (Nunc) using culture medium (ClonaCell-HY Medium A, StemCell Technologies) supplemented with 8 μg ml−1 of the TLR agonist CpG (phosphorothioate-modified oligodeoxynucleotide ZOEZOEZZZZZOEEZOEZZZT, Invitrogen), 3 μg ml−1 of Chk2 inhibitor (Sigma), 1 μg ml−1 of cyclosporine A (Sigma), and clarified supernatants from cultures of B95.8 cells (ATCC) containing Epstein–Barr virus. After 7 days, cells from each 384-well culture plate were expanded into four 96-well culture plates (Falcon) using ClonaCell-HY Medium A containing 8 μg ml−1 of CpG, 3 μg ml−1 of Chk2 inhibitor, and 107 irradiated heterologous human PBMCs (Nashville Red Cross) and cultured for an additional 4 days. Supernatants were screened in ELISA (described below) for reactivity with various ZIKV E proteins, which are described below. The minimal frequency of ZIKV E-reactive B cells was estimated based on the number of wells with E protein-reactive supernatants compared with the total number of lymphoblastoid cell line colonies in the transformation plates (calculation: E-reactive B-cell frequency = (number of wells with E-reactive supernatants) divided by (number of LCL colonies in the plate) × 100). The ectodomains of ZIKV E (H/PF/2013; GenBank Accession KJ776791) and the fusion-loop mutant E-FLM (containing four mutations: T76A, Q77G, W101R, L107R) were expressed transiently in Expi293F cells and purified as described previously7. ZIKV DIII (residues 299–407 of strain H/PF/2013), WNV DIII (residues 296–405 of strain New York 1999) and DENV-2 DIII (residues 299-410 of strain 16681) were expressed in BL21 (DE3) as inclusion bodies and refolded in vitro25. Briefly, inclusion bodies were denatured and refolded by gradual dilution into a refolding buffer (400 mM l-arginine, 100 mM Tris (pH 8.3), 2 mM EDTA, 5 and 0.5 mM reduced and oxidized glutathione) at 4 °C. Refolded proteins were purified by size-exclusion chromatography using a Superdex 75, 16/60 (GE Healthcare). Cells from wells with transformed B cells containing supernatants that exhibited reactivity to ZIKV E protein were fused with HMMA2.5 myeloma cells (gift from L. Cavacini) using an established electrofusion technique26. After fusion, hybridomas were suspended in a selection medium containing 100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT Media Supplement, Sigma), and 7 μg ml−1 ouabain (Sigma) and cultured in 384-well plates for 18 days before screening hybridomas for antibody production by ELISA. After fusion with HMMA2.5 myeloma cells, hybridomas producing ZIKV E-specific antibodies were cloned biologically by single-cell fluorescence-activated cell sorting. Hybridomas were expanded in post-fusion medium (ClonaCell-HY Medium E, STEMCELL Technologies) until 50% confluent in 75-cm2 flasks (Corning). For antibody production, cells from one 75-cm2 flask were collected with a cell scraper and expanded to four 225-cm2 flasks (Corning) in serum-free medium (Hybridoma-SFM, Life Technologies). After 21 days, supernatants were clarified by centrifugation and filtered using 0.45-μm pore size filter devices. HiTrap Protein G or HiTrap MabSelectSure columns (GE Healthcare Life Sciences) were used to purify antibodies from filtered supernatants. Total cellular RNA was extracted from pelleted cells from hybridoma clones, and an RT–PCR reaction was performed using mixtures of primers designed to amplify all heavy-chain or light-chain antibody variable regions27. The generated PCR products were purified using AMPure XP magnetic beads (Beckman Coulter) and sequenced directly using an ABI3700 automated DNA sequencer. The variable region sequences of the heavy and light chains were analysed using the IMGT/V-Quest program28, 29. Wells of microtitre plates were coated with purified, recombinant ectodomain of ZIKV E, DIII, DIII-LR mutants (DIII containing A310E and T335K mutations) or DIII of related flaviviruses DENV-2 or WNV and incubated at 4 °C overnight. In ELISA studies with purified mAbs, we used recombinant ZIKV E protein ectodomain with His tag produced in Sf9 insect cells (Meridian Life Sciences R01635). Plates were blocked with 5% skimmed milk in PBS-T for 1 h. B-cell culture supernatants or purified antibodies were added to the wells and incubated for 1 h at ambient temperature. The bound antibodies were detected using goat anti-human IgG (γ-specific) conjugated with alkaline phosphatase (Southern Biotech) and pNPP disodium salt hexahydrate substrate (Sigma). In ELISAs that assessed binding of mAbs to DIII and DIII LR mutants, we used previously described murine mAbs ZV-2 and ZV-54 (ref. 7) as controls. A goat anti-mouse IgG conjugated with alkaline phosphatase (Southern Biotech) was used for detection of these antibodies. Colour development was monitored at 405 nm in a spectrophotometer (Biotek). For determining EC , microtitre plates were coated with ZIKV E or E-FLM that eliminated interaction of fusion-loop specific antibodies. Purified antibodies were diluted serially and applied to the plates. Bound antibodies were detected as above. A nonlinear regression analysis was performed on the resulting curves using Prism (GraphPad) to calculate EC values. Fetal head and placental tissues were collected at E13.5 from groups treated with ZIKV-117 or PBS (as a negative control), homogenized in PBS (250 μl) and stored at −20 °C. ELISA plates were coated with ZIKV E protein, and thawed, clarified tissue homogenates were applied undiluted in triplicate. Bound antibodies were detected using goat anti-human IgG (Fc-specific) antibody conjugated with alkaline phosphatase. The quantity of antibody was determined by comparison with a standard curve constructed using purified ZIKV-117 in a dilution series. His -tagged ZIKV E protein was immobilized on anti-His coated biosensor tips (Pall) for 2 min on an Octet Red biosensor instrument. After measuring the baseline signal in kinetics buffer (PBS, 0.01% BSA, and 0.002% Tween 20) for 1 min, biosensor tips were immersed into the wells containing first antibody at a concentration of 10 μg ml−1 for 7 min. Biosensors then were immersed into wells containing a second mAb at a concentration of 10 μg ml−1 for 7 min. The signal obtained for binding of the second antibody in the presence of the first antibody was expressed as a percentage of the uncompeted binding of the second antibody that was derived independently. The antibodies were considered competing if the presence of first antibody reduced the signal of the second antibody to less than 30% of its maximal binding and non-competing if the signal was greater than 70%. A level of 30–70% was considered intermediate competition. Epitope mapping was performed by shotgun mutagenesis essentially as described previously6. A ZIKV prM/E protein expression construct (based on ZIKV strain SPH2015) was subjected to high-throughput alanine scanning mutagenesis to generate a comprehensive mutation library. Each residue within prM/E was changed to alanine, with alanine codons mutated to serine. In total, 672 ZIKV prM/E mutants were generated (100% coverage), sequence confirmed, and arrayed into 384-well plates. Each ZIKV prM/E mutant was transfected into HEK-293T cells and allowed to express for 22 h. Cells were fixed in 4% (v/v) paraformaldehyde (Electron Microscopy Sciences), and permeabilized with 0.1% (w/v) saponin (Sigma-Aldrich) in PBS plus calcium and magnesium (PBS++). Cells were incubated with purified mAbs diluted in PBS++, 10% normal goat serum (Sigma), and 0.1% saponin. Primary antibody screening concentrations were determined using an independent immunofluorescence titration curve against wild-type ZIKV prM/E to ensure that signals were within the linear range of detection. Antibodies were detected using 3.75 μg ml−1 of AlexaFluor488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) in 10% NGS/0.1% saponin. Cells were washed three times with PBS++/0.1% saponin followed by two washes in PBS. Mean cellular fluorescence was detected using a high-throughput flow cytometer (HTFC, Intellicyt). Antibody reactivity against each mutant prM/E clone was calculated relative to wild-type prM/E protein reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wild-type prM/E-transfected controls. Mutations within clones were identified as critical to the mAb epitope if they did not support reactivity of the test MAb, but supported reactivity of other ZIKV antibodies. This counter-screen strategy facilitates the exclusion of prM/E mutants that are locally misfolded or have an expression defect. This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (Assurance number A3381-01). Inoculations were performed under anaesthesia induced and maintained with ketamine hydrochloride and xylazine, and all efforts were made to minimize animal suffering. No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. ZIKV strain H/PF/2013 (French Polynesia, 2013) was obtained from X. de Lamballerie (Aix Marseille Université). ZIKV Brazil Paraiba 2015 was provided by S. Whitehead (Bethesda) and originally obtained from P. F. C. Vasconcelos (Instituto Evandro Cargas). ZIKV MR 766 (Uganda, 1947), Malaysia P6740 (1966), and Dakar 41519 (Senegal, 1982) were provided by the World Reference Center or Emerging Viruses and Arboviruses (R. Tesh, University of Texas Medical Branch). Nicaraguan DENV strains (DENV-1 1254-4, DENV-2 172-08, DENV-3 N2845-09, and DENV-4 N703-99) were provided generously by E. Harris (University of California, Berkeley). Virus stocks were propagated in C6/36 Aedes albopictus cells (DENV) or Vero cells (ZIKV). ZIKV Dakar 41519 (ZIKV-Dakar) was passaged twice in vivo in Rag1−/− mice (M. Gorman and M. Diamond, unpublished data) to create a mouse-adapted strain. Virus stocks were titrated by focus-forming assay (FFA) on Vero cells. All cell lines were checked regularly for mycoplasma contamination and were negative. Cell lines were authenticated at acquisition with short tandem repeat method profiling; Vero cells, though commonly misidentified in the field, were used as they are the standard cell line for flavivirus titration. Serial dilutions of mAbs were incubated with 102 FFU of different ZIKV strains (MR 766, Dakar 41519, Malaysia P6740, H/PF/2013, or Brazil Paraiba 2015) for 1 h at 37 °C. The mAb–virus complexes were added to Vero cell monolayers in 96-well plates for 90 min at 37 °C. Subsequently, cells were overlaid with 1% (w/v) methylcellulose in MEM supplemented with 4% heat-inactivated FBS. Plates were fixed 40 h later with 1% PFA in PBS for 1 h at room temperature. The plates were incubated sequentially with 500 ng ml−1 mouse anti-ZIKV (ZV-16, E.F. and M.S.D., unpublished data) and horseradish-peroxidase-conjugated goat anti-mouse IgG in PBS supplemented with 0.1% (w/v) saponin (Sigma) and 0.1% BSA. ZIKV-infected cell foci were visualized using TrueBlue peroxidase substrate (KPL) and quantitated on an ImmunoSpot 5.0.37 macroanalyzer (Cellular Technologies). C6/36 Aedes albopictus cells were inoculated with a MOI 0.01 of ZIKV (H/PF/2013) or different DENV serotypes (Nicaraguan strains DENV-1 1254-4, DENV-2 172-08, DENV-3 N2845-09, DENV-4 N703-99). At 120 h post infection, cells were fixed with 4% PFA diluted in PBS for 20 min at room temperature and permeabilized with HBSS supplemented with 10 mM HEPES, 0.1% saponin and 0.025% NaN for 10 min at room temperature. 50,000 cells were transferred to U-bottom plates and incubated for 30 min at 4 °C with 5 μg ml−1 of anti-ZIKV human mAbs or negative (hCHK-152)12, or positive (hE60)30 isotype controls. After washing, cells were incubated with Alexa-Fluor-647-conjugated goat anti-human IgG (Invitrogen) at 1:500, fixed in 1% PFA in PBS, processed on MACSQuant Analyzed (Miltenyi Biotec), and analysed using FlowJo software (Tree Star). Total RNA was extracted from hybridoma cells and genes encoding the VH and VL domains were amplified in RT–PCR using IgExp primers31. The PCR products were directly cloned into antibody expression vectors containing the constant domains of wild-type γ1 chain, LALA mutant (leucine (L) to alanine (A) substitution at positions 234 and 235) γ1 chain for the VH domains, and wild-type κ chain for the VL domain in an isothermal amplification reaction (Gibson reaction)32. Plasmids encoding the heavy and light chain were transfected into 293F cells and full-length recombinant IgG was secreted into transfected cell supernatants. Supernatants were collected and IgG purified using Protein G chromatography and eluted into PBS. The functional abrogation of the binding of the LALA variant IgG was confirmed in an ELISA binding assay with recombinant human FcγRI. The binding of wild-type ZIKV-117 or LALA antibody to FcγRI was evaluated, in comparison with the binding pattern of control antibodies (human mAb CKV063 (ref. 33) LALA mutated IgG). C57BL/6 male mice (4–5-week-old, Jackson Laboratories) were inoculated with 103 FFU of mouse-adapted ZIKV-Dakar by subcutaneous route in the footpad. One-day before infection, mice were treated with 2 mg anti-Ifnar1 mAb (MAR1-5A3, Leinco Technologies) by intraperitoneal injection. ZIKV-specific human mAb (ZIKV-117) or an isotype control (hCHK-152) was administered as a single dose at day +1 (100 μg) or day +5 (250 μg) after infection through an intraperitoneal route. Animals were monitored for 21 days. Wild-type C57BL/6 mice were bred in a specific pathogen-free facility at Washington University School of Medicine. (1) Ifnar1−/− dams, prophylaxis studies: Ifnar1−/− female and wild-type male mice were mated; at E5.5, dams were treated with a single 250 μg dose of ZIKV mAb or isotype control by intraperitoneal injection. At E6.5, mice were inoculated with 103 FFU of ZIKV Brazil Paraiba 2015 by subcutaneous injection in the footpad. (2) Wild-type dams, prophylaxis studies: wild-type female and male mice were mated; at embryonic days E5.5, dams were treated with a single 250 μg dose of ZIKV mAb or isotype control by intraperitoneal injection as well as a 1 mg injection of anti-Ifnar1 (MAR1-5A3). At E6.5, mice were inoculated with 103 FFU of mouse-adapted ZIKV-Dakar by subcutaneous injection in the footpad. At E7.5, dams received a second 1 mg dose of anti-Ifnar1 through an intraperitoneal route. (3) Wild-type dams, therapy studies: wild-type female and male mice were mated; at embryonic days E5.5, dams were treated with a 1 mg injection of anti-Ifnar1 (MAR1-5A3). At E6.5, mice were inoculated with mouse-adapted 103 FFU of ZIKV-Dakar by subcutaneous injection in the footpad. At E7.5, dams received a second 1 mg dose of anti-Ifnar1 as well as a single 250 μg dose of ZIKV mAb or isotype control through an intraperitoneal route. All animals were euthanized at E13.5, and placentas, fetuses and maternal tissues were collected. Fetus size was measured as the crown-rump length × occipitofrontal diameter of the head. ZIKV-infected tissues were weighed and homogenized with stainless steel beads in a Bullet Blender instrument (Next Advance) in 200 μl of PBS. Samples were clarified by centrifugation (2,000g for 10 min). All homogenized tissues from infected animals were stored at −20 °C. Tissue samples and serum from ZIKV-infected mice were extracted with RNeasy 96 Kit (tissues) or Viral RNA Mini Kit (serum) (Qiagen). ZIKV RNA levels were determined by TaqMan one-step quantitative reverse transcriptase PCR (qRT–PCR) on an ABI7500 Fast Instrument using published primers and conditions34. Viral burden was expressed on a log scale as viral RNA equivalents per g or ml after comparison with a standard curve produced using serial tenfold dilutions of ZIKV RNA. RNA in situ hybridization was performed with RNAscope 2.5 (Advanced Cell Diagnostics) according to the manufacturer’s instructions. PFA-fixed paraffin embedded placental sections were deparaffinized by incubation for 60 min at 60 °C. Endogenous peroxidases were quenched with H O for 10 min at room temperature. Slides were boiled for 15 min in RNAscope Target Retrieval Reagents and incubated for 30 min in RNAscope Protease Plus before probe hybridization. The probe targeting ZIKV RNA was designed and synthesized by Advanced Cell Diagnostics (catalogue number 467771). Negative (targeting bacterial gene dapB) control probes were also obtained from Advanced Cell Diagnostics (catalogue number 310043). Tissues were counterstained with Gill’s haematoxylin and visualized with standard bright-field microscopy. Collected placentas were fixed in 10% neutral buffered formalin at room temperature and embedded in paraffin. At least three placentas from different litters with the indicated treatments were sectioned and stained with haematoxylin and eosin to assess morphology. Surface area and thickness of placenta and different layers were measured using Image J software. For immunofluorescence staining on mouse placentas, deparaffinized tissues were blocked in blocking buffer (1% BSA, 0.3% Triton, PBS) for 2 h and incubated with anti-vimentin antibody (1:500, rabbit, Abcam ab92547). Secondary antibody conjugated with Alexa 488 (1:500 in PBS) was applied for 1 h at room temperature. Samples were counterstained with DAPI (4′6′-diamidino-2-phenilindole, 1:1,000 dilution). All virological data were analysed with GraphPad Prism software. Kaplan–Meier survival curves were analysed by the log rank test, and viraemia was compared using an ANOVA with a multiple comparisons test. P < 0.05 indicated statistically significant differences. All relevant data are included with the manuscript; source data for each of the main text figures is provided.


An apparatus and method for detecting microplate well surface contact and setting standoff is provided. The apparatus may include a sample probe, coupled to a spring-loaded carriage, and a sensor configured to detect when the sample probe is in contact with a surface. The sample probe is moved toward a surface of a well in a well-plate until the sample end of the sample probe contacts the surface, whereby the carriage allows the probe to be displaced. Displacement of the probe is detected by the sensor and further downward movement of the carriage is stopped. A processor records the location of the sample probe and sets standoff based on the recorded location.


A flow cytometer apparatus and methods for detecting and clearing a clog therein are disclosed. An example method for detecting a clog may include (i) detecting, via a fault detection system of a flow cytometer, a first plurality of events associated with a first aliquot from a first sample well, (ii) determining a count of the first plurality of events associated with the first aliquot, (iii) determining whether the count of the first plurality of events is below a minimum count tolerance and (iv) (a) if the count of the first plurality of events is below the minimum count tolerance, then determining that the flow cytometer has a clog, (b) if the count of the first plurality of events is equal to or above the minimum count tolerance, then detecting a second plurality of events associated with a second aliquot from a second sample well.


Trademark
Intellicyt | Date: 2016-06-17

Software for storing and managing and analyzing experiments comprising time segmented flow cytometry data; Software for cell and bead-based screening; Laboratory apparatus, namely, flow cytometers and flow-based analyzers providing cell, gene, protein, bead and particle analysis, diagnostics and drug discovery for scientific, analyzing time segmented flow cytometry data.


One embodiment of the present invention provides for a method for identifying within a single record the location of each of a plurality of samples suspected of containing particles of interest wherein the single record is obtained from a flowing stream of the plurality of samples passing through a particle analyzer. The method comprises introducing into a conduit the plurality of samples suspected of containing particles of interest wherein each ones of the plurality of samples are separated by fluid gaps to produce a plurality of samples separated by fluid gaps and wherein each of the plurality of samples further comprises marker particles. The plurality of samples separated by fluid gaps are flowed through the conduit as a flowing sample stream to a detector of a particle analyzer, which may be a flow cytometer. The particles of interest are detected as the samples pass the detector of the particle analyzer.


One embodiment of the present invention provides for a method for identifying within a single record the location of each of a plurality of samples suspected of containing particles of interest wherein the single record is obtained from a flowing stream of the plurality of samples passing through a particle analyzer. The method comprises introducing into a conduit the plurality of samples suspected of containing particles of interest wherein each ones of the plurality of samples are separated by fluid gaps to produce a plurality of samples separated by fluid gaps and wherein each of the plurality of samples further comprises marker particles. The plurality of samples separated by fluid gaps are flowed through the conduit as a flowing sample stream to a detector of a particle analyzer. The particle analyzer is for example a flow cytometer. The particles of interest when present and/or marker particles are detected as the plurality of samples pass the detector of the particle analyzer. A record over time for the particles of interest when present and/or marker particles in each of the plurality of samples are obtained in the single file once the plurality of samples pass the incident beam of light of the particle analyzer. A time position in the record is identified where particles of interest within any one of the plurality of samples would be located if present based upon the detection of marker particles present within each combined sample from the flowing stream of the plurality of samples.


Embodiments of the present invention provide a system and method for analyzing a plurality of samples comprising obtaining with an autosampler a plurality of samples from a first plate having a plurality of sample wells wherein the autosampler has a plurality of probes for sampling a set of samples and wherein each probe of the plurality of probes is in communication with a separate flow cytometer via a separate conduit. The plurality of samples comprising particles is moved into a fluid flow stream for each separate conduit. Adjacent ones of the plurality of samples are separated from each other in the fluid flow stream by a separation gas, thereby forming a gas-separated fluid flow stream. The gas-separated fluid flow stream is independently guided to and through each separate flow cytometer.


Embodiments of the present invention provide a system and method for analyzing a plurality of samples comprising obtaining with an autosampler a plurality of samples from a first plate having a plurality of sample wells wherein the autosampler has a plurality of probes for sampling a set of samples and wherein each probe of the plurality of probes is in communication with a separate flow cytometer via a separate conduit. The plurality of samples comprising particles is moved into a fluid flow stream for each separate conduit. Adjacent ones of the plurality of samples are separated from each other in the fluid flow stream by a separation gas, thereby forming a gas-separated fluid flow stream. The gas-separated fluid flow stream is independently guided to and through each separate flow cytometer.


Patent
Intellicyt | Date: 2015-06-03

A method for identifying sample boundaries of a plurality of samples is provided. The method includes moving a plurality of samples comprising particles into a fluid flow stream using a sampling probe and introducing marker beads into the fluid flow stream between adjacent samples in the plurality of samples via the sampling probe to produce a marker bead-separated plurality of samples in the fluid flow stream. Marker beads may be introduced into the fluid flow stream by introducing the marker beads from a chamber surrounding at least a portion of the sampling probe, or by introducing the marker beads from a cartridge having a pierceable membrane underlying at least one chamber, the at least one chamber containing marker beads. Sampling systems and apparatuses are also provided.

Loading Intellicyt collaborators
Loading Intellicyt collaborators