vivo optical imaging analyses  (Revvity)

Journal: PLoS Pathogens

Article Title: Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV

doi: 10.1371/journal.ppat.1008560

Figure Lengend Snippet: A) Experimental scheme of iDCgB immunization and HCMV infection in humanized mice. Mice were transplanted with CB-CD34 + cells and immunized with autologous iDCgB at weeks 6, 7, 10, 11 after HCT. HCMV infection was performed at week 17 post HCT with 1x10 6 HCMV/GLuc-infected MRC-5 cells (i.p.). From week 24 to 25, all mice were treated with granulocyte-colony stimulating factor (G-CSF, 2.5 μg/mouse/day) for 7 days in order to reactivate HCMV. B) Kinetics of human hematopoiesis in blood. The frequencies of human CD45 + (left) and CD3 + positive cells (right) were analyzed (at weeks 15, 17, 20, 24, 25) in blood of humanized mice comparing HCMV (blue) and iDCgB/HCMV cohorts (red). Human hematopoietic reconstitution was persistent and comparable for both cohorts. C) Mesenteric, axillary, inguinal, iliac and total detectable lymph nodes were quantified. Graph shows the averages with the standard deviation of the mean for HCMV (blue) and iDCgB/HCMV (red) cohorts. D) Optical imaging analysis of the bioluminescence signal detected in the torso and abdomen and displayed in photons/sec/cm 2 /sr for all mice (left panels: HCMV; right panels: iDCgB/HCMV). Data was obtained with three independent experiments using cord blood from three donors (D1, D2, D3). Signal intensity was measured with the same settings for all cohorts and mice with 300 sec exposure time, f-stop 1 and medium binning. E) Quantified bioluminescence signal displayed in photons/sec showing decrease of signal in iDCgB/HCMV cohort (red dots) compared with HCMV cohort (blue dots). ROI was set the same for all mice encompassing the whole mouse body. A significant protective effect caused by iDCgB immunization is noticeable. For statistical analysis t-Test with Welch’s correction was used and significances are indicated with ***p<0.001. Standard deviation of the mean is indicated. F) HCMV DNA viral copies were determined by quantitative PCR in LI (HCMV n = 12; iDCgB/HCMV n = 16 available) and BM (HCMV n = 8 available; iDCgB/HCMV n = 12 available). Viral copies are indicated in log 10 copies/μgDNA. For statistical analysis, t-Test with Welch’s correction was used and significances are indicated with **p<0.01 and ***p<0.001. The standard deviation of the mean is indicated. G) Total counts (# positive cells) for huCD45 + /CD4 + T cells (left) and huCD45 + /CD8 + T cells (right) measured for cells recovered from SPL (left panels) and BM (right panels) for different subtypes (N, CM, EM, TE) in the HCMV (blue dots) or the iDCgB/HCMV cohort (red dots). Representative gating example for CD45RA and CD62L staining is shown in . Each dot represents one mouse. H) Frequencies of PD-1 + CD3 + / CD4 + / CD8 + T cells (% positive cells) in LN of HCMV (blue dots) or iDCgB/HCMV cohort (red dots). I) In vitro analyses of antigen-specific T cell responses. mLN of the mice for each cohort were pooled, cohorts were analyzed separately and then the data was merged (HCMV: blue; n = 1 cohort; iDCgB/HCMV: red; n = 2 cohorts). The cells were supplemented with cytokines, stimulated with recombinant proteins (pp65, IE1 or gB) in vitro and analyzed by flow cytometry (Ø denotes no antigen stimulation). EM CD8 + or CD4 + cells were gated and IFN-γ + or TNF-α + cells were quantified. The schematic representation of the assay and exemplary gating approaches are shown in . Left panels: Detection of IFN-γ expression in CD8 + (top) or CD4 + (lower panel) T cells. Left panels: Detection of TNF-α expression in CD8 + (top) or CD4 + (lower panel) T cells. J) Total counts (# positive cells) of pan-B cells (CD19 + ), memory B cells (CD19 + CD27 + ), IgA + / IgG + / IgM + B cells, plasmablasts (PB) and plasma cells (PC) measured in HCMV (blue dots) or iDCgB/HCMV cohort (red dots). Exemplary gating approach is shown in . Each experiment was repeated at least twice with similar results. Negative binominal regression analyses were performed for all statistical determinations of the flow cytometry analysis for the total cells (# positive cells). Beta regression analysis was performed for frequencies (% positive cells). Significances are depicted with *p<0.05, **p<0.01, ***p<0.001. K) Total human IgG measured in plasma by ELISA (μg/ml) for HCMV (blue dots, n = 6 available) or iDCgB cohort (red dots, n = 9 available) (p = 0.0508 was determined with t-Test with Welch’s correction). L), M) Representative examples showing detection of IgM - IgG + positive B cells binding to gB recovered from SPL of HCMV or iDCgB/HCMV cohorts, respectively. Left panels: Gating of IgM - /IgG + cells. Right panels: Gating of gB + binding cells showing frequency within the IgM - /IgG + population. Exemplary gating approach with all markers used is shown in Supplementary Material. N) Plasma was used in strips immunoassays for the qualitative determination of IgG-reactivity against HCMV antigens (IE1, CM2, p150, pp65, gB1 and gB2). Three representative stripes are shown for iDCgB/HCMV (top, note broad cross-reactivity against multiple antigens or HCMV (bottom). Qualitative results for all the mice analyzed are shown in . O) Antinuclear antibodies (ANAs) that bind to contents of the cell nucleus were investigated. HEp-2 cells were used as a substrate to detect the auto-antibodies by indirect immunofluorescence. Human sera obtained from two subjects with autoimmunity disorders were used as reference showing bright signals in nucleus and occasionally in cytoplasm (left panels). Similar analyses performed with plasma obtained from mice in the HCMV cohort showed 6 out of 7 cases ANA positivity (middle panels). One single mouse of the 10 cases analyzed in the iDCgB/HCMV cohort showed faint immunofluorescence signals in the cytoplasm.

Article Snippet: In vivo optical imaging analyses were performed with an IVIS SpectrumCT (PerkinElmer, Waltham, MA) and all pictures were taken with a field of view C, f stop 1 and medium binning for each mouse.

Techniques: Infection, Standard Deviation, Optical Imaging, Real-time Polymerase Chain Reaction, Staining, In Vitro, Recombinant, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay, Immunofluorescence

Journal: PLoS Pathogens

Article Title: Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV

doi: 10.1371/journal.ppat.1008560

Figure Lengend Snippet: A) Kolmogorov-Smirnov (KS) test was performed on the cellular immunophenotypic biomarkers (without missing values) using all samples (incorporating both iDCgB/HCMV (n = 17); HCMV (n = 12) and in total n = 29). The sensitivity of the KS test on the sample size was evaluated by leave one-sample out (LOO), repeated for all samples. The thresholds for KS statistic and p-value were 0.3 and 0.1, respectively. The frequency of each biomarker satisfying the KS test criteria in LOO is shown. B) The sample distribution is shown for each single biomarker that satisfied the KS test thresholds and appeared with highest frequency in LOO. C) Four biomarker combination sets selected according to the pipeline (see ) and showed 73–87% accuracies to predict the iDCgB/HCMV group. D) Principal component analysis was performed for the four selected biomarkers sets (shown in c). The distribution of samples in the first principal component (PC1) is shown. E) Effects of anti-CD20 (B cell) depletion in the HCMV/GLuc infection signal. Top panels: Baseline optical imaging analyses of HCMV-infected mice (Reconstitution R1, HCMV, n = 3) or iDCgB immunized and then infected with HCMV (HCMV+iDCgB, n = 4; R1: iDCgB generated with 2 vectors; R2: iDCgB generated with a tricistronic lentiviral vector). Bottom panels: Optical imaging analyses 5 days after anti-CD20 (10 mg/kg) treatment for B cell depletion. All mice were analyzed with the same settings; the radiance of the bio-luminescence signals is indicated by the colored bar on the right side (p/sec/cm 2 /sr). F) Quantified total Flux (photons/ second, p/s) for HCMV and HCMV+iDC/gB cohorts, before and after anti-CD20 depletion. ROI was quantified for the frontal torso and abdomen and kept constant for all mice. The horizontal bars in black indicate the median values for each cohort and time of analyses. The P values were determined by t-Test. The % increase is relative to the baseline values prior to depletion (see and Tables ).

Article Snippet: In vivo optical imaging analyses were performed with an IVIS SpectrumCT (PerkinElmer, Waltham, MA) and all pictures were taken with a field of view C, f stop 1 and medium binning for each mouse.

Techniques: Biomarker Assay, Infection, Optical Imaging, Generated, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV

doi: 10.1371/journal.ppat.1008560

Figure Lengend Snippet: A) Scheme of ELISA assay using wells coated with recombinant gB protein. Monoclonal IgGs were serially diluted, transferred to wells and immune detection was performed with an HRP-conjugated antibody against human IgG. Assays were performed in duplicate independent experiments and the results were merged for plotting the data. B) OD450 measurements for reference SM5-1 monoclonal IgG (orange), Kiovig pooled IgG (green) and negative control (grey). C) OD450 measurements for monoclonal IgGs derived from iDCgB immunized mice (black) are shown. SM5-1 was included as a reference (orange). D) OD450 measurements for monoclonal IgGs derived from HCMV (blue) and iDCgB/HCMV (red) cohorts are shown. SM5-1 was included as a reference (orange). E) Scheme of ELISA assay using wells coated with protein lysates from 293T/gB cells. Monoclonal IgGs were serially diluted, transferred to wells and immune detection was performed with an HRP-conjugated antibody against human IgG. Assays were performed in duplicate independent experiments and the results were merged for plotting the data. F) OD450 measurements for reference SM5-1 monoclonal IgG against HCMV gB (orange), Kiovig pooled IgG (green) and negative control (grey). G) OD450 depicted for monoclonal antibodies derived from iDCgB immunized (black) cohort. SM5-1 (orange) measurement was included as a reference. H) ELISA (OD450) measurements for monoclonal IgG derived from HCMV (blue) and iDCgB/HCMV (red) cohort. SM5-1 (orange) was included as reference. For F) G) H) Wells coated with protein lysates obtained from control 293T/w.t. cells were included in the ELISA assay as negative control. No cross-reactivity was detectable. I) Scheme of the in vitro neutralization assay. Antibodies were serially diluted and incubated with HCMV prior to infection. TB40-GLuc viruses were pre-incubated with the antibodies for 1 h and MRC-5 cells were infected with the virus-antibody mixture. Spinoculation was performed and 1h later medium was exchanged. The catalytic activity of the secreted GLuc signal was measured in the supernatant 24h later by luminometry as relative light units (RLU). The experiment was performed as independent duplicates and the results were merged. The % neutralization (y-axis) was plotted against the log 10 μg/ml antibodies (x-axis), the neutralization curves were adjusted after non-linear regression analyses and the IC 50 values were calculated . J) % neutralization of HCMV by the reference SM5-1 IgG (light green) and Kiovig polyclonal antibodies (dark green). Infected MRC-5 cells not exposed to the mAbs were included for RLU measurements and used as 100% infection reference (Mock, grey). K) % neutralization of HCMV by mAbs DC06 and DC16 derived from the iDCgB cohort (black). SM5-1 IgG (light green) is shown as reference. L) % neutralization of HCMV by the mAb PR32 derived from a HCMV-protected mouse of the iDCgB/HCMV cohort (red). SM5-1 IgG (light green) is shown as reference. The other six cloned antibodies (DC14, DC17, CV03, CV04, PR17, PR28 and PR35) did not neutralize HCMV infection. All assays were performed in duplicate independent experiments. For ELISA and neutralization assays, measurements were taken for two and four wells, respectively. For all graphs error bars indicate standard deviation of the mean and corresponding EC 50 and IC 50 values are shown in . M) Schematic representation of the experiment to test the effects of passive immunization by adoptive antibody transfer against HCMV. For the control group, mice were infected with HCMV 16 weeks after HCT and G-CSF treatment was performed between weeks 19 and 20 after HCT. For the passive immunization cohort, mice were injected i.v. with the monoclonal antibodies (DC06 and PR32, 0.5 mg each in total 1.0 mg) on the day prior to HCMV infection. After infection, the mice were injected i.v. with the monoclonal antibodies (DC06 and PR32, 0.125 mg each in total 0.25 mg) additional seven times. N) Optical imaging analysis performed for the control HCMV cohort (left) or the cohort treated with mAbs (D06 and PR32) to test the effects of passive immunization against HCMV infection (top, “INFEC”) and after HCMV reactivation induced by G-CSF treatment (bottom, “REAC”). All mice were analyzed with the same settings; the range of the bio-luminescence signals is indicated by the colored bar on the right side (radiance: p/sec/cm 2 /sr). O) Quantified total Flux (phonons/second, p/s) for the control (CTR/HCMV, blue) or mAb-treated cohort (mABs/HCMV, red). ROI was quantified for the frontal torso and abdomen and kept constant for all mice. Data was obtained for the two time points to monitor INFEC (left) and REAC (right). P) Quantification of HCMV viral copies (log 10 copies/μg DNA) detected in liver (LI) and bone marrow (BM) comparing HCMV-infected mice (blue) and mice treated by with mAbs (red). The horizontal bars in black indicate the median values for each cohort and time of analyses. The P values were determined by t-Test. The % increase is relative to the CTR non-treated cohort (see ).

Article Snippet: In vivo optical imaging analyses were performed with an IVIS SpectrumCT (PerkinElmer, Waltham, MA) and all pictures were taken with a field of view C, f stop 1 and medium binning for each mouse.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Negative Control, Derivative Assay, Control, In Vitro, Neutralization, Incubation, Infection, Virus, Activity Assay, Clone Assay, Standard Deviation, Injection, Optical Imaging