Journal: The Journal of Experimental Medicine

Article Title: Key role of T cell defects in age-related vulnerability to West Nile virus

doi: 10.1084/jem.20090222

Figure Lengend Snippet: Functional quantitative defects in T cell activation in response to WNV infection. Infection was as in . (A) CD8 + T cells derived from spleens of old and adult mice were harvested on day 8 and analyzed for the proportion (left and middle left, representative examples; middle right, aggregate analysis of relative numbers) and absolute numbers (right, absolute numbers) of cells expressing the CD8 + NS4b-2488:K b+ phenotype. Dots denote individual mice ( n = 8) and are representative of four experiments. (B) Same as A, except that measurements show relative representation and absolute numbers of CD8 + IFN-γ + cells in response to the immunodominant NS4b-2488 CD8 epitope by ICCS at the peak of the immune response (day 8). Old mice mobilized significantly fewer numbers of IFN-γ–producing WNV-specific CD8 + T cells than their adult counterparts. Results were compiled from three independent experiments. (C) Ratio between IFN-γ + and Tet + CD8 + cells shows that many fewer Ag-specific T cells are functionally able to manufacture this cytokine in the old mice. Groups, representation, and graphics are as in A, and results are representative of two experiments. Horizontal bars indicate mean values of the unpaired Student's t test. Error bars represent SEM.

Article Snippet: Peptides NS3 1616 , NS3 2066 , and NS4b 2488-2496 (21st Century Biochemicals) were diluted in 10% H 2 O and 90% DMSO and stored at −80°C.

Techniques: Functional Assay, Activation Assay, Infection, Derivative Assay, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Key role of T cell defects in age-related vulnerability to West Nile virus

doi: 10.1084/jem.20090222

Figure Lengend Snippet: Quality of WNV-specific responses in old mice is impaired at several levels. For all panels, animals were infected with 1,000 PFU WNV 385–99 and were analyzed on day 8, unless otherwise indicated. (A) At the peak of infection, splenic CD8 cells were stimulated with class I–restricted peptides and analyzed for the expression of IFN-γ, TNF, and GzB, and results were plotted to denote percentages of cells exhibiting all three molecules (3 Fxn), two molecules (2 Fxn), or a single molecule (1 Fxn). Results depict eight animals per group and are representative of two such experiments. (B) Relative intensity of expression of IFN-γ, TNF, and GzB among CD8 + cells in response to 6 h of NS4b-2488 peptide stimulation as described in Materials and methods. After gating on CD8 + T cells, normalized mean fluorescent intensity (MFI) was obtained by subtracting the mean fluorescent intensity of the negative cell population from that of the positive cells. Data depict five adult and six old individual mice, representative of four experiments, and are shown as in . (C) Expression of CD43 on WNV-specific CD8 cells from adult and old mice was analyzed on day 8 after infection by selective gating and is expressed as percentage of Tet + CD8 + cells that are CD43 + , shown for eight animals per group and representative of three such experiments. Horizontal bars in B and C indicate mean values of the unpaired Student's t test. (D) Ex vivo cytotoxic activity of adult and old CD8 + T cells demonstrates major age-related functional defects. Adult and old mice were infected s.c. with WNV at a dose (1,000 PFU) that caused decreased survival of old mice compared with adult animals. CD8 + T cells from adult mice exhibited stronger cytotoxic activity than CD8 + T cells from old mice when assayed directly ex vivo 7 d after infection in a 6-h standard 51 Cr-release assay. Peptide-coated NS4b 2488 (10 −6 M) EL-4 cells were used as targets. Values for five mice per group with standard deviations, representative of two experiments, are shown. P-values are depicted above the graphs.

Article Snippet: Peptides NS3 1616 , NS3 2066 , and NS4b 2488-2496 (21st Century Biochemicals) were diluted in 10% H 2 O and 90% DMSO and stored at −80°C.

Techniques: Infection, Expressing, Ex Vivo, Activity Assay, Functional Assay, Release Assay

Journal: The Journal of Experimental Medicine

Article Title: Key role of T cell defects in age-related vulnerability to West Nile virus

doi: 10.1084/jem.20090222

Figure Lengend Snippet: Analysis of brain infiltrates from WNV-infected adult and old mice. The brains of old and adult C57BL/6 mice were harvested 12 d after infection with 1,000 PFU WNV 385–99 s.c., sectioned, and costained for CD3 (green) and WNV (red). (A) Representative adult (top) and old (bottom) brains are shown at medium (left) and high (right) power. CD3 T cells are stained green and WNV-infected neurons are red. Bar: (left) 50 µm; (right) 20 µm. (B) Brains of 10 old and 10 adult mice harvested 10 d after infection with WNV. To generate five independent samples per group, with sufficient cell numbers for analysis, pools of two brains each were made and cells isolated using percoll gradient. The representation of cells using mononuclear/lymphocyte gate in representative adult (left) and old (middle) mice are shown. The aggregate analysis of cell percentages in this gate are shown on the right, illustrating that there are significantly fewer cells in the lymphocyte gate in old mice. Comparison is based on at least 5 × 10 5 collected events/sample. (C) Representation (left) and aggregate analysis (right) of the percentage of CD4 and CD8 T cells within the brain of old versus adult mice as determined by flow cytometry. Significant differences were seen in representation of these cell subsets as well. (D, left) Representation of CD8 NS4b-2488 tetramer + T cells among all CD8 T cells in the brains of old mice is further reduced compared with adults (P = 0.00372). (D, right) Illustration of cumulative effects of reduced representation of total lymphocytes, CD8 cells, and CD8 NS4b-2488 Tet + /GrB + T cells in the brains of old mice compared with adults (taken as 100%) reveal an ∼12× age-related difference. Experiment is representative of three independent experiments. Horizontal bars indicate mean values of the unpaired Student's t test. Error bars represent SEM.

Article Snippet: Peptides NS3 1616 , NS3 2066 , and NS4b 2488-2496 (21st Century Biochemicals) were diluted in 10% H 2 O and 90% DMSO and stored at −80°C.

Techniques: Infection, Staining, Isolation, Comparison, Flow Cytometry

Journal: PLoS Neglected Tropical Diseases

Article Title: T-Cell Memory Responses Elicited by Yellow Fever Vaccine are Targeted to Overlapping Epitopes Containing Multiple HLA-I and -II Binding Motifs

doi: 10.1371/journal.pntd.0001938

Figure Lengend Snippet: Blood samples were obtained from 220 vaccinees. 653 peptides from Env and non-structural proteins were organized in pools and tested by ELISPOT assay. Then, 108 immunogenic peptides were selected from the pools and tested individually. In total, 16 peptides were positive in at least 10% the subjects. Statistical association showed that nine immunogenic peptides were associated with HLA class-I. Seven from these peptides could activate CD4 + depleted PBMCs from YF-17DD vaccinees ex vivo . In addition, 9–10 mers peptides covering the NS4b 77–91 were cultured in CD8 + T-cells isolated from HLA-A*02:01-positive individuals. Four epitopes (NS4b 77–85 , NS4b 75–83 , NS4b 75–84 , NS4b 76–84 ) were able to induce a specific response. Biochemical binding assays indicated that the most prevalent immunogenic peptides could bind multiple HLA-II molecules.

Article Snippet: The peptides NS4b 77–85 , NS4b 76–84 , NS4b 75–83 , NS4b 75–84 were synthesized and purified by Genescript (New Jersey, USA).

Techniques: Enzyme-linked Immunospot, Ex Vivo, Cell Culture, Isolation, Binding Assay

Journal: PLoS Neglected Tropical Diseases

Article Title: T-Cell Memory Responses Elicited by Yellow Fever Vaccine are Targeted to Overlapping Epitopes Containing Multiple HLA-I and -II Binding Motifs

doi: 10.1371/journal.pntd.0001938

Figure Lengend Snippet: Summary of screening peptide pools (first round).

Article Snippet: The peptides NS4b 77–85 , NS4b 76–84 , NS4b 75–83 , NS4b 75–84 were synthesized and purified by Genescript (New Jersey, USA).

Techniques:

Journal: PLoS Neglected Tropical Diseases

Article Title: T-Cell Memory Responses Elicited by Yellow Fever Vaccine are Targeted to Overlapping Epitopes Containing Multiple HLA-I and -II Binding Motifs

doi: 10.1371/journal.pntd.0001938

Figure Lengend Snippet: Summary of screening individual peptides (second round).

Article Snippet: The peptides NS4b 77–85 , NS4b 76–84 , NS4b 75–83 , NS4b 75–84 were synthesized and purified by Genescript (New Jersey, USA).

Techniques:

Journal: PLoS Neglected Tropical Diseases

Article Title: T-Cell Memory Responses Elicited by Yellow Fever Vaccine are Targeted to Overlapping Epitopes Containing Multiple HLA-I and -II Binding Motifs

doi: 10.1371/journal.pntd.0001938

Figure Lengend Snippet: Most frequent peptides that activated T-cells from YF-17DD vaccinees.

Article Snippet: The peptides NS4b 77–85 , NS4b 76–84 , NS4b 75–83 , NS4b 75–84 were synthesized and purified by Genescript (New Jersey, USA).

Techniques:

Journal: PLoS Neglected Tropical Diseases

Article Title: T-Cell Memory Responses Elicited by Yellow Fever Vaccine are Targeted to Overlapping Epitopes Containing Multiple HLA-I and -II Binding Motifs

doi: 10.1371/journal.pntd.0001938

Figure Lengend Snippet: Statistical HLA class I association analysis.

Article Snippet: The peptides NS4b 77–85 , NS4b 76–84 , NS4b 75–83 , NS4b 75–84 were synthesized and purified by Genescript (New Jersey, USA).

Techniques:

Journal: PLoS Neglected Tropical Diseases

Article Title: T-Cell Memory Responses Elicited by Yellow Fever Vaccine are Targeted to Overlapping Epitopes Containing Multiple HLA-I and -II Binding Motifs

doi: 10.1371/journal.pntd.0001938

Figure Lengend Snippet: Determination of memory CD8 T-cell-restricted responses against the peptides associated with HLA class I molecules.

Article Snippet: The peptides NS4b 77–85 , NS4b 76–84 , NS4b 75–83 , NS4b 75–84 were synthesized and purified by Genescript (New Jersey, USA).

Techniques:

Journal: PLoS Neglected Tropical Diseases

Article Title: T-Cell Memory Responses Elicited by Yellow Fever Vaccine are Targeted to Overlapping Epitopes Containing Multiple HLA-I and -II Binding Motifs

doi: 10.1371/journal.pntd.0001938

Figure Lengend Snippet: A . Sequences of the peptides used to identify the immunogenic nonamer epitope in NS4b. IC 50 values for IEDB-AR prediction were calculated; B . PBMCs from HLA-A2 vaccinees were cultured in the presence or absence of 9–10mer (NS4b 77–85 , NS4b 76–84 , NS4b 75–83 , NS4b 75–84 ) peptides. ELISPOT assay; C. Intracellular cytokine staining were performed. All peptides analyzed could activate CD8 + T-cells and the expansion of NS4b 76–84 -specific CD8 + T-cells was the highest among the four peptides tested. Plots gated on CD8 + T-cells for one representative donor are shown.

Article Snippet: The peptides NS4b 77–85 , NS4b 76–84 , NS4b 75–83 , NS4b 75–84 were synthesized and purified by Genescript (New Jersey, USA).

Techniques: Cell Culture, Enzyme-linked Immunospot, Staining

Journal: PLoS Neglected Tropical Diseases

Article Title: T-Cell Memory Responses Elicited by Yellow Fever Vaccine are Targeted to Overlapping Epitopes Containing Multiple HLA-I and -II Binding Motifs

doi: 10.1371/journal.pntd.0001938

Figure Lengend Snippet: Binding affinity in nanomols of the most frequent peptides that activated T-cells.

Article Snippet: The peptides NS4b 77–85 , NS4b 76–84 , NS4b 75–83 , NS4b 75–84 were synthesized and purified by Genescript (New Jersey, USA).

Techniques: Binding Assay

Journal: Viruses

Article Title: The Autophagosomes Containing Dengue Virus Proteins and Full-Length Genomic RNA Are Infectious

doi: 10.3390/v13102034

Figure Lengend Snippet: DENV2 increases LC3-II level, autophagic vesicles and viral proteins are colocalized with LC3 protein in infected lung cancer cells. ( A ) A549 cells were treated with mock, live DENV2 (MOI = 10), or heat-inactivated DENV2 (iDENV2; MOI = 10) for 36 h. Protein was extracted from the whole cell lysate and analyzed by immunoblotting for NS3 and LC3 expression. β-actin was used as the internal control. ( B ) A549 cells after infection were fixed at 36 h post-infection (p.i.) and observed under TEM. Scale = 2 µm. The white arrow represents double-membrane autophagosome in (a). Single membrane autophagic vesicles in (b). Scale bar = 0.2 µm in the enlarged picture (a). ( C ) Cells were infected with or without DENV2 (MOI = 10) for 36 h and examined for the colocalization of LC3 (green) and DENV2-Capsid, Envelope, NS1, NS3 and NS4B (red) under confocal microscopy. The yellow dots indicate colocalization of DENV2 proteins and LC3. Scale bar = 10 µm. ( D ) Quantification of the colocalization between DENV2 proteins (capsid, envelope, NS1, NS3 and NS4B) and LC3 puncta in DENV2-infected cells. The percentage of colocalization was quantified by counting the number of yellow dots in the cells and the cells containing more than five yellow dots represent colocalization-positive. Three fields of 10 cells each were countered under each experimental condition. The data shows Mean ± SD and the p values were determined by Student’s t -test analysis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were blocked with 5% skim milk for 1 h. The primary antibodies LC3B (MBL), capsid (GeneTex, Irvine, CA, USA), envelope (GeneTex), NS1 (Sigma), NS3 (GeneTex) and NS4B (GeneTex) were used to detect specific proteins.

Techniques: Infection, Western Blot, Expressing, Confocal Microscopy

Journal: Viruses

Article Title: The Autophagosomes Containing Dengue Virus Proteins and Full-Length Genomic RNA Are Infectious

doi: 10.3390/v13102034

Figure Lengend Snippet: DENV2 capsid, envelope, NS1, NS3 and NS4B proteins are abundantly detected in the purified autophagosomes of infected lung cancer A549 cells. ( A ) Cells were infected with DENV2 (MOI = 10) for 36 h followed by treatment with chloroquine (CQ, 50 μM) for another 24 h to block lysosome fusion and degradation. The autophagosome was purified by sucrose gradient centrifugation and observed under TEM. Scale bar = 100 nm. The arrow indicates the double-membrane of the purified autophagosome. ( B ) The protein expression of the capsid, NS1 and NS3 was detected by specific antibodies. Negative control (N.C.) used IgG antibody for immunogold-labeling. Immuno-gold labeled NS1 (18 nm gold beads), Capsid (12 nm gold beads) and NS3 (12 nm gold beads) in the purified autophagosomes were detected under the TEM. The quantification of gold particles for each protein inside autophagosomes was shown. Scale bar = 100 nm. * p < 0.05, ** p < 0.01, *** p < 0.001. ( C ) Lung cancer cells (A549) without DENV infection and culture for 36 h followed by blocking autophagosome and lysosome fusion with chloroquine (CQ, 50 μM) for another 24 h. Sucrose gradient centrifugation was conducted to obtain purified AP and PNS. A total of 10 μg protein from AP and PNS was loaded in SDS PAGE and analyzed by electrophoresis. The protein expression of LC3, NS3, HMGB1, Hsp 60 and Calreticulin was determined by immunoblotting. ( D ) Lung cancer cells (A549) were infected with DENV2 (MOI = 10) for 36 h followed by blocking autophagosome and lysosome fusion with CQ (50 μM) for another 24 h. Sucrose gradient centrifugation was conducted to obtain purified autophagosome (AP) and post-nucleus supernatant (PNS). A total of 10 μg protein from AP and PNS was loaded in SDS PAGE and analyzed by electrophoresis. The protein expression of LC3, LAMP1, capsid, envelope, NS1, NS3, NS4B, NS5, prM and HMGB1 was determined by immunoblotting using specific antibodies. LAMP1 is the marker of the lysosome and calreticulin is the marker of the endoplasmic reticulum.

Article Snippet: Membranes were blocked with 5% skim milk for 1 h. The primary antibodies LC3B (MBL), capsid (GeneTex, Irvine, CA, USA), envelope (GeneTex), NS1 (Sigma), NS3 (GeneTex) and NS4B (GeneTex) were used to detect specific proteins.

Techniques: Purification, Infection, Blocking Assay, Gradient Centrifugation, Expressing, Negative Control, Labeling, SDS Page, Electrophoresis, Western Blot, Marker

Journal: Viruses

Article Title: The Autophagosomes Containing Dengue Virus Proteins and Full-Length Genomic RNA Are Infectious

doi: 10.3390/v13102034

Figure Lengend Snippet: DENV2 proteins were not degraded by the conventional autophagic degradation process. ( A ) The protein expression levels of DENV2 capsid, envelope, NS3 and NS4B, as well as autophagy LC3 at 12, 24, 36 and 48 h in A549 cells with or without DENV2 (MOI = 10) infection, were investigated by immunoblotting using specific antibodies. ( B ) A549 cells were infected with or without DENV2 (MOI = 10) for 12 and 24 h followed by co-treatment with CQ (50 μM) for another 24 h. The whole-cell lysate extracted from each group was analyzed for the expression levels of DENV2 capsid, envelope, NS3 and NS4B, as well as autophagy LC3 and p62 proteins by immunoblotting using specific antibodies. β-actin was used as the internal control.

Article Snippet: Membranes were blocked with 5% skim milk for 1 h. The primary antibodies LC3B (MBL), capsid (GeneTex, Irvine, CA, USA), envelope (GeneTex), NS1 (Sigma), NS3 (GeneTex) and NS4B (GeneTex) were used to detect specific proteins.

Techniques: Expressing, Infection, Western Blot