Journal: PLoS ONE

Article Title: Immunogenicity, Protective Efficacy, and Non-Replicative Status of the HSV-2 Vaccine Candidate HSV529 in Mice and Guinea Pigs

doi: 10.1371/journal.pone.0121518

Figure Lengend Snippet: BALB/c mice (n = 10/group) were immunized with HSV529 (10 4 CCID 50 , 10 5 CCID 50 , or 10 6 CCID 50 ) or PBS by the i.m. route on days 0 and 21. Sera were collected on days 21 (D21; n = 10) and 41 (D41; n = 5). (A) HSV-2-specific IgG1 and IgG2a antibody titers in the sera were determined by ELISA using a lysate prepared from HSV-2-infected Vero cells and secondary antibodies specific for mouse IgG1 and IgG2a. (B) HSV-2 neutralizing antibodies in the sera were measured by preincubating dilutions of heat-inactivated sera with 100 CCID 50 of live HSV-2 (strain G) virus for 1 hour prior to infection of Vero cell cultures. Infected cells were detected with anti-HSV glycoprotein D antibodies. The serum dilution that neutralized 50% of the virus (SN 50 ) was determined by plotting the neutralization activity versus the serum dilutions. Splenic lymphocytes secreting IFNγ (C) or IL-5 (D) in response to ex vivo stimulation with heat-inactivated HSV-2 (strain G) were counted using an ELISPOT assay. Error bars represent standard error of the mean.

Article Snippet: The plates were washed and the locations where cells secreting INFγ or IL-5 during incubation were stained with a biotinylated anti-mouse IFNγ or IL-5 antibody (BD Pharmigen, Le Pont de Claix, France), a streptavidin-horseradish peroxidase conjugate (Southern Biotechnology), and a chromogenic substrate (amino ethyl carbazole; Sigma-Aldrich, St. Louis, MO).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Neutralization, Activity Assay, Ex Vivo, Enzyme-linked Immunospot

Journal: The Journal of Biological Chemistry

Article Title: Peptide-Major Histocompatibility Complex Dimensions Control Proximal Kinase-Phosphatase Balance during T Cell Activation

doi: 10.1074/jbc.M109.039966

Figure Lengend Snippet: T cell receptor triggering and effector responses are abrogated by pMHC elongation in a murine system. A , schematic representation of native and elongated versions of D b SCT containing the indicated membrane-proximal IgSF spacers, tethered to the plasma membrane by the native H-2D b stalk and transmembrane segments. B , surface expression levels of SCT constructs in TAP2-deficient CHO cells. C , primed transgenic T cells expressing the F5 TCR (10 4 T cells/well) were incubated with varying numbers of SCT-expressing CHO cells (denoted as APC/F5 ratio). Culture supernatants were assayed for IL-2 by ELISA after 24 h of incubation. A representative plot of three independent experiments is shown. The error bars represent ± S.D. D , F5/APC conjugates were incubated for 2 min at 37 °C before fixing and stained for phosphotyrosine and H-2D b (SCTs). The conjugates were imaged by confocal immunofluorescence microscopy (Fig. S1A ). Untransfected CHO cells were used to determine basal phosphorylation levels. Phosphotyrosine accumulation at the T cell interface was quantified as the ratio of interface/noninterface fluorescence ( black bars ). At least 25 conjugates were randomly chosen for analysis. Accumulation of SCTs was measured using a similar approach ( supplemental Fig. S1 B ). As a control, conjugates of B3Z hybridomas expressing the noncognate OT1 TCR and CHO cells expressing D b SCT were used. Accumulation of SCT at the interface was quantified for 19–25 randomly chosen conjugates for each SCT ( purple bars ). The error bars represent S.E. The dashed line represents enrichment ratio of 1 (no enrichment) E , fully differentiated F5 CTL were assessed for sensitivity to elongated pMHC expressed at high levels (as in B ) on CHO cells. Cytotoxic responses of F5 CTL to SCTs was tested in a 6-h 51 Cr release assay using CHO cells expressing native and maximally elongated (SCT-CD4) SCTs as targets. The data are representative of two independent experiments. The error bars represent ± S.D. F , cytotoxic responses by F5 CTL to low levels (∼50-fold lower than cells shown in B ) of SCT expression ( inset ). The data are representative of two independent experiments. G , cytolytic activity of F5 CTL and IL-2 release by F5 RAG −/− T cells in response to high levels of SCT expression (as in B ) in the presence of 0–10 μ m PP2 was measured as in C and E . The data are expressed as the percentages of reduction in response relative to responses in the absence of PP2. The concentration of PP2 that gave 50% inhibition (IC 50 ) was derived from error-weighted sigmoidal fitting of the data. The error bars represent ± S.D.

Article Snippet: For IL-2 measurements, culture supernatants and mouse recombinant IL-2 standards (Sigma) were incubated in microtitre plates coated with a capture anti-mouse IL-2 antibody (BD Pharmingen) for 2 h at 37 °C, washed, and incubated for another 1 h with a biotinylated detection anti-mouse IL-2 antibody (BD Pharmingen).

Techniques: Expressing, Construct, Transgenic Assay, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Microscopy, Fluorescence, Release Assay, Activity Assay, Concentration Assay, Inhibition, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Peptide-Major Histocompatibility Complex Dimensions Control Proximal Kinase-Phosphatase Balance during T Cell Activation

doi: 10.1074/jbc.M109.039966

Figure Lengend Snippet: Elongation of a human pMHC (gagSLY/HLA-A2) abrogates activation of G10 T cells expressing cognate TCR. A , schematic representation of single-chain constructs of native and elongated single-chain versions of HLA-A2/gag and corresponding coreceptor binding mutants ( yellow stars ). Constructs were stably expressed in TAP2-deficient CHO cells and sorted for comparable expression for use as surrogate antigen-presenting cells ( supplemental Fig. S3 A ). Also shown are soluble forms of native and elongated constructs comprising the extracellular portion followed by a C-terminal biotin acceptor site ( red dot ) and His 6 tag ( green line ). B , activation of primed G10 T cells was assessed by incubating 10 4 G10 cells/well with varying numbers of SCT-expressing APC (denoted as APC/G10 ratio) and IFNγ release measured by ELISA of culture supernatants after 8 h incubation. A representative plot of three independent experiments is shown. C , the human T cell clone SLY10 was used to assess responses to SCTs with mutated coreceptor-binding sites. Varying numbers of SCT-expressing CHO cells were incubated with 10 4 SLY10 cells/well, and MIP1b release was measured by ELISA of culture supernatants after 24 h of incubation. D , coreceptor binding to native and elongated SCTs was assessed by surface plasmon resonance (BIAcore). Monomeric biotinylated SCTs were immobilized by coupling to streptavidin conjugated CM5 flow cells (∼500 reference units). Binding curves were obtained by injecting CD8αα at a range of concentrations (0.9–154 μ m ) over flow cell surfaces immobilized with native or elongated SCTs. Immobilized biotinylated polyclonal anti-hamster IgG antibody was used as a control surface. The experiments were performed at 25 °C at a flow rate of 5 μl/min. Plateau response units were plotted against CD8αα concentration, and K D values were obtained by nonlinear curve fitting. The data are representative of two independent experiments. E , binding of native and elongated SCTs to G10 TCR was compared by surface plasmon resonance as described for D . The binding curves were obtained by injecting G10 TCR (0.1–7 μ m ) over immobilized SCTs, HLA-A2/NY-ESO, in addition to an irrelevant antibody-bound control surface.

Article Snippet: For IL-2 measurements, culture supernatants and mouse recombinant IL-2 standards (Sigma) were incubated in microtitre plates coated with a capture anti-mouse IL-2 antibody (BD Pharmingen) for 2 h at 37 °C, washed, and incubated for another 1 h with a biotinylated detection anti-mouse IL-2 antibody (BD Pharmingen).

Techniques: Activation Assay, Expressing, Construct, Binding Assay, Stable Transfection, Enzyme-linked Immunosorbent Assay, Incubation, SPR Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Peptide-Major Histocompatibility Complex Dimensions Control Proximal Kinase-Phosphatase Balance during T Cell Activation

doi: 10.1074/jbc.M109.039966

Figure Lengend Snippet: TCR·CD3 triggering but not clustering is diminished by pMHC elongation. A , clean poly- l -lysine coated coverslips were incubated at 37 °C for 2 h with 50 μg/ml streptavidin/PBS. Biotinylated SCTs were immobilized on coverslips at comparable densities (Fig. S3 ). G10 cells were placed on SCT-coated coverslips and incubated for 1 min at 37 °C. The cells were fixed and permeabilized before serial staining CD3, Zap70, and CD45. The images were acquired using uniform settings between experiments. The cells adhered poorly on uncoated coverslips; therefore coverslips coated with biotinylated anti-HLA antibody was used as a control surface (without cognate TCR ligands). The scale bar represents 4 μm. B–D , the mean intensities for CD3, Zap70, and CD45 accumulation at the T cell interface was quantified. For a better appreciation of regions of TCR clustering pseudocolor scaled images of the CD3 distribution are shown (CD3 Scale) in A and E . The regions of CD3 clustering were arbitrarily defined by thresholding at two times the mean CD3 fluorescence intensity at the T cell interface (corresponding to the yellow regions in the pseudocolored scale images) and expressed as percentages of total CD3 interface fluorescence (Fig. S4 ). E , G10 cells were incubated with 10 μ m PP2 for 30 min prior to placing on to SCT-coated coverslips. Preparation and labeling were performed as described above. The scale bar represents 4 μm. The horizontal black bars represent the means. Statistical significance was determined by analysis of variance with correction for multiple comparisons. ns , p > 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: For IL-2 measurements, culture supernatants and mouse recombinant IL-2 standards (Sigma) were incubated in microtitre plates coated with a capture anti-mouse IL-2 antibody (BD Pharmingen) for 2 h at 37 °C, washed, and incubated for another 1 h with a biotinylated detection anti-mouse IL-2 antibody (BD Pharmingen).

Techniques: Incubation, Staining, Fluorescence, Labeling

Journal:

Article Title: Inhibition of endogenous thioredoxin in the heart increases oxidative stress and cardiac hypertrophy

doi: 10.1172/JCI200317700

Figure Lengend Snippet: The activities of the Ras–Raf-1–ERK pathway, p38-MAPK, and p46/p54–JNK1 were examined in Tg-DN-Trx1 mice and NTg littermates. (a) Some mice were subjected to aortic banding for 2 weeks. Activities of p42/p44–ERK (a), Raf-1 (b), p38-MAPK (e), and p46/p54–JNK1 (f) were determined using anti-phosphospecific Ab’s. The same filter was reprobed with respective non-phosphospecific Ab. (e) Cell extracts prepared from cardiac myocytes overexpressing mammalian sterile 20–like kinase 1 (Mst1) were used as positive control (P/C). The gel picture is representative of three to ten experiments in each immunoblot analysis. (c) The activity of Ras was determined using the Ras-binding domain in Raf-1 coupled with agarose. (d) S-thiolation of Ras was determined as described in Methods. COS-7 cells grown in 60-mm dishes were transfected with the indicated expression plasmids (2 μg). Cells were incubated with biotinylated cysteine (0.5 mM), and S-thiolated proteins were isolated by streptavidin-Sepharose. Samples were subjected to immunoblot analyses with anti-Ras Ab. AS-Trx1, antisense Trx1.

Article Snippet: After washing, biotinylated secondary Ab (anti-mouse IgG; BD Pharmingen) was applied for 1 hour, followed by streptavidin-HRP (BD Pharmingen) for 30 minutes at room temperature.

Techniques: Positive Control, Western Blot, Activity Assay, Binding Assay, Transfection, Expressing, Incubation, Isolation

Journal:

Article Title: Interleukin-6 Enhances Production of Anti-OspC Immunoglobulin G2b Borreliacidal Antibody

doi: 10.1128/IAI.69.7.4268-4275.2001

Figure Lengend Snippet: Titers of borreliacidal antibody in supernatants obtained from cultures of lymph node cells containing B. burgdorferi isolate 50772 and 1, 10, or 100 μg of anti-IL-6 after incubation for 7 days. Lymph node cells were obtained from C3H/HeJ mice 28 days after vaccination. Control cultures were treated with a goat isotype-nonspecific IgG antibody.

Article Snippet: They were then washed five times with PBS-T and incubated for 45 min at 21°C with 50 μl of biotinylated rat anti-murine IL-6 (MP5-32C11 clone; PharMingen) diluted in PBS-FBS to 1 μg/ml.

Techniques: Incubation

Journal:

Article Title: Interleukin-6 Enhances Production of Anti-OspC Immunoglobulin G2b Borreliacidal Antibody

doi: 10.1128/IAI.69.7.4268-4275.2001

Figure Lengend Snippet: Immunoblots of B. burgdorferi isolate 50772 after treatment with supernatant obtained from cultures of lymph node cells containing B. burgdorferi isolate 50772 and 100 μg of anti-murine IL-6. Supernatant was collected on day 7 of incubation. Lymph node cells were obtained from C3H/HeJ mice 28 or 35 days after vaccination. Control cultures were treated with a goat isotype-nonspecific IgG antibody.

Article Snippet: They were then washed five times with PBS-T and incubated for 45 min at 21°C with 50 μl of biotinylated rat anti-murine IL-6 (MP5-32C11 clone; PharMingen) diluted in PBS-FBS to 1 μg/ml.

Techniques: Western Blot, Incubation

Journal: Human Gene Therapy

Article Title: Nonintegrating Lentiviral Vectors Can Effectively Deliver Ovalbumin Antigen for Induction of Antitumor Immunity

doi: 10.1089/hum.2009.012

Figure Lengend Snippet: Nonintegrating lentiviral vector expressing OVA antigen was comparable to its integrating counterpart in inducing antigen-specific CTLs and humoral responses in vivo. (A–G) B6 mice were immunized with either FKOVA/VSVG(IN+) or FKOVA/VSVG(IN–) via footpad injections. The amount of viral particles used for each immunization was quantified to be 900 ng of p24. Mice without immunization (No LV) were included as a control. Mice were analyzed 2 weeks postimmunization. (A) T cells were collected from spleens and examined for the presence of OVAp1-specific CD8+ T cells by tetramer staining. (B) Spleen cells from (A) were restimulated with OVAp1 for 6 hr and IFN-γ secretion was evaluated by intracellular staining. (C) IgG serum specific for OVA was assessed by ELISA. (D–G) On restimulation with OVAp1 or OVAp2, spleen cells from (A) were analyzed by ELISPOT to measure IFN-γ or IL-2 spot-forming cells.

Article Snippet: On the third day, biotinylated anti-mouse IFN-γ or IL-2 antibodies (BD Biosciences) were added to the plate, followed by the addition of streptavidin–alkaline phosphatase (Millipore) and BCIP/NBTplus substrate (Chemicon International, Temecula, CA) to develop spots.

Techniques: Plasmid Preparation, Expressing, In Vivo, Staining, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Human Vaccines & Immunotherapeutics

Article Title: Intranasal immunization with a single dose of the fusion protein formulated with a combination adjuvant induces long-term protective immunity against respiratory syncytial virus

doi: 10.1080/21645515.2017.1349584

Figure Lengend Snippet: RSV ΔF-specific systemic humoral immune responses in mice. (A) Serum IgG1 and IgG2a titers before challenge (B/C), (B) Serum IgG1 and IgG2a titers after challenge (A/C) with RSV, (C) Serum VN antibody titers determined before (B/C) and after (A/C) RSV challenge, and (D) Numbers of IFN-γ and IL-5 secreting splenocytes determined in response to in vitro restimulation with ΔF protein. BALB/c mice were immunized once IN with ΔF formulated with TriAdj and challenged 3 weeks later with RSV. Control groups were immunized with PBS and challenged with RSV (Placebo) or mock-challenged (Placebo/mock). ELISA titers are expressed as the reciprocal of the highest dilution resulting in a value of two standard deviations above the negative control serum. Virus neutralization titers are expressed as the highest dilution of serum that resulted in <50% of cells displaying cytopathic effects. Cytokine secreting cell numbers are expressed as the difference in the number of spots between ΔF-stimulated wells and medium-control wells. Bars indicate median values with interquartile ranges. **P < 0.01; ***P < 0.001.

Article Snippet: Briefly, splenocytes and LN cells were stimulated with ΔF protein (2 µg/ml) and spots were developed with biotinylated IFN-γ- or IL-5-specific goat anti-mouse IgG (BD Biosciences, 554410, 554397) or biotinylated goat anti-mouse IgA-specific antibody, followed by AP-conjugated streptavidin and BCIP/NBT (Sigma-Aldrich, B5655) as the substrate.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Negative Control, Neutralization

Journal: Human Vaccines & Immunotherapeutics

Article Title: Intranasal immunization with a single dose of the fusion protein formulated with a combination adjuvant induces long-term protective immunity against respiratory syncytial virus

doi: 10.1080/21645515.2017.1349584

Figure Lengend Snippet: Mucosal immune responses to RSV ΔF protein in mice after challenge with RSV. The IgA titers (A), virus titers (B), percentages of ΔF-specific CD8 + T cells (C), and ΔF-specific IFN-γ secreting CD8 + T cells (D) were measured in the lung after RSV challenge. Mice were immunized and challenged as described in the legend for Fig. 1 . ELISA titers are expressed as the reciprocal of the highest dilution resulting in a value of two standard deviations above the negative control serum. Virus replication in the lungs is expressed as pfu per gram of lung tissue. The proportion of RSV F-specific CD8 + T cells was determined in the lung by KYKNAVTEL-MHC I pentamer staining. IFN-γ expression by CD8 + T cells was determined by intracellular cytokine staining. Bars represent median values with interquartile ranges. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Briefly, splenocytes and LN cells were stimulated with ΔF protein (2 µg/ml) and spots were developed with biotinylated IFN-γ- or IL-5-specific goat anti-mouse IgG (BD Biosciences, 554410, 554397) or biotinylated goat anti-mouse IgA-specific antibody, followed by AP-conjugated streptavidin and BCIP/NBT (Sigma-Aldrich, B5655) as the substrate.

Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Staining, Expressing

Journal: Human Vaccines & Immunotherapeutics

Article Title: Intranasal immunization with a single dose of the fusion protein formulated with a combination adjuvant induces long-term protective immunity against respiratory syncytial virus

doi: 10.1080/21645515.2017.1349584

Figure Lengend Snippet: Systemic immune responses to RSV ΔF protein. (A) Serum IgG1 and IgG2a titers before challenge (B/C), (B) Serum IgG1 and IgG2a titers after RSV challenge (A/C), (C) Serum VN antibody titers after RSV challenge (A/C), and (D) Numbers of IFN-γ and IL-5 secreting splenocytes determined in response to in vitro restimulation with ΔF protein. BALB/c mice were immunized once IN with ΔF formulated with TriAdj and challenged with RSV on day 150. Control groups were immunized with PBS and challenged with RSV (Placebo) or mock-challenged (Placebo/mock). ELISA titers are expressed as the reciprocal of the highest dilution resulting in a value of two standard deviations above the negative control serum. Virus neutralization titers are expressed as the highest dilution of serum that resulted in <50% of cells displaying cytopathic effects. Cytokine secreting cell numbers are expressed as the difference in the number of spots between ΔF-stimulated wells and medium-control wells. Bars indicate median values with interquartile ranges. ***P < 0.001.

Article Snippet: Briefly, splenocytes and LN cells were stimulated with ΔF protein (2 µg/ml) and spots were developed with biotinylated IFN-γ- or IL-5-specific goat anti-mouse IgG (BD Biosciences, 554410, 554397) or biotinylated goat anti-mouse IgA-specific antibody, followed by AP-conjugated streptavidin and BCIP/NBT (Sigma-Aldrich, B5655) as the substrate.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Negative Control, Neutralization