post hsv 1 infection  (Thermo Fisher)

Journal: NPJ Vaccines

Article Title: Model of vaccine efficacy against HSV-2 superinfection of HSV-1 seropositive mice demonstrates protection by antibodies mediating cellular cytotoxicity

doi: 10.1038/s41541-020-0184-7

Figure Lengend Snippet: a Female (closed symbols) and male (open symbols) C57BL6 mice were infected intranasally with 5 × 10 4 pfu/mouse of HSV-1 B 3 × 1.1. Mice were monitored over 20 days for signs of disease and distress. At day 14 post-primary challenge, mice were bled retro-orbitally to obtain immune serum, which was tested for b total HSV-1-specific IgG by ELISA (1:10,000 dilution) and c fold-induction of FcγRIV activation (mFcγRIV ADCC Reporter Bioassay, Promega). Data points are shown with symbols to indicate vaccination groups to which each mouse was subsequently assigned; the mean is indicated. Panel a , n = 75 females and 20 males; Panel b , n = 67 females and 16 males across four independent experiments.

Article Snippet: Serum was obtained on day 14 post-HSV-1 infection and 1 week following boost vaccination and assayed for a total HSV-2 IgG by ELISA (1:90,000 dilution); b neutralizing titer; and c fold-induction of FcγRIV activation (mFcγRIV ADCC Reporter Bioassay, Promega).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: NPJ Vaccines

Article Title: Model of vaccine efficacy against HSV-2 superinfection of HSV-1 seropositive mice demonstrates protection by antibodies mediating cellular cytotoxicity

doi: 10.1038/s41541-020-0184-7

Figure Lengend Snippet: Beginning on day 21-post-HSV-1 challenge, female (closed symbols) and male (open symbols) mice were prime-boost vaccinated at 3-week intervals with 5 × 10 6 pfu/mouse ΔgD-2; 5 µg of recombinant gD-2 with alum-MPL (rgD-2/alum-MPL) or an uninfected VD60 lysate. Additional controls included naïve age-matched mice vaccinated with ΔgD-2 or VD60 lysate. Serum was obtained on day 14 post-HSV-1 infection and one week following boost vaccination and assayed for a total HSV-2 IgG by ELISA (1:90,000 dilution); b neutralizing titer; and c fold-induction of FcγRIV activation (mFcγRIV ADCC Reporter Bioassay, Promega). d Total HSV-1-specific (left) and HSV-2-specific (right) IgG isotypes were assessed before (left) and after (right) boost vaccination. The asterisks (****) indicate p < 0.0001 by paired t -test comparing the post-vaccine versus post-HSV-1 antibody responses, ns indicates not significant. Data are shown as mean + SEM. The number of animals per group for Panels a – c were: HSV-1 seropositive (HSV-1 + ), ΔgD-2 n = 46; HSV-1 + , rgD-2/alum-MPL n = 10; HSV-1 + , VD60 n = 23 ; Naïve, ΔgD-2 n = 20; Naïve, VD60 n = 20. For Panel d , the number of animals was 10 per vaccination group. Mice were from three independent experiments.

Article Snippet: Serum was obtained on day 14 post-HSV-1 infection and 1 week following boost vaccination and assayed for a total HSV-2 IgG by ELISA (1:90,000 dilution); b neutralizing titer; and c fold-induction of FcγRIV activation (mFcγRIV ADCC Reporter Bioassay, Promega).

Techniques: Recombinant, Infection, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: NPJ Vaccines

Article Title: Model of vaccine efficacy against HSV-2 superinfection of HSV-1 seropositive mice demonstrates protection by antibodies mediating cellular cytotoxicity

doi: 10.1038/s41541-020-0184-7

Figure Lengend Snippet: Three weeks following boost vaccination, female (closed symbols) and male (open symbols) mice were challenged on the skin with 100 × LD90 (5 × 10 6 pfu/mouse) of HSV-2 clinical isolate, SD90. a Disease scores (blinded) and b percentage survival following HSV-2 challenge. HSV-1 + , ΔgD-2 n = 46; HSV-1 + , rgD-2/alum-MPL n = 10; HSV-1 + , VD60 n = 23; Naïve, ΔgD-2 n = 20; Naïve, VD60 n = 20 from three independent experiments. Survival was compared by Gehan–Breslow-Wilcoxon test (* p < 0.05, **** p < 0.0001, and ns not significant).

Article Snippet: Serum was obtained on day 14 post-HSV-1 infection and 1 week following boost vaccination and assayed for a total HSV-2 IgG by ELISA (1:90,000 dilution); b neutralizing titer; and c fold-induction of FcγRIV activation (mFcγRIV ADCC Reporter Bioassay, Promega).

Techniques:

Journal: NPJ Vaccines

Article Title: Model of vaccine efficacy against HSV-2 superinfection of HSV-1 seropositive mice demonstrates protection by antibodies mediating cellular cytotoxicity

doi: 10.1038/s41541-020-0184-7

Figure Lengend Snippet: At the time of death or sacrifice (D14 post-HSV-2 challenge), trigeminal ( a ) and sacral ( b ) ganglia were isolated from female (closed symbols) and male (open symbols) mice, and HSV-1 and HSV-2 DNA quantified by qPCR using serotype-specific primers. Mice that succumbed to HSV-2 superinfection are indicated by a crossed-through symbol in b . HSV-1 + , ΔgD-2 n = 25; HSV-1 + , rgD-2-alum/MPL n = 10; HSV-1 + , VD60 n = 15; Naïve, ΔgD-2 n = 10; Naïve, VD60 n = 8 from two independent experiments. The quantity of viral DNA detected was compared by ANOVA with correction for multiple comparison (** p < 0.01, **** p < 0.001, ns not significant), and means are indicated.

Article Snippet: Serum was obtained on day 14 post-HSV-1 infection and 1 week following boost vaccination and assayed for a total HSV-2 IgG by ELISA (1:90,000 dilution); b neutralizing titer; and c fold-induction of FcγRIV activation (mFcγRIV ADCC Reporter Bioassay, Promega).

Techniques: Isolation

Journal: NPJ Vaccines

Article Title: Model of vaccine efficacy against HSV-2 superinfection of HSV-1 seropositive mice demonstrates protection by antibodies mediating cellular cytotoxicity

doi: 10.1038/s41541-020-0184-7

Figure Lengend Snippet: Female C57BL6 mice were infected intranasally with 5 × 10 4 pfu/mouse of HSV-1 B 3 × 1.1. Mice were monitored over 20 days for signs of disease and distress. At day 14 post-primary challenge, mice were bled retro-orbitally to obtain immune serum, which was tested for total HSV-1 specific IgG by ELISA and mice were assigned to vaccination groups based on HSV-1-specific IgG titers. Beginning on day 21-post-HSV-1 challenge, mice were prime-boost vaccinated at 3-week intervals with 5 × 10 6 pfu/mouse dl5-29 or an uninfected VD60 lysate. Serum was obtained on day 14 post-HSV-1 infection and 1 week following boost vaccination and assayed for a total HSV-2 IgG by ELISA (1:90,000 dilution); b neutralizing titer; and c fold-induction of FcγRIV activation (mFcγRIV ADCC Reporter Bioassay, Promega). Three weeks following boost vaccination, mice were challenged on the skin with 100 × LD90 (5 × 10 6 pfu/mouse) of HSV-2 clinical isolate, SD90. d Percentage survival following HSV-2 challenge. e At the time of death or sacrifice (D14 post-HSV-2 challenge), sacral ganglia were isolated and HSV-2 DNA was quantified by qPCR using serotype-specific primers. Mice that succumbed to HSV-2 superinfection are indicated by a crossed-through symbol. a – c Paired t -test comparing the post-vaccine versus post-HSV-1 antibody responses; d Gehan–Breslow-Wilcoxon test; e Fisher’s Exact Test. The asterisks (****) indicate p < 0.0001. ** p < 0.01; ns indicates not significant. Data are shown as mean + SEM. n = 10 for dl5-29 and n = 5 for VD60.

Article Snippet: Serum was obtained on day 14 post-HSV-1 infection and 1 week following boost vaccination and assayed for a total HSV-2 IgG by ELISA (1:90,000 dilution); b neutralizing titer; and c fold-induction of FcγRIV activation (mFcγRIV ADCC Reporter Bioassay, Promega).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Activation Assay, Isolation

Journal: Molecular cell

Article Title: Topoisomerase 2β-dependent nuclear DNA damage shapes extracellular growth factor responses via dynamic AKT phosphorylation to control virus latency

doi: 10.1016/j.molcel.2019.02.032

Figure Lengend Snippet: (A) Schematic for establishment of HSV-1 latency and reactivation in rat SCG-derived neuron cultures. Dissociated SCG were seeded and cultured for 7d in the presence of NGF and anti-mitotic agents to remove non-neuronal cells. Cells were then infected with HSV-1 GFP-Us11 in the presence of ACV for 6 d to allow the virus to establish a non-replicating infection. After ACV removal, cultures were treated with inhibitors or an shRNA-expressing lentivirus. GFP fluorescence was monitored in live cells and quantified (n=30).

Article Snippet: After 6 days post HSV-1 infection, ACV was removed and cultures were induced to reactivate by treatment with LY294002 (20 μM, 20hrs, Calbiochem), NU7441 (1 μM, 20hrs, TOCRIS), Mirin (100 μM, 20hrs, TOCRIS), KU55933 (10 μM, Calbiochem), Etoposide (10 μM, 8hrs, Calbiochem), or Bleomycin (10 μM, 8hrs, Calbiochem).

Techniques: Derivative Assay, Cell Culture, Infection, shRNA, Expressing, Fluorescence

Journal: Molecular cell

Article Title: Topoisomerase 2β-dependent nuclear DNA damage shapes extracellular growth factor responses via dynamic AKT phosphorylation to control virus latency

doi: 10.1016/j.molcel.2019.02.032

Figure Lengend Snippet: (A) Reactivation assay comparing the response of HSV-1 GFP-Us11 infected SCG neurons treated with either NU4771 (1 μM, 20 h), Mirin (100 μM, 20 h), LY294002 (20 μM, 20 h) or DMSO control, and then maintained for 6 d in fresh media. Data was plotted (n=6) and scored on successive days with mean ±s.e.m.

Article Snippet: After 6 days post HSV-1 infection, ACV was removed and cultures were induced to reactivate by treatment with LY294002 (20 μM, 20hrs, Calbiochem), NU7441 (1 μM, 20hrs, TOCRIS), Mirin (100 μM, 20hrs, TOCRIS), KU55933 (10 μM, Calbiochem), Etoposide (10 μM, 8hrs, Calbiochem), or Bleomycin (10 μM, 8hrs, Calbiochem).

Techniques: Infection

Journal: Molecular cell

Article Title: Topoisomerase 2β-dependent nuclear DNA damage shapes extracellular growth factor responses via dynamic AKT phosphorylation to control virus latency

doi: 10.1016/j.molcel.2019.02.032

Figure Lengend Snippet: (A) Diagram of mTORC1 signaling induced by both the NGF-mediated TrkA/PI3K/PDK1/AKT/TSC pathway and DNA damage (and DNA repair inhibition) activation, leading to the maintenance of HSV-1 latency. Rheb is depicted in GDP- and GTP-bound forms. The Rheb (Q64L) mutant is a constitutively active protein that acts at the point shown (stabilization of the GTP-bound form) to bypass upstream AKT inhibition in order to activate downstream mTORC1 signaling.

Article Snippet: After 6 days post HSV-1 infection, ACV was removed and cultures were induced to reactivate by treatment with LY294002 (20 μM, 20hrs, Calbiochem), NU7441 (1 μM, 20hrs, TOCRIS), Mirin (100 μM, 20hrs, TOCRIS), KU55933 (10 μM, Calbiochem), Etoposide (10 μM, 8hrs, Calbiochem), or Bleomycin (10 μM, 8hrs, Calbiochem).

Techniques: Inhibition, Activation Assay, Mutagenesis

Journal: Molecular cell

Article Title: Topoisomerase 2β-dependent nuclear DNA damage shapes extracellular growth factor responses via dynamic AKT phosphorylation to control virus latency

doi: 10.1016/j.molcel.2019.02.032

Figure Lengend Snippet: (A) Working model of how extracellular growth factor and TOP2β-mediated nuclear DNA damage signaling collaborate to sustain AKT-mTORC1 activation and maintain HSV-1 latency. NGF-dependent activation of TrKA leads to sustained AKT Thr308 phosphorylation. This step is critical for AKT to be phosphorylated on Ser473 by DNA-PK. DNA-PK is then activated from transient DSBs generated by TOP2βcc intermediates acted on by TDP2, which can occur at promoters of early-response host genes during normal neuronal activity (Madabhushi et al., 2015). The MRN complex is critical for the processing of TOP2βcc for both DNA repair (Hoa et al., 2016) and activation of DNA-PK (our study). We speculate that AKT undergoes continuous nucleocytoplasmic shuttling. AKT is likely phosphorylated on Thr308 at the plasma membrane by PDK1. This step is critical for initiating AKT nuclear translocation and phosphorylation by DNA-PK on Ser473, but negatively regulated by PHLPP1, leading to its nuclear export and activation of cytoplasmic AKT-mTORC1 canonical pathway. Continuous AKT-mTORC1 signaling is crucial for the maintenance of HSV-1 latency. Thus, sustained activation of the AKT-mTORC1 signaling axis in neurons requires consolidating two independent signals generated from potentially different cellular compartments (nuclear and plasma membrane/cytoplasm). During latency, histones at HSV-1 lytic promoters remain in a repressed state. Neuronal stress stimuli that trigger DLK/JIP3-mediated activation of JNK contribute to a histone methyl/phospho switch (Cliffe et al., 2015), and subsequent replacement by euchromatin-associated marks (not pictured).

Article Snippet: After 6 days post HSV-1 infection, ACV was removed and cultures were induced to reactivate by treatment with LY294002 (20 μM, 20hrs, Calbiochem), NU7441 (1 μM, 20hrs, TOCRIS), Mirin (100 μM, 20hrs, TOCRIS), KU55933 (10 μM, Calbiochem), Etoposide (10 μM, 8hrs, Calbiochem), or Bleomycin (10 μM, 8hrs, Calbiochem).

Techniques: Activation Assay, Generated, Activity Assay, Translocation Assay

Journal: Molecular cell

Article Title: Topoisomerase 2β-dependent nuclear DNA damage shapes extracellular growth factor responses via dynamic AKT phosphorylation to control virus latency

doi: 10.1016/j.molcel.2019.02.032

Figure Lengend Snippet: (A) Schematic for establishment of HSV-1 latency and reactivation in rat SCG-derived neuron cultures. Dissociated SCG were seeded and cultured for 7d in the presence of NGF and anti-mitotic agents to remove non-neuronal cells. Cells were then infected with HSV-1 GFP-Us11 in the presence of ACV for 6 d to allow the virus to establish a non-replicating infection. After ACV removal, cultures were treated with inhibitors or an shRNA-expressing lentivirus. GFP fluorescence was monitored in live cells and quantified (n=30).

Article Snippet: After 6 days post HSV-1 infection, ACV was removed and cultures were induced to reactivate by treatment with LY294002 (20 μM, 20hrs, Calbiochem), NU7441 (1 μM, 20hrs, TOCRIS), Mirin (100 μM, 20hrs, TOCRIS), KU55933 (10 μM, Calbiochem), Etoposide (10 μM, 8hrs, Calbiochem), or Bleomycin (10 μM, 8hrs, Calbiochem).

Techniques: Derivative Assay, Cell Culture, Infection, Virus, shRNA, Expressing, Fluorescence

Journal: Molecular cell

Article Title: Topoisomerase 2β-dependent nuclear DNA damage shapes extracellular growth factor responses via dynamic AKT phosphorylation to control virus latency

doi: 10.1016/j.molcel.2019.02.032

Figure Lengend Snippet: (A) Reactivation assay comparing the response of HSV-1 GFP-Us11 infected SCG neurons treated with either NU4771 (1 μM, 20 h), Mirin (100 μM, 20 h), LY294002 (20 μM, 20 h) or DMSO control, and then maintained for 6 d in fresh media. Data was plotted (n=6) and scored on successive days with mean ±s.e.m.

Article Snippet: After 6 days post HSV-1 infection, ACV was removed and cultures were induced to reactivate by treatment with LY294002 (20 μM, 20hrs, Calbiochem), NU7441 (1 μM, 20hrs, TOCRIS), Mirin (100 μM, 20hrs, TOCRIS), KU55933 (10 μM, Calbiochem), Etoposide (10 μM, 8hrs, Calbiochem), or Bleomycin (10 μM, 8hrs, Calbiochem).

Techniques: Infection

Journal: Molecular cell

Article Title: Topoisomerase 2β-dependent nuclear DNA damage shapes extracellular growth factor responses via dynamic AKT phosphorylation to control virus latency

doi: 10.1016/j.molcel.2019.02.032

Figure Lengend Snippet: (A) Diagram of mTORC1 signaling induced by both the NGF-mediated TrkA/PI3K/PDK1/AKT/TSC pathway and DNA damage (and DNA repair inhibition) activation, leading to the maintenance of HSV-1 latency. Rheb is depicted in GDP- and GTP-bound forms. The Rheb (Q64L) mutant is a constitutively active protein that acts at the point shown (stabilization of the GTP-bound form) to bypass upstream AKT inhibition in order to activate downstream mTORC1 signaling.

Article Snippet: After 6 days post HSV-1 infection, ACV was removed and cultures were induced to reactivate by treatment with LY294002 (20 μM, 20hrs, Calbiochem), NU7441 (1 μM, 20hrs, TOCRIS), Mirin (100 μM, 20hrs, TOCRIS), KU55933 (10 μM, Calbiochem), Etoposide (10 μM, 8hrs, Calbiochem), or Bleomycin (10 μM, 8hrs, Calbiochem).

Techniques: Inhibition, Activation Assay, Mutagenesis

Journal: Molecular cell

Article Title: Topoisomerase 2β-dependent nuclear DNA damage shapes extracellular growth factor responses via dynamic AKT phosphorylation to control virus latency

doi: 10.1016/j.molcel.2019.02.032

Figure Lengend Snippet: (A) Working model of how extracellular growth factor and TOP2β-mediated nuclear DNA damage signaling collaborate to sustain AKT-mTORC1 activation and maintain HSV-1 latency. NGF-dependent activation of TrKA leads to sustained AKT Thr308 phosphorylation. This step is critical for AKT to be phosphorylated on Ser473 by DNA-PK. DNA-PK is then activated from transient DSBs generated by TOP2βcc intermediates acted on by TDP2, which can occur at promoters of early-response host genes during normal neuronal activity (Madabhushi et al., 2015). The MRN complex is critical for the processing of TOP2βcc for both DNA repair (Hoa et al., 2016) and activation of DNA-PK (our study). We speculate that AKT undergoes continuous nucleocytoplasmic shuttling. AKT is likely phosphorylated on Thr308 at the plasma membrane by PDK1. This step is critical for initiating AKT nuclear translocation and phosphorylation by DNA-PK on Ser473, but negatively regulated by PHLPP1, leading to its nuclear export and activation of cytoplasmic AKT-mTORC1 canonical pathway. Continuous AKT-mTORC1 signaling is crucial for the maintenance of HSV-1 latency. Thus, sustained activation of the AKT-mTORC1 signaling axis in neurons requires consolidating two independent signals generated from potentially different cellular compartments (nuclear and plasma membrane/cytoplasm). During latency, histones at HSV-1 lytic promoters remain in a repressed state. Neuronal stress stimuli that trigger DLK/JIP3-mediated activation of JNK contribute to a histone methyl/phospho switch (Cliffe et al., 2015), and subsequent replacement by euchromatin-associated marks (not pictured).

Article Snippet: After 6 days post HSV-1 infection, ACV was removed and cultures were induced to reactivate by treatment with LY294002 (20 μM, 20hrs, Calbiochem), NU7441 (1 μM, 20hrs, TOCRIS), Mirin (100 μM, 20hrs, TOCRIS), KU55933 (10 μM, Calbiochem), Etoposide (10 μM, 8hrs, Calbiochem), or Bleomycin (10 μM, 8hrs, Calbiochem).

Techniques: Activation Assay, Generated, Activity Assay, Membrane, Translocation Assay