recombinant hiv 1 reverse transcriptase standard curve  (Worthington Biochemical)


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    Worthington Biochemical recombinant hiv 1 reverse transcriptase standard curve
    Longitudinal non-invasive bioluminescent imaging of <t>HIV-1</t> acute infection, suppression, and recrudescent infection in the Hu-BLT mouse group placed on cART 12 days post-infection. (A) Bioluminescent imaging of spreading infection of Hu-BLT Mouse #3 infected with 1 x 10 6 IUs of Q23.BG505.Nluc T/F reporter virus and placed on a daily cART regimen comprised of daily i.p. cART injections of Truvada and Isentress 12 days post-infection. (B) Whole animal ex vivo necroscopic analysis of rebounding infection in Hu-BLT Mouse #3 five days following cART cessation. (C) Plasma reverse transcriptase activity from Hu-BLT Mouse #3 over the course of the 40 day imaging period. Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (dotted line).
    Recombinant Hiv 1 Reverse Transcriptase Standard Curve, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice"

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    Journal: bioRxiv

    doi: 10.1101/745125

    Longitudinal non-invasive bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in the Hu-BLT mouse group placed on cART 12 days post-infection. (A) Bioluminescent imaging of spreading infection of Hu-BLT Mouse #3 infected with 1 x 10 6 IUs of Q23.BG505.Nluc T/F reporter virus and placed on a daily cART regimen comprised of daily i.p. cART injections of Truvada and Isentress 12 days post-infection. (B) Whole animal ex vivo necroscopic analysis of rebounding infection in Hu-BLT Mouse #3 five days following cART cessation. (C) Plasma reverse transcriptase activity from Hu-BLT Mouse #3 over the course of the 40 day imaging period. Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (dotted line).
    Figure Legend Snippet: Longitudinal non-invasive bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in the Hu-BLT mouse group placed on cART 12 days post-infection. (A) Bioluminescent imaging of spreading infection of Hu-BLT Mouse #3 infected with 1 x 10 6 IUs of Q23.BG505.Nluc T/F reporter virus and placed on a daily cART regimen comprised of daily i.p. cART injections of Truvada and Isentress 12 days post-infection. (B) Whole animal ex vivo necroscopic analysis of rebounding infection in Hu-BLT Mouse #3 five days following cART cessation. (C) Plasma reverse transcriptase activity from Hu-BLT Mouse #3 over the course of the 40 day imaging period. Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (dotted line).

    Techniques Used: Imaging, Infection, Ex Vivo, Activity Assay

    In vitro HIV-1 T/F reporter virus reporter gene stability in primary CD4+ T cells. (A) NL43.R5.GFP reporter virus design. Purple regions correspond to duplicated 3’-LTR sequence flanking the reporter gene. (B, C) Stability of GFP and Nluc reporter viruses in primary CD4 T cells. To force the reporter viruses to continuously spread to new cells, fresh autologous uninfected T cells were added every 48 hours so that the percent of p24+ cells in the total culture was maintained at the same value for each experiment. HIV-1 T/F and NL43.R5.GFP reporter virus GFP and p24 expression 2 days and 8 days post-initiation (B). FACS data is representative of 3-6 independent experiments. Reporter expression was determined via flow cytometry and displayed as the percentage of double-positive HIV-1 and GFP co-expressing cells in the total p24+ population for GFP expressing reporter viruses and the total fold Nluc-derived light units above an Efiverenz negative control for the BG505.Nluc* reporter virus (C). Data in (C) shown as the mean +/-SEM of 3-6 independent donor primary CD4+ T cell preparations.
    Figure Legend Snippet: In vitro HIV-1 T/F reporter virus reporter gene stability in primary CD4+ T cells. (A) NL43.R5.GFP reporter virus design. Purple regions correspond to duplicated 3’-LTR sequence flanking the reporter gene. (B, C) Stability of GFP and Nluc reporter viruses in primary CD4 T cells. To force the reporter viruses to continuously spread to new cells, fresh autologous uninfected T cells were added every 48 hours so that the percent of p24+ cells in the total culture was maintained at the same value for each experiment. HIV-1 T/F and NL43.R5.GFP reporter virus GFP and p24 expression 2 days and 8 days post-initiation (B). FACS data is representative of 3-6 independent experiments. Reporter expression was determined via flow cytometry and displayed as the percentage of double-positive HIV-1 and GFP co-expressing cells in the total p24+ population for GFP expressing reporter viruses and the total fold Nluc-derived light units above an Efiverenz negative control for the BG505.Nluc* reporter virus (C). Data in (C) shown as the mean +/-SEM of 3-6 independent donor primary CD4+ T cell preparations.

    Techniques Used: In Vitro, Sequencing, Expressing, FACS, Flow Cytometry, Derivative Assay, Negative Control

    RNA viral load assay and SG-PERT RT activity assay sensitivities. (A) eripheral blood mononuclear cell (PBMC) derived HIV-1 JR-CSF viral supernatant was stored in separate aliquots of equal volume in order to compare the sensitivity of the Quantitect qRT-PCR viral load assay and the SG-PERT reverse transcriptase activity qPCR assay in parallel. (B) The Quantitect qRT-PCR viral load assay and the SG-PERT reverse transcriptase activity qPCR assay was run in parallel with viral RNA eluate and HIV-1 supernatant serially diluted until the limit of detection for each assay was reached. Data shown as the average cycle threshold (Cq) values determined from two technical replicates at each dilution. The limit of detection was defined as the Cq value at which the linear range of the assay ended. Absolute quantification of HIV-1 particles was determined from a viral RNA standard curve run in parallel with the Quantitect qRT-PCR viral load assay.
    Figure Legend Snippet: RNA viral load assay and SG-PERT RT activity assay sensitivities. (A) eripheral blood mononuclear cell (PBMC) derived HIV-1 JR-CSF viral supernatant was stored in separate aliquots of equal volume in order to compare the sensitivity of the Quantitect qRT-PCR viral load assay and the SG-PERT reverse transcriptase activity qPCR assay in parallel. (B) The Quantitect qRT-PCR viral load assay and the SG-PERT reverse transcriptase activity qPCR assay was run in parallel with viral RNA eluate and HIV-1 supernatant serially diluted until the limit of detection for each assay was reached. Data shown as the average cycle threshold (Cq) values determined from two technical replicates at each dilution. The limit of detection was defined as the Cq value at which the linear range of the assay ended. Absolute quantification of HIV-1 particles was determined from a viral RNA standard curve run in parallel with the Quantitect qRT-PCR viral load assay.

    Techniques Used: RNA Viral-load Assay, RT Activity Assay, Derivative Assay, Quantitative RT-PCR, Viral-load Assay, Activity Assay, Real-time Polymerase Chain Reaction

    Confocal immunofluorescence microscopy of cleared HIV-1 Q23.BG505.Nluc infected spleen tissue during recrudescent infection. (A) Nluc expressing spleen tissue from Hu-HSC mouse #14 infected with BG505.Nluc* reporter virus. Nluc expressing spleen is enclosed in red boxes at whole-animal and tissue resolutions, respectively. Spleen tissue was surgically removed 17 days following cART withdrawal and was subsequently fixed, cleared, and immunostained for confocal microscopy to identify HIV-1 p24+ cells. (B-C) Representative confocal slices of Nluc expressing spleen tissue from (A). Tissues were immunostained for HIV-1 p24 (green), human CD3+ T cells (magenta), human CD68+ macrophages (red), and stained with DAPI to identify nuclei (cyan). HIV-1 p24 was associated with human CD3+ T-cells (B) and human CD68+ macrophages (C) within the same piece of Nluc expressing spleen tissue. (D-F) Immunostaining of representative Nluc expressing cells in spleen tissue from BG505.Nluc* infected Hu-HSC mouse #14 (D) and Hu-HSC mouse #15 (F). Nluc expression (red) colocalizes with HIV-1 p24 (green) and CD3 (magenta) in spleen tissue isolated from both Hu-HSC mice.
    Figure Legend Snippet: Confocal immunofluorescence microscopy of cleared HIV-1 Q23.BG505.Nluc infected spleen tissue during recrudescent infection. (A) Nluc expressing spleen tissue from Hu-HSC mouse #14 infected with BG505.Nluc* reporter virus. Nluc expressing spleen is enclosed in red boxes at whole-animal and tissue resolutions, respectively. Spleen tissue was surgically removed 17 days following cART withdrawal and was subsequently fixed, cleared, and immunostained for confocal microscopy to identify HIV-1 p24+ cells. (B-C) Representative confocal slices of Nluc expressing spleen tissue from (A). Tissues were immunostained for HIV-1 p24 (green), human CD3+ T cells (magenta), human CD68+ macrophages (red), and stained with DAPI to identify nuclei (cyan). HIV-1 p24 was associated with human CD3+ T-cells (B) and human CD68+ macrophages (C) within the same piece of Nluc expressing spleen tissue. (D-F) Immunostaining of representative Nluc expressing cells in spleen tissue from BG505.Nluc* infected Hu-HSC mouse #14 (D) and Hu-HSC mouse #15 (F). Nluc expression (red) colocalizes with HIV-1 p24 (green) and CD3 (magenta) in spleen tissue isolated from both Hu-HSC mice.

    Techniques Used: Immunofluorescence, Microscopy, Infection, Expressing, Confocal Microscopy, Staining, Immunostaining, Isolation, Mouse Assay

    Longitudinal imaging of HIV-1 acute infection, cART suppression, and infection recrudescence in Hu-HSC mice placed on ART 6 days post-infection. (A) Longitudinal bioluminescent imaging of spreading infection in Hu-HSC mice infected with 1 x 10 6 infectious units (IUs) of BG505.Nluc* T/F reporter virus and placed on a daily ART regimen comprised of daily i.p. ART injections of Truvada and Isentress 6 days post-infection (n=2). (B) Quantification of plasma reverse transcriptase activity from the mice in (A). Plasma reverse transcriptase activity in serum samples obtained every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A) approximately two weeks following ART cessation.
    Figure Legend Snippet: Longitudinal imaging of HIV-1 acute infection, cART suppression, and infection recrudescence in Hu-HSC mice placed on ART 6 days post-infection. (A) Longitudinal bioluminescent imaging of spreading infection in Hu-HSC mice infected with 1 x 10 6 infectious units (IUs) of BG505.Nluc* T/F reporter virus and placed on a daily ART regimen comprised of daily i.p. ART injections of Truvada and Isentress 6 days post-infection (n=2). (B) Quantification of plasma reverse transcriptase activity from the mice in (A). Plasma reverse transcriptase activity in serum samples obtained every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A) approximately two weeks following ART cessation.

    Techniques Used: Imaging, Infection, Mouse Assay, Activity Assay, Ex Vivo

    Non-invasive bioluminescent imaging of longitudinal HIV-1 Q23.BG505 infection in humanized mice. (A) Longitudinal imaging of Hu-PBL mice (n=6) infected intraperitonially (i.p.) with 1 x 10 7 infectious units (IUs) of HIV-1 BG505.Nluc* reporter virus. Data representative of six independently infected animals. (B) Longitudinal imaging of productive HIV-1 infection in Hu-HSC mice (n=2) infected i.p. with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus. (C) Quantification of bioluminescent signal measured over a 15 day period in BG505.Nluc* infected Hu-PBL mice. Average total animal flux was calculated by taking the mean of the total animal flux measured from both the ventral and lateral imaging orientations, excluding the cranial region to avoid signal artifacts arising from the Nano-glo substrate injection site. Data displayed as the mean +/-SEM from six independent experiments. (D, E) Correlative analysis of average total animal flux with the increase of peripheral HIV-1 infected cells (p24+CD3+CD8-cells) measured by flow cytometry (D) and plasma reserve transcriptase activity (RT U) in the infected Hu-PBL mice in (C) measured by the reserve transcriptase SG-PERT activity qPCR assay (E). Upper and lower bounds of the SEM is displayed as gray shaded regions above and below the mean value at each day measured. Pearson correlation calculated from (D) the average values of peripheral HIV-1 infected cells and average total animal flux and (E) plasma reverse transcriptase activity and average total animal flux.
    Figure Legend Snippet: Non-invasive bioluminescent imaging of longitudinal HIV-1 Q23.BG505 infection in humanized mice. (A) Longitudinal imaging of Hu-PBL mice (n=6) infected intraperitonially (i.p.) with 1 x 10 7 infectious units (IUs) of HIV-1 BG505.Nluc* reporter virus. Data representative of six independently infected animals. (B) Longitudinal imaging of productive HIV-1 infection in Hu-HSC mice (n=2) infected i.p. with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus. (C) Quantification of bioluminescent signal measured over a 15 day period in BG505.Nluc* infected Hu-PBL mice. Average total animal flux was calculated by taking the mean of the total animal flux measured from both the ventral and lateral imaging orientations, excluding the cranial region to avoid signal artifacts arising from the Nano-glo substrate injection site. Data displayed as the mean +/-SEM from six independent experiments. (D, E) Correlative analysis of average total animal flux with the increase of peripheral HIV-1 infected cells (p24+CD3+CD8-cells) measured by flow cytometry (D) and plasma reserve transcriptase activity (RT U) in the infected Hu-PBL mice in (C) measured by the reserve transcriptase SG-PERT activity qPCR assay (E). Upper and lower bounds of the SEM is displayed as gray shaded regions above and below the mean value at each day measured. Pearson correlation calculated from (D) the average values of peripheral HIV-1 infected cells and average total animal flux and (E) plasma reverse transcriptase activity and average total animal flux.

    Techniques Used: Imaging, Infection, Mouse Assay, Injection, Flow Cytometry, Activity Assay, Real-time Polymerase Chain Reaction

    Functional characterization of HIV-1 T/F full-length reporter viruses. (A) HIV-1 T/F full-length reporter virus design. (B-C) Western blot analysis of Nef protein expression in HEK293 producer cell lysates transfected with TRJO.c (B) and Q23.BG505 (C) derived T/F full-length reporter virus plasmid DNA. Equal amounts of p55 Gag were loaded onto each lane to assess the relative Nef expression. (D) Surface CD4 surface expression on p24+ Jurkat CCR5+ JLTRGFP.R5 cells infected with HIV-1 T/F wild-type or HIV-1 T/F reporter virus 48 hours after initiating infection. Data shown as mean +/-SD of 3 technical replicates with significance calculated from a one-way ANOVA. (E) Single-round infectivity of HIV-1 T/F reporter viruses on TZM-bl cells cells in the presence of 10 nM of the protease inhibitor saquinavir to block virus spreading (n=3). Infectivity for each virus was determined by measuring the fold firefly luciferase expression from infected TZM-bl cells above uninfected negative controls and normalizing to reverse transcriptase units (RT U). Data displayed is average fold HIV-1 T/F reporter virus infectivity over TRJO.c and Q23.BG505 wild-type virus +/-SEM from three independent experiments. (F) HIV-1 T/F reporter virus spread in primary CD4+ T cells (n=3). Each sample was set to the same level of p24 positive cells and then allowed to spread for four days in culture. Data displayed as the fold HIV-1 T/F reporter virus infection (as the value of % p24) over wild-type +/-the SEM from three independent CD4+ T cell preparations.
    Figure Legend Snippet: Functional characterization of HIV-1 T/F full-length reporter viruses. (A) HIV-1 T/F full-length reporter virus design. (B-C) Western blot analysis of Nef protein expression in HEK293 producer cell lysates transfected with TRJO.c (B) and Q23.BG505 (C) derived T/F full-length reporter virus plasmid DNA. Equal amounts of p55 Gag were loaded onto each lane to assess the relative Nef expression. (D) Surface CD4 surface expression on p24+ Jurkat CCR5+ JLTRGFP.R5 cells infected with HIV-1 T/F wild-type or HIV-1 T/F reporter virus 48 hours after initiating infection. Data shown as mean +/-SD of 3 technical replicates with significance calculated from a one-way ANOVA. (E) Single-round infectivity of HIV-1 T/F reporter viruses on TZM-bl cells cells in the presence of 10 nM of the protease inhibitor saquinavir to block virus spreading (n=3). Infectivity for each virus was determined by measuring the fold firefly luciferase expression from infected TZM-bl cells above uninfected negative controls and normalizing to reverse transcriptase units (RT U). Data displayed is average fold HIV-1 T/F reporter virus infectivity over TRJO.c and Q23.BG505 wild-type virus +/-SEM from three independent experiments. (F) HIV-1 T/F reporter virus spread in primary CD4+ T cells (n=3). Each sample was set to the same level of p24 positive cells and then allowed to spread for four days in culture. Data displayed as the fold HIV-1 T/F reporter virus infection (as the value of % p24) over wild-type +/-the SEM from three independent CD4+ T cell preparations.

    Techniques Used: Functional Assay, Western Blot, Expressing, Transfection, Derivative Assay, Plasmid Preparation, Infection, Protease Inhibitor, Blocking Assay, Luciferase

    Q23.BG505 T/F reporter virus reporter gene expression kinetics in infected CEMSS.NKr.R5 cells. (A) GFP reporter gene expression at different times points during the initial 24 hours of infection in CEMSS.NKr.R5 cells with or without the addition of 1 μM Efavirenz. Data is representative of three independent experiments. (B-C) Percent of single positive (p24+ GFP-) and double-positive (p24+ GFP+) cells over 24 hours in CEMSS.NKr.R5 cells infected with (B) BG505.GFP or (C) BG505.GFP* T/F reporter viruses. Data displayed as the mean +/-SEM of three independent experiments (n=3). (D-E) The fraction of double-positive cells in the total HIV-1 infected (p24+) population 18 hours and 24 hours post-infection in CEMSS.NKr.R5 cells infected with (D) BG505.GFP or (E) BG505.GFP* T/F reporter viruses.
    Figure Legend Snippet: Q23.BG505 T/F reporter virus reporter gene expression kinetics in infected CEMSS.NKr.R5 cells. (A) GFP reporter gene expression at different times points during the initial 24 hours of infection in CEMSS.NKr.R5 cells with or without the addition of 1 μM Efavirenz. Data is representative of three independent experiments. (B-C) Percent of single positive (p24+ GFP-) and double-positive (p24+ GFP+) cells over 24 hours in CEMSS.NKr.R5 cells infected with (B) BG505.GFP or (C) BG505.GFP* T/F reporter viruses. Data displayed as the mean +/-SEM of three independent experiments (n=3). (D-E) The fraction of double-positive cells in the total HIV-1 infected (p24+) population 18 hours and 24 hours post-infection in CEMSS.NKr.R5 cells infected with (D) BG505.GFP or (E) BG505.GFP* T/F reporter viruses.

    Techniques Used: Expressing, Infection

    In vivo reporter gene stability in Hu-PBL mice. (A-C) GFP reporter gene stability in p24+ CD3+ PBMCs extracted from the peripheral blood of Hu-PBL mice infected intraperitonially (i.p.) with 1 x 10 7 infectious units (IUs) HIV-1 T/F reporter virus. (A) Representative FACS plots gated on CD3+ PBMCs extracted from BG505.GFP* infected Hu-PBL mice (n=4). Data is representative of four individual Hu-PBL mice. (B-C) Average GFP reporter gene stability in Hu-PBL mice infected with 1 x 10 7 infectious units (IUs) of BG505.GFP* (n=4) (B), and BG505.GFP (n=4) (C) T/F reporter virus for 14-16 days. Data displayed as the percentage of GFP and p24 double-positive cells in the total p24+ population. A line crosses the average percent GFP expressing cells within the total p24 + cell population for mice analyzed at each time point.
    Figure Legend Snippet: In vivo reporter gene stability in Hu-PBL mice. (A-C) GFP reporter gene stability in p24+ CD3+ PBMCs extracted from the peripheral blood of Hu-PBL mice infected intraperitonially (i.p.) with 1 x 10 7 infectious units (IUs) HIV-1 T/F reporter virus. (A) Representative FACS plots gated on CD3+ PBMCs extracted from BG505.GFP* infected Hu-PBL mice (n=4). Data is representative of four individual Hu-PBL mice. (B-C) Average GFP reporter gene stability in Hu-PBL mice infected with 1 x 10 7 infectious units (IUs) of BG505.GFP* (n=4) (B), and BG505.GFP (n=4) (C) T/F reporter virus for 14-16 days. Data displayed as the percentage of GFP and p24 double-positive cells in the total p24+ population. A line crosses the average percent GFP expressing cells within the total p24 + cell population for mice analyzed at each time point.

    Techniques Used: In Vivo, Mouse Assay, Infection, FACS, Expressing

    Longitudinal imaging of HIV-1 acute infection, suppression, and recrudescent infection in Hu-HSC mice placed on ART 9 days post-infection. (A) Longitudinal bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in Hu-HSC mice infected with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus and placed on daily i.p. ART injections of Truvada and Isentress 9 days post-infection (n=2). Red star denotes the timepoint and Hu-HSC mouse that exhibited a transient increase in plasma reverse transcriptase activity during ART treatment. (B) Quantification of plasma reverse transcriptase activity from the animals in (A). Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A), approximately two weeks following ART cessation.
    Figure Legend Snippet: Longitudinal imaging of HIV-1 acute infection, suppression, and recrudescent infection in Hu-HSC mice placed on ART 9 days post-infection. (A) Longitudinal bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in Hu-HSC mice infected with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus and placed on daily i.p. ART injections of Truvada and Isentress 9 days post-infection (n=2). Red star denotes the timepoint and Hu-HSC mouse that exhibited a transient increase in plasma reverse transcriptase activity during ART treatment. (B) Quantification of plasma reverse transcriptase activity from the animals in (A). Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A), approximately two weeks following ART cessation.

    Techniques Used: Imaging, Infection, Mouse Assay, Activity Assay, Ex Vivo

    2) Product Images from "Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice"

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008161

    Confocal immunofluorescence microscopy of cleared Q23.BG505.Nluc infected spleen tissue during recrudescent infection. (A) Nluc expressing spleen tissue from Hu-HSC mouse #14 infected with Q23.BG505.Nluc T/F reporter virus. Nluc expressing spleen is enclosed in red boxes at whole-animal and tissue resolutions, respectively. Spleen tissue was surgically removed 17 days following cART withdrawal and was subsequently fixed, cleared, and immunostained for confocal microscopy to identify HIV-1 p24 + cells. (B-C) Representative confocal slices of Nluc expressing spleen tissue from (A). Tissues were immunostained for HIV-1 p24 (green), human CD3 + T cells (magenta), human CD68 + macrophages (red), and stained with DAPI to identify nuclei (cyan). HIV-1 p24 was associated with human CD3 + T-cells (B) and human CD68 + macrophages (C) within the same piece of Nluc expressing spleen tissue. (D-E) Immunostaining of representative Nluc expressing cells in spleen tissue from BG505.Nluc* infected Hu-HSC mouse #14 (D) and Hu-HSC mouse #15 (E). Nluc expression (red) colocalizes with HIV-1 p24 (green) and CD3 (magenta) in spleen tissue isolated from both Hu-HSC mice.
    Figure Legend Snippet: Confocal immunofluorescence microscopy of cleared Q23.BG505.Nluc infected spleen tissue during recrudescent infection. (A) Nluc expressing spleen tissue from Hu-HSC mouse #14 infected with Q23.BG505.Nluc T/F reporter virus. Nluc expressing spleen is enclosed in red boxes at whole-animal and tissue resolutions, respectively. Spleen tissue was surgically removed 17 days following cART withdrawal and was subsequently fixed, cleared, and immunostained for confocal microscopy to identify HIV-1 p24 + cells. (B-C) Representative confocal slices of Nluc expressing spleen tissue from (A). Tissues were immunostained for HIV-1 p24 (green), human CD3 + T cells (magenta), human CD68 + macrophages (red), and stained with DAPI to identify nuclei (cyan). HIV-1 p24 was associated with human CD3 + T-cells (B) and human CD68 + macrophages (C) within the same piece of Nluc expressing spleen tissue. (D-E) Immunostaining of representative Nluc expressing cells in spleen tissue from BG505.Nluc* infected Hu-HSC mouse #14 (D) and Hu-HSC mouse #15 (E). Nluc expression (red) colocalizes with HIV-1 p24 (green) and CD3 (magenta) in spleen tissue isolated from both Hu-HSC mice.

    Techniques Used: Immunofluorescence, Microscopy, Infection, Expressing, Confocal Microscopy, Staining, Immunostaining, Isolation, Mouse Assay

    Functional characterization of HIV-1 T/F full-length reporter viruses. (A) HIV-1 T/F full-length reporter virus design. (B-C) Western blot analysis of Nef protein expression in HEK293 producer cell lysates transfected with TRJO.c (B) and Q23.BG505 (C) derived T/F full-length reporter virus plasmid DNA. Equal amounts of p55 Gag were loaded onto each lane to assess the relative Nef expression. (D) Surface CD4 surface expression on p24+ Jurkat CCR5 + JLTRGFP.R5 cells infected with HIV-1 T/F wild-type or HIV-1 T/F reporter virus 48 hours after initiating infection. Data shown as mean +/- SD of 3 technical replicates with significance calculated from a one-way ANOVA. (E) Single-round infectivity of HIV-1 T/F reporter viruses on TZM-bl cells in the presence of 10 nM of the protease inhibitor saquinavir to block virus spreading (n = 3). Infectivity for each virus was determined by measuring the fold firefly luciferase expression from infected TZM-bl cells above uninfected negative controls and normalizing to reverse transcriptase units (RT U). Data displayed is average fold HIV-1 T/F reporter virus infectivity over TRJO.c and Q23.BG505 wild-type virus +/- SEM from three independent experiments. (F) HIV-1 T/F reporter virus spread in primary CD4 + T cells (n = 3). Each sample was set to the same level of p24 positive cells and then allowed to spread for four days in culture. Data displayed as the fold HIV-1 T/F reporter virus infection (as the value of % p24) over wild-type +/- the SEM from three independent CD4 + T cell preparations.
    Figure Legend Snippet: Functional characterization of HIV-1 T/F full-length reporter viruses. (A) HIV-1 T/F full-length reporter virus design. (B-C) Western blot analysis of Nef protein expression in HEK293 producer cell lysates transfected with TRJO.c (B) and Q23.BG505 (C) derived T/F full-length reporter virus plasmid DNA. Equal amounts of p55 Gag were loaded onto each lane to assess the relative Nef expression. (D) Surface CD4 surface expression on p24+ Jurkat CCR5 + JLTRGFP.R5 cells infected with HIV-1 T/F wild-type or HIV-1 T/F reporter virus 48 hours after initiating infection. Data shown as mean +/- SD of 3 technical replicates with significance calculated from a one-way ANOVA. (E) Single-round infectivity of HIV-1 T/F reporter viruses on TZM-bl cells in the presence of 10 nM of the protease inhibitor saquinavir to block virus spreading (n = 3). Infectivity for each virus was determined by measuring the fold firefly luciferase expression from infected TZM-bl cells above uninfected negative controls and normalizing to reverse transcriptase units (RT U). Data displayed is average fold HIV-1 T/F reporter virus infectivity over TRJO.c and Q23.BG505 wild-type virus +/- SEM from three independent experiments. (F) HIV-1 T/F reporter virus spread in primary CD4 + T cells (n = 3). Each sample was set to the same level of p24 positive cells and then allowed to spread for four days in culture. Data displayed as the fold HIV-1 T/F reporter virus infection (as the value of % p24) over wild-type +/- the SEM from three independent CD4 + T cell preparations.

    Techniques Used: Functional Assay, Western Blot, Expressing, Transfection, Derivative Assay, Plasmid Preparation, Infection, Protease Inhibitor, Blocking Assay, Luciferase

    Non-invasive bioluminescent imaging of longitudinal Q23.BG505.Nluc infection in humanized mice. (A) Longitudinal imaging of Hu-PBL mice (n = 6) infected intraperitonially (i.p.) with 1 x 10 7 infectious units (IUs) of BG505.Nluc* reporter virus. Data representative of six independently infected animals. (B) Longitudinal imaging of productive HIV-1 infection in Hu-HSC mice (n = 2) infected i.p. with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus. (C) Quantification of bioluminescent signal measured over a 15-day period in BG505.Nluc* infected Hu-PBL mice. Average total animal flux was calculated by taking the mean of the total animal flux measured from both the ventral and lateral imaging orientations, excluding the cranial region to avoid signal artifacts arising from the Nano-glo substrate injection site. Data displayed as the mean +/- SEM from six independent experiments. (D, E) Correlative analysis of average total animal flux with the increase of peripheral HIV-1 infected cells (p24 + CD3 + CD8 - cells) measured by flow cytometry (D) and plasma reserve transcriptase activity (RT U) in the infected Hu-PBL mice in (C) measured by the reserve transcriptase SG-PERT activity qPCR assay (E). Upper and lower bounds of the SEM is displayed as gray shaded regions above and below the mean value at each day measured. Pearson correlations was calculated from the average values of peripheral HIV-1 infected cells and average total animal flux and plasma reverse transcriptase activity and average total animal flux.
    Figure Legend Snippet: Non-invasive bioluminescent imaging of longitudinal Q23.BG505.Nluc infection in humanized mice. (A) Longitudinal imaging of Hu-PBL mice (n = 6) infected intraperitonially (i.p.) with 1 x 10 7 infectious units (IUs) of BG505.Nluc* reporter virus. Data representative of six independently infected animals. (B) Longitudinal imaging of productive HIV-1 infection in Hu-HSC mice (n = 2) infected i.p. with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus. (C) Quantification of bioluminescent signal measured over a 15-day period in BG505.Nluc* infected Hu-PBL mice. Average total animal flux was calculated by taking the mean of the total animal flux measured from both the ventral and lateral imaging orientations, excluding the cranial region to avoid signal artifacts arising from the Nano-glo substrate injection site. Data displayed as the mean +/- SEM from six independent experiments. (D, E) Correlative analysis of average total animal flux with the increase of peripheral HIV-1 infected cells (p24 + CD3 + CD8 - cells) measured by flow cytometry (D) and plasma reserve transcriptase activity (RT U) in the infected Hu-PBL mice in (C) measured by the reserve transcriptase SG-PERT activity qPCR assay (E). Upper and lower bounds of the SEM is displayed as gray shaded regions above and below the mean value at each day measured. Pearson correlations was calculated from the average values of peripheral HIV-1 infected cells and average total animal flux and plasma reverse transcriptase activity and average total animal flux.

    Techniques Used: Imaging, Infection, Mouse Assay, Injection, Flow Cytometry, Cytometry, Activity Assay, Real-time Polymerase Chain Reaction

    Longitudinal imaging of HIV-1 acute infection, cART suppression, and recrudescent infection in Hu-HSC mice placed on ART 9 days post-infection. (A) Longitudinal bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in Hu-HSC mice infected with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus and placed on daily i.p. ART injections of Truvada and Isentress 9 days post-infection (n = 2). Red star denotes the timepoint and Hu-HSC mouse that exhibited a transient increase in plasma reverse transcriptase activity during ART treatment. (B) Quantification of plasma reverse transcriptase activity (above) and Nluc signal as average total animal flux (below) from the animals in (A). Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A), approximately two weeks following cART cessation.
    Figure Legend Snippet: Longitudinal imaging of HIV-1 acute infection, cART suppression, and recrudescent infection in Hu-HSC mice placed on ART 9 days post-infection. (A) Longitudinal bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in Hu-HSC mice infected with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus and placed on daily i.p. ART injections of Truvada and Isentress 9 days post-infection (n = 2). Red star denotes the timepoint and Hu-HSC mouse that exhibited a transient increase in plasma reverse transcriptase activity during ART treatment. (B) Quantification of plasma reverse transcriptase activity (above) and Nluc signal as average total animal flux (below) from the animals in (A). Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A), approximately two weeks following cART cessation.

    Techniques Used: Imaging, Infection, Mouse Assay, Activity Assay, Ex Vivo

    Longitudinal imaging of HIV-1 acute infection, cART suppression, and infection recrudescence in Hu-HSC mice placed on ART 6 days post-infection. (A) Longitudinal bioluminescent imaging of spreading infection in Hu-HSC mice infected with 1 x 10 6 infectious units (IUs) of BG505.Nluc* T/F reporter virus and placed on a daily ART regimen comprised of daily i.p. ART injections of Truvada and Isentress 6 days post-infection (n = 2). (B) Quantification of plasma reverse transcriptase activity (above) and Nluc signal shown as average total animal flux (below) from the mice in (A). Plasma reverse transcriptase activity in serum samples obtained every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A) approximately two weeks following cART cessation.
    Figure Legend Snippet: Longitudinal imaging of HIV-1 acute infection, cART suppression, and infection recrudescence in Hu-HSC mice placed on ART 6 days post-infection. (A) Longitudinal bioluminescent imaging of spreading infection in Hu-HSC mice infected with 1 x 10 6 infectious units (IUs) of BG505.Nluc* T/F reporter virus and placed on a daily ART regimen comprised of daily i.p. ART injections of Truvada and Isentress 6 days post-infection (n = 2). (B) Quantification of plasma reverse transcriptase activity (above) and Nluc signal shown as average total animal flux (below) from the mice in (A). Plasma reverse transcriptase activity in serum samples obtained every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A) approximately two weeks following cART cessation.

    Techniques Used: Imaging, Infection, Mouse Assay, Activity Assay, Ex Vivo

    In vitro HIV-1 T/F reporter virus reporter gene stability in primary CD4+ T cells. (A) NL43.R5.GFP reporter virus design. Purple regions correspond to duplicated 3’-LTR sequence flanking the reporter gene. (B, C) Stability of GFP and Nluc reporter viruses in primary CD4 T cells. To force the reporter viruses to continuously spread to new cells, fresh autologous uninfected T cells were added every 48 hours so that the percent of p24 + cells in the total culture was maintained at the same value for each experiment. HIV-1 T/F and NL43.R5.GFP reporter virus GFP and p24 expression 2 days and 8 days post-initiation (B). FACS data is representative of 3–6 independent experiments. Reporter expression was determined via flow cytometry and displayed as the percentage of double-positive HIV-1 and GFP co-expressing cells in the total p24 + population for GFP expressing reporter viruses and the total fold Nluc-derived light units above an Efiverenz negative control for the BG505.Nluc* reporter virus (C). Data in (C) shown as the mean +/- SEM of 3–6 independent donor primary CD4 + T cell preparations.
    Figure Legend Snippet: In vitro HIV-1 T/F reporter virus reporter gene stability in primary CD4+ T cells. (A) NL43.R5.GFP reporter virus design. Purple regions correspond to duplicated 3’-LTR sequence flanking the reporter gene. (B, C) Stability of GFP and Nluc reporter viruses in primary CD4 T cells. To force the reporter viruses to continuously spread to new cells, fresh autologous uninfected T cells were added every 48 hours so that the percent of p24 + cells in the total culture was maintained at the same value for each experiment. HIV-1 T/F and NL43.R5.GFP reporter virus GFP and p24 expression 2 days and 8 days post-initiation (B). FACS data is representative of 3–6 independent experiments. Reporter expression was determined via flow cytometry and displayed as the percentage of double-positive HIV-1 and GFP co-expressing cells in the total p24 + population for GFP expressing reporter viruses and the total fold Nluc-derived light units above an Efiverenz negative control for the BG505.Nluc* reporter virus (C). Data in (C) shown as the mean +/- SEM of 3–6 independent donor primary CD4 + T cell preparations.

    Techniques Used: In Vitro, Sequencing, Expressing, FACS, Flow Cytometry, Cytometry, Derivative Assay, Negative Control

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    Worthington Biochemical recombinant hiv 1 reverse transcriptase standard curve
    Longitudinal non-invasive bioluminescent imaging of <t>HIV-1</t> acute infection, suppression, and recrudescent infection in the Hu-BLT mouse group placed on cART 12 days post-infection. (A) Bioluminescent imaging of spreading infection of Hu-BLT Mouse #3 infected with 1 x 10 6 IUs of Q23.BG505.Nluc T/F reporter virus and placed on a daily cART regimen comprised of daily i.p. cART injections of Truvada and Isentress 12 days post-infection. (B) Whole animal ex vivo necroscopic analysis of rebounding infection in Hu-BLT Mouse #3 five days following cART cessation. (C) Plasma reverse transcriptase activity from Hu-BLT Mouse #3 over the course of the 40 day imaging period. Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (dotted line).
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    Longitudinal non-invasive bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in the Hu-BLT mouse group placed on cART 12 days post-infection. (A) Bioluminescent imaging of spreading infection of Hu-BLT Mouse #3 infected with 1 x 10 6 IUs of Q23.BG505.Nluc T/F reporter virus and placed on a daily cART regimen comprised of daily i.p. cART injections of Truvada and Isentress 12 days post-infection. (B) Whole animal ex vivo necroscopic analysis of rebounding infection in Hu-BLT Mouse #3 five days following cART cessation. (C) Plasma reverse transcriptase activity from Hu-BLT Mouse #3 over the course of the 40 day imaging period. Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (dotted line).

    Journal: bioRxiv

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    doi: 10.1101/745125

    Figure Lengend Snippet: Longitudinal non-invasive bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in the Hu-BLT mouse group placed on cART 12 days post-infection. (A) Bioluminescent imaging of spreading infection of Hu-BLT Mouse #3 infected with 1 x 10 6 IUs of Q23.BG505.Nluc T/F reporter virus and placed on a daily cART regimen comprised of daily i.p. cART injections of Truvada and Isentress 12 days post-infection. (B) Whole animal ex vivo necroscopic analysis of rebounding infection in Hu-BLT Mouse #3 five days following cART cessation. (C) Plasma reverse transcriptase activity from Hu-BLT Mouse #3 over the course of the 40 day imaging period. Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (dotted line).

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: Imaging, Infection, Ex Vivo, Activity Assay

    In vitro HIV-1 T/F reporter virus reporter gene stability in primary CD4+ T cells. (A) NL43.R5.GFP reporter virus design. Purple regions correspond to duplicated 3’-LTR sequence flanking the reporter gene. (B, C) Stability of GFP and Nluc reporter viruses in primary CD4 T cells. To force the reporter viruses to continuously spread to new cells, fresh autologous uninfected T cells were added every 48 hours so that the percent of p24+ cells in the total culture was maintained at the same value for each experiment. HIV-1 T/F and NL43.R5.GFP reporter virus GFP and p24 expression 2 days and 8 days post-initiation (B). FACS data is representative of 3-6 independent experiments. Reporter expression was determined via flow cytometry and displayed as the percentage of double-positive HIV-1 and GFP co-expressing cells in the total p24+ population for GFP expressing reporter viruses and the total fold Nluc-derived light units above an Efiverenz negative control for the BG505.Nluc* reporter virus (C). Data in (C) shown as the mean +/-SEM of 3-6 independent donor primary CD4+ T cell preparations.

    Journal: bioRxiv

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    doi: 10.1101/745125

    Figure Lengend Snippet: In vitro HIV-1 T/F reporter virus reporter gene stability in primary CD4+ T cells. (A) NL43.R5.GFP reporter virus design. Purple regions correspond to duplicated 3’-LTR sequence flanking the reporter gene. (B, C) Stability of GFP and Nluc reporter viruses in primary CD4 T cells. To force the reporter viruses to continuously spread to new cells, fresh autologous uninfected T cells were added every 48 hours so that the percent of p24+ cells in the total culture was maintained at the same value for each experiment. HIV-1 T/F and NL43.R5.GFP reporter virus GFP and p24 expression 2 days and 8 days post-initiation (B). FACS data is representative of 3-6 independent experiments. Reporter expression was determined via flow cytometry and displayed as the percentage of double-positive HIV-1 and GFP co-expressing cells in the total p24+ population for GFP expressing reporter viruses and the total fold Nluc-derived light units above an Efiverenz negative control for the BG505.Nluc* reporter virus (C). Data in (C) shown as the mean +/-SEM of 3-6 independent donor primary CD4+ T cell preparations.

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: In Vitro, Sequencing, Expressing, FACS, Flow Cytometry, Derivative Assay, Negative Control

    RNA viral load assay and SG-PERT RT activity assay sensitivities. (A) eripheral blood mononuclear cell (PBMC) derived HIV-1 JR-CSF viral supernatant was stored in separate aliquots of equal volume in order to compare the sensitivity of the Quantitect qRT-PCR viral load assay and the SG-PERT reverse transcriptase activity qPCR assay in parallel. (B) The Quantitect qRT-PCR viral load assay and the SG-PERT reverse transcriptase activity qPCR assay was run in parallel with viral RNA eluate and HIV-1 supernatant serially diluted until the limit of detection for each assay was reached. Data shown as the average cycle threshold (Cq) values determined from two technical replicates at each dilution. The limit of detection was defined as the Cq value at which the linear range of the assay ended. Absolute quantification of HIV-1 particles was determined from a viral RNA standard curve run in parallel with the Quantitect qRT-PCR viral load assay.

    Journal: bioRxiv

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    doi: 10.1101/745125

    Figure Lengend Snippet: RNA viral load assay and SG-PERT RT activity assay sensitivities. (A) eripheral blood mononuclear cell (PBMC) derived HIV-1 JR-CSF viral supernatant was stored in separate aliquots of equal volume in order to compare the sensitivity of the Quantitect qRT-PCR viral load assay and the SG-PERT reverse transcriptase activity qPCR assay in parallel. (B) The Quantitect qRT-PCR viral load assay and the SG-PERT reverse transcriptase activity qPCR assay was run in parallel with viral RNA eluate and HIV-1 supernatant serially diluted until the limit of detection for each assay was reached. Data shown as the average cycle threshold (Cq) values determined from two technical replicates at each dilution. The limit of detection was defined as the Cq value at which the linear range of the assay ended. Absolute quantification of HIV-1 particles was determined from a viral RNA standard curve run in parallel with the Quantitect qRT-PCR viral load assay.

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: RNA Viral-load Assay, RT Activity Assay, Derivative Assay, Quantitative RT-PCR, Viral-load Assay, Activity Assay, Real-time Polymerase Chain Reaction

    Confocal immunofluorescence microscopy of cleared HIV-1 Q23.BG505.Nluc infected spleen tissue during recrudescent infection. (A) Nluc expressing spleen tissue from Hu-HSC mouse #14 infected with BG505.Nluc* reporter virus. Nluc expressing spleen is enclosed in red boxes at whole-animal and tissue resolutions, respectively. Spleen tissue was surgically removed 17 days following cART withdrawal and was subsequently fixed, cleared, and immunostained for confocal microscopy to identify HIV-1 p24+ cells. (B-C) Representative confocal slices of Nluc expressing spleen tissue from (A). Tissues were immunostained for HIV-1 p24 (green), human CD3+ T cells (magenta), human CD68+ macrophages (red), and stained with DAPI to identify nuclei (cyan). HIV-1 p24 was associated with human CD3+ T-cells (B) and human CD68+ macrophages (C) within the same piece of Nluc expressing spleen tissue. (D-F) Immunostaining of representative Nluc expressing cells in spleen tissue from BG505.Nluc* infected Hu-HSC mouse #14 (D) and Hu-HSC mouse #15 (F). Nluc expression (red) colocalizes with HIV-1 p24 (green) and CD3 (magenta) in spleen tissue isolated from both Hu-HSC mice.

    Journal: bioRxiv

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    doi: 10.1101/745125

    Figure Lengend Snippet: Confocal immunofluorescence microscopy of cleared HIV-1 Q23.BG505.Nluc infected spleen tissue during recrudescent infection. (A) Nluc expressing spleen tissue from Hu-HSC mouse #14 infected with BG505.Nluc* reporter virus. Nluc expressing spleen is enclosed in red boxes at whole-animal and tissue resolutions, respectively. Spleen tissue was surgically removed 17 days following cART withdrawal and was subsequently fixed, cleared, and immunostained for confocal microscopy to identify HIV-1 p24+ cells. (B-C) Representative confocal slices of Nluc expressing spleen tissue from (A). Tissues were immunostained for HIV-1 p24 (green), human CD3+ T cells (magenta), human CD68+ macrophages (red), and stained with DAPI to identify nuclei (cyan). HIV-1 p24 was associated with human CD3+ T-cells (B) and human CD68+ macrophages (C) within the same piece of Nluc expressing spleen tissue. (D-F) Immunostaining of representative Nluc expressing cells in spleen tissue from BG505.Nluc* infected Hu-HSC mouse #14 (D) and Hu-HSC mouse #15 (F). Nluc expression (red) colocalizes with HIV-1 p24 (green) and CD3 (magenta) in spleen tissue isolated from both Hu-HSC mice.

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: Immunofluorescence, Microscopy, Infection, Expressing, Confocal Microscopy, Staining, Immunostaining, Isolation, Mouse Assay

    Longitudinal imaging of HIV-1 acute infection, cART suppression, and infection recrudescence in Hu-HSC mice placed on ART 6 days post-infection. (A) Longitudinal bioluminescent imaging of spreading infection in Hu-HSC mice infected with 1 x 10 6 infectious units (IUs) of BG505.Nluc* T/F reporter virus and placed on a daily ART regimen comprised of daily i.p. ART injections of Truvada and Isentress 6 days post-infection (n=2). (B) Quantification of plasma reverse transcriptase activity from the mice in (A). Plasma reverse transcriptase activity in serum samples obtained every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A) approximately two weeks following ART cessation.

    Journal: bioRxiv

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    doi: 10.1101/745125

    Figure Lengend Snippet: Longitudinal imaging of HIV-1 acute infection, cART suppression, and infection recrudescence in Hu-HSC mice placed on ART 6 days post-infection. (A) Longitudinal bioluminescent imaging of spreading infection in Hu-HSC mice infected with 1 x 10 6 infectious units (IUs) of BG505.Nluc* T/F reporter virus and placed on a daily ART regimen comprised of daily i.p. ART injections of Truvada and Isentress 6 days post-infection (n=2). (B) Quantification of plasma reverse transcriptase activity from the mice in (A). Plasma reverse transcriptase activity in serum samples obtained every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A) approximately two weeks following ART cessation.

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: Imaging, Infection, Mouse Assay, Activity Assay, Ex Vivo

    Non-invasive bioluminescent imaging of longitudinal HIV-1 Q23.BG505 infection in humanized mice. (A) Longitudinal imaging of Hu-PBL mice (n=6) infected intraperitonially (i.p.) with 1 x 10 7 infectious units (IUs) of HIV-1 BG505.Nluc* reporter virus. Data representative of six independently infected animals. (B) Longitudinal imaging of productive HIV-1 infection in Hu-HSC mice (n=2) infected i.p. with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus. (C) Quantification of bioluminescent signal measured over a 15 day period in BG505.Nluc* infected Hu-PBL mice. Average total animal flux was calculated by taking the mean of the total animal flux measured from both the ventral and lateral imaging orientations, excluding the cranial region to avoid signal artifacts arising from the Nano-glo substrate injection site. Data displayed as the mean +/-SEM from six independent experiments. (D, E) Correlative analysis of average total animal flux with the increase of peripheral HIV-1 infected cells (p24+CD3+CD8-cells) measured by flow cytometry (D) and plasma reserve transcriptase activity (RT U) in the infected Hu-PBL mice in (C) measured by the reserve transcriptase SG-PERT activity qPCR assay (E). Upper and lower bounds of the SEM is displayed as gray shaded regions above and below the mean value at each day measured. Pearson correlation calculated from (D) the average values of peripheral HIV-1 infected cells and average total animal flux and (E) plasma reverse transcriptase activity and average total animal flux.

    Journal: bioRxiv

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    doi: 10.1101/745125

    Figure Lengend Snippet: Non-invasive bioluminescent imaging of longitudinal HIV-1 Q23.BG505 infection in humanized mice. (A) Longitudinal imaging of Hu-PBL mice (n=6) infected intraperitonially (i.p.) with 1 x 10 7 infectious units (IUs) of HIV-1 BG505.Nluc* reporter virus. Data representative of six independently infected animals. (B) Longitudinal imaging of productive HIV-1 infection in Hu-HSC mice (n=2) infected i.p. with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus. (C) Quantification of bioluminescent signal measured over a 15 day period in BG505.Nluc* infected Hu-PBL mice. Average total animal flux was calculated by taking the mean of the total animal flux measured from both the ventral and lateral imaging orientations, excluding the cranial region to avoid signal artifacts arising from the Nano-glo substrate injection site. Data displayed as the mean +/-SEM from six independent experiments. (D, E) Correlative analysis of average total animal flux with the increase of peripheral HIV-1 infected cells (p24+CD3+CD8-cells) measured by flow cytometry (D) and plasma reserve transcriptase activity (RT U) in the infected Hu-PBL mice in (C) measured by the reserve transcriptase SG-PERT activity qPCR assay (E). Upper and lower bounds of the SEM is displayed as gray shaded regions above and below the mean value at each day measured. Pearson correlation calculated from (D) the average values of peripheral HIV-1 infected cells and average total animal flux and (E) plasma reverse transcriptase activity and average total animal flux.

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: Imaging, Infection, Mouse Assay, Injection, Flow Cytometry, Activity Assay, Real-time Polymerase Chain Reaction

    Functional characterization of HIV-1 T/F full-length reporter viruses. (A) HIV-1 T/F full-length reporter virus design. (B-C) Western blot analysis of Nef protein expression in HEK293 producer cell lysates transfected with TRJO.c (B) and Q23.BG505 (C) derived T/F full-length reporter virus plasmid DNA. Equal amounts of p55 Gag were loaded onto each lane to assess the relative Nef expression. (D) Surface CD4 surface expression on p24+ Jurkat CCR5+ JLTRGFP.R5 cells infected with HIV-1 T/F wild-type or HIV-1 T/F reporter virus 48 hours after initiating infection. Data shown as mean +/-SD of 3 technical replicates with significance calculated from a one-way ANOVA. (E) Single-round infectivity of HIV-1 T/F reporter viruses on TZM-bl cells cells in the presence of 10 nM of the protease inhibitor saquinavir to block virus spreading (n=3). Infectivity for each virus was determined by measuring the fold firefly luciferase expression from infected TZM-bl cells above uninfected negative controls and normalizing to reverse transcriptase units (RT U). Data displayed is average fold HIV-1 T/F reporter virus infectivity over TRJO.c and Q23.BG505 wild-type virus +/-SEM from three independent experiments. (F) HIV-1 T/F reporter virus spread in primary CD4+ T cells (n=3). Each sample was set to the same level of p24 positive cells and then allowed to spread for four days in culture. Data displayed as the fold HIV-1 T/F reporter virus infection (as the value of % p24) over wild-type +/-the SEM from three independent CD4+ T cell preparations.

    Journal: bioRxiv

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    doi: 10.1101/745125

    Figure Lengend Snippet: Functional characterization of HIV-1 T/F full-length reporter viruses. (A) HIV-1 T/F full-length reporter virus design. (B-C) Western blot analysis of Nef protein expression in HEK293 producer cell lysates transfected with TRJO.c (B) and Q23.BG505 (C) derived T/F full-length reporter virus plasmid DNA. Equal amounts of p55 Gag were loaded onto each lane to assess the relative Nef expression. (D) Surface CD4 surface expression on p24+ Jurkat CCR5+ JLTRGFP.R5 cells infected with HIV-1 T/F wild-type or HIV-1 T/F reporter virus 48 hours after initiating infection. Data shown as mean +/-SD of 3 technical replicates with significance calculated from a one-way ANOVA. (E) Single-round infectivity of HIV-1 T/F reporter viruses on TZM-bl cells cells in the presence of 10 nM of the protease inhibitor saquinavir to block virus spreading (n=3). Infectivity for each virus was determined by measuring the fold firefly luciferase expression from infected TZM-bl cells above uninfected negative controls and normalizing to reverse transcriptase units (RT U). Data displayed is average fold HIV-1 T/F reporter virus infectivity over TRJO.c and Q23.BG505 wild-type virus +/-SEM from three independent experiments. (F) HIV-1 T/F reporter virus spread in primary CD4+ T cells (n=3). Each sample was set to the same level of p24 positive cells and then allowed to spread for four days in culture. Data displayed as the fold HIV-1 T/F reporter virus infection (as the value of % p24) over wild-type +/-the SEM from three independent CD4+ T cell preparations.

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: Functional Assay, Western Blot, Expressing, Transfection, Derivative Assay, Plasmid Preparation, Infection, Protease Inhibitor, Blocking Assay, Luciferase

    Longitudinal imaging of HIV-1 acute infection, suppression, and recrudescent infection in Hu-HSC mice placed on ART 9 days post-infection. (A) Longitudinal bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in Hu-HSC mice infected with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus and placed on daily i.p. ART injections of Truvada and Isentress 9 days post-infection (n=2). Red star denotes the timepoint and Hu-HSC mouse that exhibited a transient increase in plasma reverse transcriptase activity during ART treatment. (B) Quantification of plasma reverse transcriptase activity from the animals in (A). Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A), approximately two weeks following ART cessation.

    Journal: bioRxiv

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    doi: 10.1101/745125

    Figure Lengend Snippet: Longitudinal imaging of HIV-1 acute infection, suppression, and recrudescent infection in Hu-HSC mice placed on ART 9 days post-infection. (A) Longitudinal bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in Hu-HSC mice infected with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus and placed on daily i.p. ART injections of Truvada and Isentress 9 days post-infection (n=2). Red star denotes the timepoint and Hu-HSC mouse that exhibited a transient increase in plasma reverse transcriptase activity during ART treatment. (B) Quantification of plasma reverse transcriptase activity from the animals in (A). Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A), approximately two weeks following ART cessation.

    Article Snippet: All samples were run simultaneously and concurrently with an recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: Imaging, Infection, Mouse Assay, Activity Assay, Ex Vivo

    Confocal immunofluorescence microscopy of cleared Q23.BG505.Nluc infected spleen tissue during recrudescent infection. (A) Nluc expressing spleen tissue from Hu-HSC mouse #14 infected with Q23.BG505.Nluc T/F reporter virus. Nluc expressing spleen is enclosed in red boxes at whole-animal and tissue resolutions, respectively. Spleen tissue was surgically removed 17 days following cART withdrawal and was subsequently fixed, cleared, and immunostained for confocal microscopy to identify HIV-1 p24 + cells. (B-C) Representative confocal slices of Nluc expressing spleen tissue from (A). Tissues were immunostained for HIV-1 p24 (green), human CD3 + T cells (magenta), human CD68 + macrophages (red), and stained with DAPI to identify nuclei (cyan). HIV-1 p24 was associated with human CD3 + T-cells (B) and human CD68 + macrophages (C) within the same piece of Nluc expressing spleen tissue. (D-E) Immunostaining of representative Nluc expressing cells in spleen tissue from BG505.Nluc* infected Hu-HSC mouse #14 (D) and Hu-HSC mouse #15 (E). Nluc expression (red) colocalizes with HIV-1 p24 (green) and CD3 (magenta) in spleen tissue isolated from both Hu-HSC mice.

    Journal: PLoS Pathogens

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    doi: 10.1371/journal.ppat.1008161

    Figure Lengend Snippet: Confocal immunofluorescence microscopy of cleared Q23.BG505.Nluc infected spleen tissue during recrudescent infection. (A) Nluc expressing spleen tissue from Hu-HSC mouse #14 infected with Q23.BG505.Nluc T/F reporter virus. Nluc expressing spleen is enclosed in red boxes at whole-animal and tissue resolutions, respectively. Spleen tissue was surgically removed 17 days following cART withdrawal and was subsequently fixed, cleared, and immunostained for confocal microscopy to identify HIV-1 p24 + cells. (B-C) Representative confocal slices of Nluc expressing spleen tissue from (A). Tissues were immunostained for HIV-1 p24 (green), human CD3 + T cells (magenta), human CD68 + macrophages (red), and stained with DAPI to identify nuclei (cyan). HIV-1 p24 was associated with human CD3 + T-cells (B) and human CD68 + macrophages (C) within the same piece of Nluc expressing spleen tissue. (D-E) Immunostaining of representative Nluc expressing cells in spleen tissue from BG505.Nluc* infected Hu-HSC mouse #14 (D) and Hu-HSC mouse #15 (E). Nluc expression (red) colocalizes with HIV-1 p24 (green) and CD3 (magenta) in spleen tissue isolated from both Hu-HSC mice.

    Article Snippet: All samples were run simultaneously and concurrently with a recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: Immunofluorescence, Microscopy, Infection, Expressing, Confocal Microscopy, Staining, Immunostaining, Isolation, Mouse Assay

    Functional characterization of HIV-1 T/F full-length reporter viruses. (A) HIV-1 T/F full-length reporter virus design. (B-C) Western blot analysis of Nef protein expression in HEK293 producer cell lysates transfected with TRJO.c (B) and Q23.BG505 (C) derived T/F full-length reporter virus plasmid DNA. Equal amounts of p55 Gag were loaded onto each lane to assess the relative Nef expression. (D) Surface CD4 surface expression on p24+ Jurkat CCR5 + JLTRGFP.R5 cells infected with HIV-1 T/F wild-type or HIV-1 T/F reporter virus 48 hours after initiating infection. Data shown as mean +/- SD of 3 technical replicates with significance calculated from a one-way ANOVA. (E) Single-round infectivity of HIV-1 T/F reporter viruses on TZM-bl cells in the presence of 10 nM of the protease inhibitor saquinavir to block virus spreading (n = 3). Infectivity for each virus was determined by measuring the fold firefly luciferase expression from infected TZM-bl cells above uninfected negative controls and normalizing to reverse transcriptase units (RT U). Data displayed is average fold HIV-1 T/F reporter virus infectivity over TRJO.c and Q23.BG505 wild-type virus +/- SEM from three independent experiments. (F) HIV-1 T/F reporter virus spread in primary CD4 + T cells (n = 3). Each sample was set to the same level of p24 positive cells and then allowed to spread for four days in culture. Data displayed as the fold HIV-1 T/F reporter virus infection (as the value of % p24) over wild-type +/- the SEM from three independent CD4 + T cell preparations.

    Journal: PLoS Pathogens

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    doi: 10.1371/journal.ppat.1008161

    Figure Lengend Snippet: Functional characterization of HIV-1 T/F full-length reporter viruses. (A) HIV-1 T/F full-length reporter virus design. (B-C) Western blot analysis of Nef protein expression in HEK293 producer cell lysates transfected with TRJO.c (B) and Q23.BG505 (C) derived T/F full-length reporter virus plasmid DNA. Equal amounts of p55 Gag were loaded onto each lane to assess the relative Nef expression. (D) Surface CD4 surface expression on p24+ Jurkat CCR5 + JLTRGFP.R5 cells infected with HIV-1 T/F wild-type or HIV-1 T/F reporter virus 48 hours after initiating infection. Data shown as mean +/- SD of 3 technical replicates with significance calculated from a one-way ANOVA. (E) Single-round infectivity of HIV-1 T/F reporter viruses on TZM-bl cells in the presence of 10 nM of the protease inhibitor saquinavir to block virus spreading (n = 3). Infectivity for each virus was determined by measuring the fold firefly luciferase expression from infected TZM-bl cells above uninfected negative controls and normalizing to reverse transcriptase units (RT U). Data displayed is average fold HIV-1 T/F reporter virus infectivity over TRJO.c and Q23.BG505 wild-type virus +/- SEM from three independent experiments. (F) HIV-1 T/F reporter virus spread in primary CD4 + T cells (n = 3). Each sample was set to the same level of p24 positive cells and then allowed to spread for four days in culture. Data displayed as the fold HIV-1 T/F reporter virus infection (as the value of % p24) over wild-type +/- the SEM from three independent CD4 + T cell preparations.

    Article Snippet: All samples were run simultaneously and concurrently with a recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: Functional Assay, Western Blot, Expressing, Transfection, Derivative Assay, Plasmid Preparation, Infection, Protease Inhibitor, Blocking Assay, Luciferase

    Non-invasive bioluminescent imaging of longitudinal Q23.BG505.Nluc infection in humanized mice. (A) Longitudinal imaging of Hu-PBL mice (n = 6) infected intraperitonially (i.p.) with 1 x 10 7 infectious units (IUs) of BG505.Nluc* reporter virus. Data representative of six independently infected animals. (B) Longitudinal imaging of productive HIV-1 infection in Hu-HSC mice (n = 2) infected i.p. with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus. (C) Quantification of bioluminescent signal measured over a 15-day period in BG505.Nluc* infected Hu-PBL mice. Average total animal flux was calculated by taking the mean of the total animal flux measured from both the ventral and lateral imaging orientations, excluding the cranial region to avoid signal artifacts arising from the Nano-glo substrate injection site. Data displayed as the mean +/- SEM from six independent experiments. (D, E) Correlative analysis of average total animal flux with the increase of peripheral HIV-1 infected cells (p24 + CD3 + CD8 - cells) measured by flow cytometry (D) and plasma reserve transcriptase activity (RT U) in the infected Hu-PBL mice in (C) measured by the reserve transcriptase SG-PERT activity qPCR assay (E). Upper and lower bounds of the SEM is displayed as gray shaded regions above and below the mean value at each day measured. Pearson correlations was calculated from the average values of peripheral HIV-1 infected cells and average total animal flux and plasma reverse transcriptase activity and average total animal flux.

    Journal: PLoS Pathogens

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    doi: 10.1371/journal.ppat.1008161

    Figure Lengend Snippet: Non-invasive bioluminescent imaging of longitudinal Q23.BG505.Nluc infection in humanized mice. (A) Longitudinal imaging of Hu-PBL mice (n = 6) infected intraperitonially (i.p.) with 1 x 10 7 infectious units (IUs) of BG505.Nluc* reporter virus. Data representative of six independently infected animals. (B) Longitudinal imaging of productive HIV-1 infection in Hu-HSC mice (n = 2) infected i.p. with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus. (C) Quantification of bioluminescent signal measured over a 15-day period in BG505.Nluc* infected Hu-PBL mice. Average total animal flux was calculated by taking the mean of the total animal flux measured from both the ventral and lateral imaging orientations, excluding the cranial region to avoid signal artifacts arising from the Nano-glo substrate injection site. Data displayed as the mean +/- SEM from six independent experiments. (D, E) Correlative analysis of average total animal flux with the increase of peripheral HIV-1 infected cells (p24 + CD3 + CD8 - cells) measured by flow cytometry (D) and plasma reserve transcriptase activity (RT U) in the infected Hu-PBL mice in (C) measured by the reserve transcriptase SG-PERT activity qPCR assay (E). Upper and lower bounds of the SEM is displayed as gray shaded regions above and below the mean value at each day measured. Pearson correlations was calculated from the average values of peripheral HIV-1 infected cells and average total animal flux and plasma reverse transcriptase activity and average total animal flux.

    Article Snippet: All samples were run simultaneously and concurrently with a recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: Imaging, Infection, Mouse Assay, Injection, Flow Cytometry, Cytometry, Activity Assay, Real-time Polymerase Chain Reaction

    Longitudinal imaging of HIV-1 acute infection, cART suppression, and recrudescent infection in Hu-HSC mice placed on ART 9 days post-infection. (A) Longitudinal bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in Hu-HSC mice infected with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus and placed on daily i.p. ART injections of Truvada and Isentress 9 days post-infection (n = 2). Red star denotes the timepoint and Hu-HSC mouse that exhibited a transient increase in plasma reverse transcriptase activity during ART treatment. (B) Quantification of plasma reverse transcriptase activity (above) and Nluc signal as average total animal flux (below) from the animals in (A). Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A), approximately two weeks following cART cessation.

    Journal: PLoS Pathogens

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    doi: 10.1371/journal.ppat.1008161

    Figure Lengend Snippet: Longitudinal imaging of HIV-1 acute infection, cART suppression, and recrudescent infection in Hu-HSC mice placed on ART 9 days post-infection. (A) Longitudinal bioluminescent imaging of HIV-1 acute infection, suppression, and recrudescent infection in Hu-HSC mice infected with 1 x 10 6 IUs of BG505.Nluc* T/F reporter virus and placed on daily i.p. ART injections of Truvada and Isentress 9 days post-infection (n = 2). Red star denotes the timepoint and Hu-HSC mouse that exhibited a transient increase in plasma reverse transcriptase activity during ART treatment. (B) Quantification of plasma reverse transcriptase activity (above) and Nluc signal as average total animal flux (below) from the animals in (A). Plasma reverse transcriptase activity in serum samples taken every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A), approximately two weeks following cART cessation.

    Article Snippet: All samples were run simultaneously and concurrently with a recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: Imaging, Infection, Mouse Assay, Activity Assay, Ex Vivo

    Longitudinal imaging of HIV-1 acute infection, cART suppression, and infection recrudescence in Hu-HSC mice placed on ART 6 days post-infection. (A) Longitudinal bioluminescent imaging of spreading infection in Hu-HSC mice infected with 1 x 10 6 infectious units (IUs) of BG505.Nluc* T/F reporter virus and placed on a daily ART regimen comprised of daily i.p. ART injections of Truvada and Isentress 6 days post-infection (n = 2). (B) Quantification of plasma reverse transcriptase activity (above) and Nluc signal shown as average total animal flux (below) from the mice in (A). Plasma reverse transcriptase activity in serum samples obtained every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A) approximately two weeks following cART cessation.

    Journal: PLoS Pathogens

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    doi: 10.1371/journal.ppat.1008161

    Figure Lengend Snippet: Longitudinal imaging of HIV-1 acute infection, cART suppression, and infection recrudescence in Hu-HSC mice placed on ART 6 days post-infection. (A) Longitudinal bioluminescent imaging of spreading infection in Hu-HSC mice infected with 1 x 10 6 infectious units (IUs) of BG505.Nluc* T/F reporter virus and placed on a daily ART regimen comprised of daily i.p. ART injections of Truvada and Isentress 6 days post-infection (n = 2). (B) Quantification of plasma reverse transcriptase activity (above) and Nluc signal shown as average total animal flux (below) from the mice in (A). Plasma reverse transcriptase activity in serum samples obtained every six days over the course of the imaging period was measured via the SG-PERT reverse transcriptase activity assay and described as reverse transcriptase activity units / mL above endogenous uninfected background levels (shown as a dotted line). (C) Whole animal ex vivo necroscopic analysis of recrudescent infection in Hu-HSC mice from (A) approximately two weeks following cART cessation.

    Article Snippet: All samples were run simultaneously and concurrently with a recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: Imaging, Infection, Mouse Assay, Activity Assay, Ex Vivo

    In vitro HIV-1 T/F reporter virus reporter gene stability in primary CD4+ T cells. (A) NL43.R5.GFP reporter virus design. Purple regions correspond to duplicated 3’-LTR sequence flanking the reporter gene. (B, C) Stability of GFP and Nluc reporter viruses in primary CD4 T cells. To force the reporter viruses to continuously spread to new cells, fresh autologous uninfected T cells were added every 48 hours so that the percent of p24 + cells in the total culture was maintained at the same value for each experiment. HIV-1 T/F and NL43.R5.GFP reporter virus GFP and p24 expression 2 days and 8 days post-initiation (B). FACS data is representative of 3–6 independent experiments. Reporter expression was determined via flow cytometry and displayed as the percentage of double-positive HIV-1 and GFP co-expressing cells in the total p24 + population for GFP expressing reporter viruses and the total fold Nluc-derived light units above an Efiverenz negative control for the BG505.Nluc* reporter virus (C). Data in (C) shown as the mean +/- SEM of 3–6 independent donor primary CD4 + T cell preparations.

    Journal: PLoS Pathogens

    Article Title: Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

    doi: 10.1371/journal.ppat.1008161

    Figure Lengend Snippet: In vitro HIV-1 T/F reporter virus reporter gene stability in primary CD4+ T cells. (A) NL43.R5.GFP reporter virus design. Purple regions correspond to duplicated 3’-LTR sequence flanking the reporter gene. (B, C) Stability of GFP and Nluc reporter viruses in primary CD4 T cells. To force the reporter viruses to continuously spread to new cells, fresh autologous uninfected T cells were added every 48 hours so that the percent of p24 + cells in the total culture was maintained at the same value for each experiment. HIV-1 T/F and NL43.R5.GFP reporter virus GFP and p24 expression 2 days and 8 days post-initiation (B). FACS data is representative of 3–6 independent experiments. Reporter expression was determined via flow cytometry and displayed as the percentage of double-positive HIV-1 and GFP co-expressing cells in the total p24 + population for GFP expressing reporter viruses and the total fold Nluc-derived light units above an Efiverenz negative control for the BG505.Nluc* reporter virus (C). Data in (C) shown as the mean +/- SEM of 3–6 independent donor primary CD4 + T cell preparations.

    Article Snippet: All samples were run simultaneously and concurrently with a recombinant HIV-1 reverse transcriptase standard curve (Worthington Biochemical Corporation, Lakewood, NJ).

    Techniques: In Vitro, Sequencing, Expressing, FACS, Flow Cytometry, Cytometry, Derivative Assay, Negative Control