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Therapeutic Strategy for the Prevention of Pseudorabies Virus Infection in C57BL/6 Mice by 3D8 scFv with Intrinsic Nuclease Activity.

Lee G, Cho S, Hoang PM, Kim D, Lee Y, Kil EJ, Byun SJ, Lee TK, Kim DH, Kim S, Lee S - Mol. Cells (2015)

Bottom Line: The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays.Intraperitoneal injection of 5 and 10 μg 3D8 scFv resulted in no detectable toxicity.The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Korea.

ABSTRACT
3D8 single chain variable fragment (scFv) is a recombinant monoclonal antibody with nuclease activity that was originally isolated from autoimmune-prone MRL mice. In a previous study, we analyzed the nuclease activity of 3D8 scFv and determined that a HeLa cell line expressing 3D8 scFv conferred resistance to herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). In this study, we demonstrate that 3D8 scFv could be delivered to target tissues and cells where it exerted a therapeutic effect against PRV. PRV was inoculated via intramuscular injection, and 3D8 scFv was injected intraperitoneally. The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays. Intraperitoneal injection of 5 and 10 μg 3D8 scFv resulted in no detectable toxicity. The survival rate in C57BL/6 mice was 9% after intramuscular injection of 10 LD50 PRV. In contrast, the 3D8 scFv-injected C57BL/6 mice showed survival rates of 57% (5 μg) and 47% (10 μg). The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.

No MeSH data available.


Related in: MedlinePlus

The 3D8 single chain variable fragment (scFv) protein penetrates tissues (liver, muscle, lung and brain) and cells (B16F10 and C6). (A) FITC-labeled 3D8 scFv protein was injected intraperitoneally at 10 μg/20 g mouse into C57BL/6 mice. The organs (liver, muscle, lung and brain) of all mice were collected at 0, 20, 40, 60, 120, and 180 min, and measured at 494 nm via flow cytometry. Fluorescence density (a.u.: arbitrary unit) of three independent trials is shown. (B) Penetration of 3D8 scFv into cultured cells (B16F10 and C6) was evaluated by immunocytochemistry with confocal microscopy. 3D8 scFv was visualized by TRITC (rhodamine)-labeled anti-3D8 polyclonal scFv antibody. Upper panels: PBS treatment, bottom panels: 3D8 scFv treatment.
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f2-molce-38-9-773: The 3D8 single chain variable fragment (scFv) protein penetrates tissues (liver, muscle, lung and brain) and cells (B16F10 and C6). (A) FITC-labeled 3D8 scFv protein was injected intraperitoneally at 10 μg/20 g mouse into C57BL/6 mice. The organs (liver, muscle, lung and brain) of all mice were collected at 0, 20, 40, 60, 120, and 180 min, and measured at 494 nm via flow cytometry. Fluorescence density (a.u.: arbitrary unit) of three independent trials is shown. (B) Penetration of 3D8 scFv into cultured cells (B16F10 and C6) was evaluated by immunocytochemistry with confocal microscopy. 3D8 scFv was visualized by TRITC (rhodamine)-labeled anti-3D8 polyclonal scFv antibody. Upper panels: PBS treatment, bottom panels: 3D8 scFv treatment.

Mentions: The relative quantities of endotoxin-free 3D8 scFv were measured in muscle, liver, lung, and brain tissues after the initial injection to determine if i.p. injected 3D8 scFv penetrates to target tissues. FITC-labeled 3D8 scFv protein was injected i.p. at 10 μg/20 g mouse. Total protein from each of the tissue samples was subsequently prepared at time intervals of 0, 20, 40, 60, 120, and 180 min, and the levels of FITC-3D8 scFv were measured by flow cytometry at an excitation wavelength of 494 nm (Fig. 2A). The 3D8 scFv protein was detected in all tissues 20 min after injection, and the protein level peaked at 60 min post-injection (20–30 fluorescence density). The protein level was maintained at a fluorescence density of approximately 15–20 for up to 120 min after injection. These findings demonstrate that the injected 3D8 scFv protein was transported through the bloodstream to reach target tissues, and that was stably maintained in these cells for at least a short time (Fig. 2A). This experiment was conducted using different mice, and the same results were consistently observed in all cases.


Therapeutic Strategy for the Prevention of Pseudorabies Virus Infection in C57BL/6 Mice by 3D8 scFv with Intrinsic Nuclease Activity.

Lee G, Cho S, Hoang PM, Kim D, Lee Y, Kil EJ, Byun SJ, Lee TK, Kim DH, Kim S, Lee S - Mol. Cells (2015)

The 3D8 single chain variable fragment (scFv) protein penetrates tissues (liver, muscle, lung and brain) and cells (B16F10 and C6). (A) FITC-labeled 3D8 scFv protein was injected intraperitoneally at 10 μg/20 g mouse into C57BL/6 mice. The organs (liver, muscle, lung and brain) of all mice were collected at 0, 20, 40, 60, 120, and 180 min, and measured at 494 nm via flow cytometry. Fluorescence density (a.u.: arbitrary unit) of three independent trials is shown. (B) Penetration of 3D8 scFv into cultured cells (B16F10 and C6) was evaluated by immunocytochemistry with confocal microscopy. 3D8 scFv was visualized by TRITC (rhodamine)-labeled anti-3D8 polyclonal scFv antibody. Upper panels: PBS treatment, bottom panels: 3D8 scFv treatment.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588720&req=5

f2-molce-38-9-773: The 3D8 single chain variable fragment (scFv) protein penetrates tissues (liver, muscle, lung and brain) and cells (B16F10 and C6). (A) FITC-labeled 3D8 scFv protein was injected intraperitoneally at 10 μg/20 g mouse into C57BL/6 mice. The organs (liver, muscle, lung and brain) of all mice were collected at 0, 20, 40, 60, 120, and 180 min, and measured at 494 nm via flow cytometry. Fluorescence density (a.u.: arbitrary unit) of three independent trials is shown. (B) Penetration of 3D8 scFv into cultured cells (B16F10 and C6) was evaluated by immunocytochemistry with confocal microscopy. 3D8 scFv was visualized by TRITC (rhodamine)-labeled anti-3D8 polyclonal scFv antibody. Upper panels: PBS treatment, bottom panels: 3D8 scFv treatment.
Mentions: The relative quantities of endotoxin-free 3D8 scFv were measured in muscle, liver, lung, and brain tissues after the initial injection to determine if i.p. injected 3D8 scFv penetrates to target tissues. FITC-labeled 3D8 scFv protein was injected i.p. at 10 μg/20 g mouse. Total protein from each of the tissue samples was subsequently prepared at time intervals of 0, 20, 40, 60, 120, and 180 min, and the levels of FITC-3D8 scFv were measured by flow cytometry at an excitation wavelength of 494 nm (Fig. 2A). The 3D8 scFv protein was detected in all tissues 20 min after injection, and the protein level peaked at 60 min post-injection (20–30 fluorescence density). The protein level was maintained at a fluorescence density of approximately 15–20 for up to 120 min after injection. These findings demonstrate that the injected 3D8 scFv protein was transported through the bloodstream to reach target tissues, and that was stably maintained in these cells for at least a short time (Fig. 2A). This experiment was conducted using different mice, and the same results were consistently observed in all cases.

Bottom Line: The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays.Intraperitoneal injection of 5 and 10 μg 3D8 scFv resulted in no detectable toxicity.The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Korea.

ABSTRACT
3D8 single chain variable fragment (scFv) is a recombinant monoclonal antibody with nuclease activity that was originally isolated from autoimmune-prone MRL mice. In a previous study, we analyzed the nuclease activity of 3D8 scFv and determined that a HeLa cell line expressing 3D8 scFv conferred resistance to herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). In this study, we demonstrate that 3D8 scFv could be delivered to target tissues and cells where it exerted a therapeutic effect against PRV. PRV was inoculated via intramuscular injection, and 3D8 scFv was injected intraperitoneally. The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays. Intraperitoneal injection of 5 and 10 μg 3D8 scFv resulted in no detectable toxicity. The survival rate in C57BL/6 mice was 9% after intramuscular injection of 10 LD50 PRV. In contrast, the 3D8 scFv-injected C57BL/6 mice showed survival rates of 57% (5 μg) and 47% (10 μg). The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.

No MeSH data available.


Related in: MedlinePlus