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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

3D8 scFv protein exerts antiviral therapeutic effects. (A) Absence of viral transcript expression (pseudorabies virus [PRV] gpD) in mice treated with the 3D8 scFv, as shown by reverse transcription-polymerase chain reaction analysis results. RNA samples from various organs (liver, muscle, lung, and brain) of different mouse groups were analyzed for PRV gpD expression. M: Mock-treated group; D challenged with 10 LD50 PRV followed by PBS treatment; E challenged with 10 LD50 PRV followed by 5 μg 3D8 scFv treatment; F challenged with 10LD50 PRV followed by 10 μg 3D8 scFv treatment. Both surviving (O) and dead (X) mice were analyzed for PRV gpD expression in each group. Data from surviving mice in groups E and F are provided in (a) and data from the dead mice in groups E and F are shown in (b). (B) Absence of viral protein expression (PRV gpD) in mice treated with 3D8 scFv as detected via immunohistochemistry. Brain cerebellum tissues of M, D, and E mouse groups were stained with anti-PRV monoclonal antibody visualized with biotinylated anti-mouse secondary antibody followed by DAB staining. Magnification: 100× for upper panel, 400× for bottom panel. Presence of PRV gpD protein (shown as brown staining) is detectable only in the Purkinje layer cells of the group D mice.
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f4-molce-38-9-773: 3D8 scFv protein exerts antiviral therapeutic effects. (A) Absence of viral transcript expression (pseudorabies virus [PRV] gpD) in mice treated with the 3D8 scFv, as shown by reverse transcription-polymerase chain reaction analysis results. RNA samples from various organs (liver, muscle, lung, and brain) of different mouse groups were analyzed for PRV gpD expression. M: Mock-treated group; D challenged with 10 LD50 PRV followed by PBS treatment; E challenged with 10 LD50 PRV followed by 5 μg 3D8 scFv treatment; F challenged with 10LD50 PRV followed by 10 μg 3D8 scFv treatment. Both surviving (O) and dead (X) mice were analyzed for PRV gpD expression in each group. Data from surviving mice in groups E and F are provided in (a) and data from the dead mice in groups E and F are shown in (b). (B) Absence of viral protein expression (PRV gpD) in mice treated with 3D8 scFv as detected via immunohistochemistry. Brain cerebellum tissues of M, D, and E mouse groups were stained with anti-PRV monoclonal antibody visualized with biotinylated anti-mouse secondary antibody followed by DAB staining. Magnification: 100× for upper panel, 400× for bottom panel. Presence of PRV gpD protein (shown as brown staining) is detectable only in the Purkinje layer cells of the group D mice.

Mentions: Viral replication and gene and protein expression levels were measured in each group using RT-PCR and immunohistochemistry to confirm that the observed difference in survival rate after treatment with 3D8 scFv was the result of reduced viral pathogenicity. The RT-PCR analyses conducted on RNA isolated from the groups described in Fig. 4 showed that the viral transcript glycoprotein D (gpD) was readily detected in the RNA samples isolated from dead mice in the groups treated with 3D8 scFv (groups E and F). However, no gpD transcripts were detected in samples isolated from survivors of the same groups (Fig. 4A). Similarly, the gpD protein, as measured by immunohistochemistry, was detectable only in the mice that died from viral infection, whereas the protein was not detected in any of the surviving mice, regardless of their 3D8 scFv treatment status (Fig. 4B).


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)

3D8 scFv protein exerts antiviral therapeutic effects. (A) Absence of viral transcript expression (pseudorabies virus [PRV] gpD) in mice treated with the 3D8 scFv, as shown by reverse transcription-polymerase chain reaction analysis results. RNA samples from various organs (liver, muscle, lung, and brain) of different mouse groups were analyzed for PRV gpD expression. M: Mock-treated group; D challenged with 10 LD50 PRV followed by PBS treatment; E challenged with 10 LD50 PRV followed by 5 μg 3D8 scFv treatment; F challenged with 10LD50 PRV followed by 10 μg 3D8 scFv treatment. Both surviving (O) and dead (X) mice were analyzed for PRV gpD expression in each group. Data from surviving mice in groups E and F are provided in (a) and data from the dead mice in groups E and F are shown in (b). (B) Absence of viral protein expression (PRV gpD) in mice treated with 3D8 scFv as detected via immunohistochemistry. Brain cerebellum tissues of M, D, and E mouse groups were stained with anti-PRV monoclonal antibody visualized with biotinylated anti-mouse secondary antibody followed by DAB staining. Magnification: 100× for upper panel, 400× for bottom panel. Presence of PRV gpD protein (shown as brown staining) is detectable only in the Purkinje layer cells of the group D mice.
© Copyright Policy
Related In: Results  -  Collection

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

f4-molce-38-9-773: 3D8 scFv protein exerts antiviral therapeutic effects. (A) Absence of viral transcript expression (pseudorabies virus [PRV] gpD) in mice treated with the 3D8 scFv, as shown by reverse transcription-polymerase chain reaction analysis results. RNA samples from various organs (liver, muscle, lung, and brain) of different mouse groups were analyzed for PRV gpD expression. M: Mock-treated group; D challenged with 10 LD50 PRV followed by PBS treatment; E challenged with 10 LD50 PRV followed by 5 μg 3D8 scFv treatment; F challenged with 10LD50 PRV followed by 10 μg 3D8 scFv treatment. Both surviving (O) and dead (X) mice were analyzed for PRV gpD expression in each group. Data from surviving mice in groups E and F are provided in (a) and data from the dead mice in groups E and F are shown in (b). (B) Absence of viral protein expression (PRV gpD) in mice treated with 3D8 scFv as detected via immunohistochemistry. Brain cerebellum tissues of M, D, and E mouse groups were stained with anti-PRV monoclonal antibody visualized with biotinylated anti-mouse secondary antibody followed by DAB staining. Magnification: 100× for upper panel, 400× for bottom panel. Presence of PRV gpD protein (shown as brown staining) is detectable only in the Purkinje layer cells of the group D mice.
Mentions: Viral replication and gene and protein expression levels were measured in each group using RT-PCR and immunohistochemistry to confirm that the observed difference in survival rate after treatment with 3D8 scFv was the result of reduced viral pathogenicity. The RT-PCR analyses conducted on RNA isolated from the groups described in Fig. 4 showed that the viral transcript glycoprotein D (gpD) was readily detected in the RNA samples isolated from dead mice in the groups treated with 3D8 scFv (groups E and F). However, no gpD transcripts were detected in samples isolated from survivors of the same groups (Fig. 4A). Similarly, the gpD protein, as measured by immunohistochemistry, was detectable only in the mice that died from viral infection, whereas the protein was not detected in any of the surviving mice, regardless of their 3D8 scFv treatment status (Fig. 4B).

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