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The Engineering of a Novel Ligand in gH Confers to HSV an Expanded Tropism Independent of gD Activation by Its Receptors.

Gatta V, Petrovic B, Campadelli-Fiume G - PLoS Pathog. (2015)

Bottom Line: With respect to the design of oncolytic-HSVs, previous retargeting strategies were based exclusively on insertion in gD of ligands to cancer-specific receptors.The current findings show that (i) gH accepts a heterologous ligand.Thus, gH represents an additional tool for the design of fully-virulent oncolytic-HSVs retargeted to cancer receptors and detargeted from gD receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy.

ABSTRACT
Herpes simplex virus (HSV) enters cells by means of four essential glycoproteins - gD, gH/gL, gB, activated in a cascade fashion by gD binding to one of its receptors, nectin1 and HVEM. We report that the engineering in gH of a heterologous ligand - a single-chain antibody (scFv) to the cancer-specific HER2 receptor - expands the HSV tropism to cells which express HER2 as the sole receptor. The significance of this finding is twofold. It impacts on our understanding of HSV entry mechanism and the design of retargeted oncolytic-HSVs. Specifically, entry of the recombinant viruses carrying the scFv-HER2-gH chimera into HER2+ cells occurred in the absence of gD receptors, or upon deletion of key residues in gD that constitute the nectin1/HVEM binding sites. In essence, the scFv in gH substituted for gD-mediated activation and rendered a functional gD non-essential for entry via HER2. The activation of the gH moiety in the chimera was carried out by the scFv in cis, not in trans as it occurs with wt-gD. With respect to the design of oncolytic-HSVs, previous retargeting strategies were based exclusively on insertion in gD of ligands to cancer-specific receptors. The current findings show that (i) gH accepts a heterologous ligand. The viruses retargeted via gH (ii) do not require the gD-dependent activation, and (iii) replicate and kill cells at high efficiency. Thus, gH represents an additional tool for the design of fully-virulent oncolytic-HSVs retargeted to cancer receptors and detargeted from gD receptors.

No MeSH data available.


Related in: MedlinePlus

R-VG803 infects cells that express HER2 as the sole receptor (J-HER2 cells) in a HER2-dependent manner, and progeny virus spreads in these cells.J cells express no receptor for wt-HSV. J-HER2, J-Nectin, J-HVEM only express the indicated receptor. (A) The indicated cells were infected with R-VG803 (2 PFU/cell as titrated in SK-OV-3), and monitored for red fluorescence microscopy. (B) J-HER2 cells were infected with R-VG803 (0.01 PFU/cell), overlaid with medium containing the neutralizing MAb 52S (ascites fluid 1:10,000) [45], and monitored daily for red fluorescence. Pictures of a same plaque are shown. (C, D) Trastuzumab inhibits R-VG803 infection of J-HER2 cells. J-HER2 cells were infected with R-VG803 in the presence of trastuzumab (trastuz) (28 μg/ml, final concentration) or control IgGs (28 μg/ml, final concentration). Infection was monitored by fluorescence microscopy (C), or flow cytometry (D). All pictures in panel A were taken with an exposure time of 0.6 sec. The whole pictures showing SK-OV-3- and HFF14-infected cells were adjusted as follows; increase in brightness 25%, increase in contrast 50%. Panel B pictures were adjusted as follows, increase in brightness 20%, increase in contrast 30%.
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ppat.1004907.g002: R-VG803 infects cells that express HER2 as the sole receptor (J-HER2 cells) in a HER2-dependent manner, and progeny virus spreads in these cells.J cells express no receptor for wt-HSV. J-HER2, J-Nectin, J-HVEM only express the indicated receptor. (A) The indicated cells were infected with R-VG803 (2 PFU/cell as titrated in SK-OV-3), and monitored for red fluorescence microscopy. (B) J-HER2 cells were infected with R-VG803 (0.01 PFU/cell), overlaid with medium containing the neutralizing MAb 52S (ascites fluid 1:10,000) [45], and monitored daily for red fluorescence. Pictures of a same plaque are shown. (C, D) Trastuzumab inhibits R-VG803 infection of J-HER2 cells. J-HER2 cells were infected with R-VG803 in the presence of trastuzumab (trastuz) (28 μg/ml, final concentration) or control IgGs (28 μg/ml, final concentration). Infection was monitored by fluorescence microscopy (C), or flow cytometry (D). All pictures in panel A were taken with an exposure time of 0.6 sec. The whole pictures showing SK-OV-3- and HFF14-infected cells were adjusted as follows; increase in brightness 25%, increase in contrast 50%. Panel B pictures were adjusted as follows, increase in brightness 20%, increase in contrast 30%.

Mentions: Initially, we engineered R-VG803. To test whether it can use HER2 as an entry receptor, we made use of J-HER2 cells. The parental J cells express no receptor for gD, hence cannot activate gD, and are not infected by wt-HSV [7]. J-HER2 cells transgenically express HER2 as the sole receptor [43]. As controls, we included J-nectin and J-HVEM cells, which transgenically express nectin1 or HVEM as receptors and are infected by wt-HSV [7], and a panel of human and animal cells, which express the human or animal nectin1/HVEM. The panel included CHO, BHK, keratinocytic HaCaT, human fibroblastic HFF14, epithelial HeLa, the neuronal SK-N-SH cells, and the HER2-positive SK-OV-3 cancer cells. As shown in Fig 2A, R-VG803 infected J-HER2 cells. The infection of J-nectin1, J-HVEM, and of the animal and human cells (Fig 2A) was not surprising, given that R-VG803 encodes a wt-gD. Furthermore, R-VG803 could perform cell-to-cell spread in J-HER2 cells. Cells were infected at 0.01 PFU/cell, overlaid with medium containing MAb 52S (ascites fluid 1:10,000). At day 1 infection involved single cells. In the following day infection involved clusters of cells (Fig 2B).


The Engineering of a Novel Ligand in gH Confers to HSV an Expanded Tropism Independent of gD Activation by Its Receptors.

Gatta V, Petrovic B, Campadelli-Fiume G - PLoS Pathog. (2015)

R-VG803 infects cells that express HER2 as the sole receptor (J-HER2 cells) in a HER2-dependent manner, and progeny virus spreads in these cells.J cells express no receptor for wt-HSV. J-HER2, J-Nectin, J-HVEM only express the indicated receptor. (A) The indicated cells were infected with R-VG803 (2 PFU/cell as titrated in SK-OV-3), and monitored for red fluorescence microscopy. (B) J-HER2 cells were infected with R-VG803 (0.01 PFU/cell), overlaid with medium containing the neutralizing MAb 52S (ascites fluid 1:10,000) [45], and monitored daily for red fluorescence. Pictures of a same plaque are shown. (C, D) Trastuzumab inhibits R-VG803 infection of J-HER2 cells. J-HER2 cells were infected with R-VG803 in the presence of trastuzumab (trastuz) (28 μg/ml, final concentration) or control IgGs (28 μg/ml, final concentration). Infection was monitored by fluorescence microscopy (C), or flow cytometry (D). All pictures in panel A were taken with an exposure time of 0.6 sec. The whole pictures showing SK-OV-3- and HFF14-infected cells were adjusted as follows; increase in brightness 25%, increase in contrast 50%. Panel B pictures were adjusted as follows, increase in brightness 20%, increase in contrast 30%.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440635&req=5

ppat.1004907.g002: R-VG803 infects cells that express HER2 as the sole receptor (J-HER2 cells) in a HER2-dependent manner, and progeny virus spreads in these cells.J cells express no receptor for wt-HSV. J-HER2, J-Nectin, J-HVEM only express the indicated receptor. (A) The indicated cells were infected with R-VG803 (2 PFU/cell as titrated in SK-OV-3), and monitored for red fluorescence microscopy. (B) J-HER2 cells were infected with R-VG803 (0.01 PFU/cell), overlaid with medium containing the neutralizing MAb 52S (ascites fluid 1:10,000) [45], and monitored daily for red fluorescence. Pictures of a same plaque are shown. (C, D) Trastuzumab inhibits R-VG803 infection of J-HER2 cells. J-HER2 cells were infected with R-VG803 in the presence of trastuzumab (trastuz) (28 μg/ml, final concentration) or control IgGs (28 μg/ml, final concentration). Infection was monitored by fluorescence microscopy (C), or flow cytometry (D). All pictures in panel A were taken with an exposure time of 0.6 sec. The whole pictures showing SK-OV-3- and HFF14-infected cells were adjusted as follows; increase in brightness 25%, increase in contrast 50%. Panel B pictures were adjusted as follows, increase in brightness 20%, increase in contrast 30%.
Mentions: Initially, we engineered R-VG803. To test whether it can use HER2 as an entry receptor, we made use of J-HER2 cells. The parental J cells express no receptor for gD, hence cannot activate gD, and are not infected by wt-HSV [7]. J-HER2 cells transgenically express HER2 as the sole receptor [43]. As controls, we included J-nectin and J-HVEM cells, which transgenically express nectin1 or HVEM as receptors and are infected by wt-HSV [7], and a panel of human and animal cells, which express the human or animal nectin1/HVEM. The panel included CHO, BHK, keratinocytic HaCaT, human fibroblastic HFF14, epithelial HeLa, the neuronal SK-N-SH cells, and the HER2-positive SK-OV-3 cancer cells. As shown in Fig 2A, R-VG803 infected J-HER2 cells. The infection of J-nectin1, J-HVEM, and of the animal and human cells (Fig 2A) was not surprising, given that R-VG803 encodes a wt-gD. Furthermore, R-VG803 could perform cell-to-cell spread in J-HER2 cells. Cells were infected at 0.01 PFU/cell, overlaid with medium containing MAb 52S (ascites fluid 1:10,000). At day 1 infection involved single cells. In the following day infection involved clusters of cells (Fig 2B).

Bottom Line: With respect to the design of oncolytic-HSVs, previous retargeting strategies were based exclusively on insertion in gD of ligands to cancer-specific receptors.The current findings show that (i) gH accepts a heterologous ligand.Thus, gH represents an additional tool for the design of fully-virulent oncolytic-HSVs retargeted to cancer receptors and detargeted from gD receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy.

ABSTRACT
Herpes simplex virus (HSV) enters cells by means of four essential glycoproteins - gD, gH/gL, gB, activated in a cascade fashion by gD binding to one of its receptors, nectin1 and HVEM. We report that the engineering in gH of a heterologous ligand - a single-chain antibody (scFv) to the cancer-specific HER2 receptor - expands the HSV tropism to cells which express HER2 as the sole receptor. The significance of this finding is twofold. It impacts on our understanding of HSV entry mechanism and the design of retargeted oncolytic-HSVs. Specifically, entry of the recombinant viruses carrying the scFv-HER2-gH chimera into HER2+ cells occurred in the absence of gD receptors, or upon deletion of key residues in gD that constitute the nectin1/HVEM binding sites. In essence, the scFv in gH substituted for gD-mediated activation and rendered a functional gD non-essential for entry via HER2. The activation of the gH moiety in the chimera was carried out by the scFv in cis, not in trans as it occurs with wt-gD. With respect to the design of oncolytic-HSVs, previous retargeting strategies were based exclusively on insertion in gD of ligands to cancer-specific receptors. The current findings show that (i) gH accepts a heterologous ligand. The viruses retargeted via gH (ii) do not require the gD-dependent activation, and (iii) replicate and kill cells at high efficiency. Thus, gH represents an additional tool for the design of fully-virulent oncolytic-HSVs retargeted to cancer receptors and detargeted from gD receptors.

No MeSH data available.


Related in: MedlinePlus