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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-VG809 infects cells that express HER2, fails to infect cells via gD receptors, and progeny virus spreads in J-HER2 cells.(A) The indicated cells were infected with R-VG809 (20 PFU/cell as titrated in SK-OV-3), and monitored for red fluorescence microscopy at 24 h after infection. (B) J-HER2 cells were infected with R-VG809 (0.01 PFU/cell), overlaid with medium containing the neutralizing MAb 52S to gH (ascites fluid, 1:10,000), and monitored daily for red fluorescence. Daily pictures of a same plaque are shown. (C, D) Trastuzumab inhibits R-VG809 infection of J-HER2 cells. J-HER2 cells were infected with R-VG809 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). Panel B pictures were adjusted as follows, increase in brightness 20%, increase in contrast 30%.
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ppat.1004907.g004: R-VG809 infects cells that express HER2, fails to infect cells via gD receptors, and progeny virus spreads in J-HER2 cells.(A) The indicated cells were infected with R-VG809 (20 PFU/cell as titrated in SK-OV-3), and monitored for red fluorescence microscopy at 24 h after infection. (B) J-HER2 cells were infected with R-VG809 (0.01 PFU/cell), overlaid with medium containing the neutralizing MAb 52S to gH (ascites fluid, 1:10,000), and monitored daily for red fluorescence. Daily pictures of a same plaque are shown. (C, D) Trastuzumab inhibits R-VG809 infection of J-HER2 cells. J-HER2 cells were infected with R-VG809 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). Panel B pictures were adjusted as follows, increase in brightness 20%, increase in contrast 30%.

Mentions: Inasmuch as R-VG803 infects J-HER2 cells independently of gD receptors and of neutralizing MAb to gD, we reasoned that it might be possible to engineer a recombinant carrying the scFv-HER2 in gH and the deletion of receptors’ binding sites from gD. We deleted the AA 6–38 region. R-VG809 failed to infect not only J-HVEM cells, but also J-nectin cells, and did not infect or infected very little the panel of animal and human cell lines employed above. It infected efficiently J-HER2, CHO-HER2 and SK-OV-3 cells (Fig 4A). In summary, R-VG809 exhibited a redirected tropism, strikingly different from that of R-VG803 (compare Fig 4A with Fig 2A). R-VG809 was also capable of cell-to-cell spread in J-HER2 cells (Fig 4B). Further validation that R-VG809 uses HER2 as portal of entry was provided by inhibition with trastuzumab (Fig 4C and 4D).


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-VG809 infects cells that express HER2, fails to infect cells via gD receptors, and progeny virus spreads in J-HER2 cells.(A) The indicated cells were infected with R-VG809 (20 PFU/cell as titrated in SK-OV-3), and monitored for red fluorescence microscopy at 24 h after infection. (B) J-HER2 cells were infected with R-VG809 (0.01 PFU/cell), overlaid with medium containing the neutralizing MAb 52S to gH (ascites fluid, 1:10,000), and monitored daily for red fluorescence. Daily pictures of a same plaque are shown. (C, D) Trastuzumab inhibits R-VG809 infection of J-HER2 cells. J-HER2 cells were infected with R-VG809 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). Panel B pictures were adjusted as follows, increase in brightness 20%, increase in contrast 30%.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440635&req=5

ppat.1004907.g004: R-VG809 infects cells that express HER2, fails to infect cells via gD receptors, and progeny virus spreads in J-HER2 cells.(A) The indicated cells were infected with R-VG809 (20 PFU/cell as titrated in SK-OV-3), and monitored for red fluorescence microscopy at 24 h after infection. (B) J-HER2 cells were infected with R-VG809 (0.01 PFU/cell), overlaid with medium containing the neutralizing MAb 52S to gH (ascites fluid, 1:10,000), and monitored daily for red fluorescence. Daily pictures of a same plaque are shown. (C, D) Trastuzumab inhibits R-VG809 infection of J-HER2 cells. J-HER2 cells were infected with R-VG809 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). Panel B pictures were adjusted as follows, increase in brightness 20%, increase in contrast 30%.
Mentions: Inasmuch as R-VG803 infects J-HER2 cells independently of gD receptors and of neutralizing MAb to gD, we reasoned that it might be possible to engineer a recombinant carrying the scFv-HER2 in gH and the deletion of receptors’ binding sites from gD. We deleted the AA 6–38 region. R-VG809 failed to infect not only J-HVEM cells, but also J-nectin cells, and did not infect or infected very little the panel of animal and human cell lines employed above. It infected efficiently J-HER2, CHO-HER2 and SK-OV-3 cells (Fig 4A). In summary, R-VG809 exhibited a redirected tropism, strikingly different from that of R-VG803 (compare Fig 4A with Fig 2A). R-VG809 was also capable of cell-to-cell spread in J-HER2 cells (Fig 4B). Further validation that R-VG809 uses HER2 as portal of entry was provided by inhibition with trastuzumab (Fig 4C and 4D).

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