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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

Receptor usage by R-VG803 and R-VG809 in SK-OV-3 cells, detected through inhibition of infection by trastuzumab, MAb HD1, or combination thereof.The indicated viruses were preincubated with HD1 (1.5 μg IgG/ml, final concentration) and then allowed to infect SK-OV-3 cells. When indicated, cells were pretreated with trastuzumab (28 μg/ml, final concentration), or control IgGs. Extent of infection was quantified 24 h later by means of flow cytometry, and expressed as percentage relative to cells infected with untreated virus, or untreated cells. Each value represents the average of three independent experiments ± S.D. (B, C) R-VG803 and R-VG809 infection of SK-OV-3 (B) and J-HER2 (C) cells is inhibited by MAb 52S to gH. The indicated virions were preincubated with MAb 52S (ascites fluid 1:25), or mouse IgGs for 1 h, prior to infection of SK-OV-3 or J-HER2 cells (2 or 0.3 PFU/cell, respectively), until harvesting at 24 h after infection. Infection was quantified by flow cytometry and expressed as % of cells infected with untreated virions. Each value represents the average of three independent experiments ± S.D.
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ppat.1004907.g003: Receptor usage by R-VG803 and R-VG809 in SK-OV-3 cells, detected through inhibition of infection by trastuzumab, MAb HD1, or combination thereof.The indicated viruses were preincubated with HD1 (1.5 μg IgG/ml, final concentration) and then allowed to infect SK-OV-3 cells. When indicated, cells were pretreated with trastuzumab (28 μg/ml, final concentration), or control IgGs. Extent of infection was quantified 24 h later by means of flow cytometry, and expressed as percentage relative to cells infected with untreated virus, or untreated cells. Each value represents the average of three independent experiments ± S.D. (B, C) R-VG803 and R-VG809 infection of SK-OV-3 (B) and J-HER2 (C) cells is inhibited by MAb 52S to gH. The indicated virions were preincubated with MAb 52S (ascites fluid 1:25), or mouse IgGs for 1 h, prior to infection of SK-OV-3 or J-HER2 cells (2 or 0.3 PFU/cell, respectively), until harvesting at 24 h after infection. Infection was quantified by flow cytometry and expressed as % of cells infected with untreated virions. Each value represents the average of three independent experiments ± S.D.

Mentions: We analysed the receptor usage in cells that express both sets of receptors, HER2 and nectin1/HVEM, exemplified by SK-OV-3 cells. The question was whether one receptor was preferentially used over the other, or each one was used alternatively. In the latter case, we expected that a block in the access to one of the two sets of receptors—e.g. HER2—should result in low extent of inhibition, whereas the simultaneous block to both sets of receptors should result in strong inhibition. The latter was indeed the case. As controls, we included the two retargeted viruses R-LM113 and R-LM249, and wt R-LM5. R-LM113 is detargeted from natural gD receptors [33,43], even though the AA 6–38 deletion in gD removes only some residues implicated in the nectin1-binding site, in addition to the entire HVEM binding site. The nectin1 binding site is widespread in the molecule, and includes the Ig-folded core and portions located between AA 35–38, 199–201, 214–217, 219–221 [12,13,34]. SK-OV-3 cells were infected with the indicated viruses, in the presence of trastuzumab, MAb HD1 to gD, or both. Fig 3A shows that trastuzumab or HD1 exerted almost no inhibition on R-VG803 when given singly, but practically abolished infection when given together. In contrast, R-LM113 and R-LM249 were inhibited by trastuzumab alone. Thus, R-VG803 can use alternatively HER2 or nectin1/HVEM to infect SK-OV-3 cells. Usage of one or the other portals of entry by R-VG803 depends on the spectrum of receptors displayed by the cells.


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)

Receptor usage by R-VG803 and R-VG809 in SK-OV-3 cells, detected through inhibition of infection by trastuzumab, MAb HD1, or combination thereof.The indicated viruses were preincubated with HD1 (1.5 μg IgG/ml, final concentration) and then allowed to infect SK-OV-3 cells. When indicated, cells were pretreated with trastuzumab (28 μg/ml, final concentration), or control IgGs. Extent of infection was quantified 24 h later by means of flow cytometry, and expressed as percentage relative to cells infected with untreated virus, or untreated cells. Each value represents the average of three independent experiments ± S.D. (B, C) R-VG803 and R-VG809 infection of SK-OV-3 (B) and J-HER2 (C) cells is inhibited by MAb 52S to gH. The indicated virions were preincubated with MAb 52S (ascites fluid 1:25), or mouse IgGs for 1 h, prior to infection of SK-OV-3 or J-HER2 cells (2 or 0.3 PFU/cell, respectively), until harvesting at 24 h after infection. Infection was quantified by flow cytometry and expressed as % of cells infected with untreated virions. Each value represents the average of three independent experiments ± S.D.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440635&req=5

ppat.1004907.g003: Receptor usage by R-VG803 and R-VG809 in SK-OV-3 cells, detected through inhibition of infection by trastuzumab, MAb HD1, or combination thereof.The indicated viruses were preincubated with HD1 (1.5 μg IgG/ml, final concentration) and then allowed to infect SK-OV-3 cells. When indicated, cells were pretreated with trastuzumab (28 μg/ml, final concentration), or control IgGs. Extent of infection was quantified 24 h later by means of flow cytometry, and expressed as percentage relative to cells infected with untreated virus, or untreated cells. Each value represents the average of three independent experiments ± S.D. (B, C) R-VG803 and R-VG809 infection of SK-OV-3 (B) and J-HER2 (C) cells is inhibited by MAb 52S to gH. The indicated virions were preincubated with MAb 52S (ascites fluid 1:25), or mouse IgGs for 1 h, prior to infection of SK-OV-3 or J-HER2 cells (2 or 0.3 PFU/cell, respectively), until harvesting at 24 h after infection. Infection was quantified by flow cytometry and expressed as % of cells infected with untreated virions. Each value represents the average of three independent experiments ± S.D.
Mentions: We analysed the receptor usage in cells that express both sets of receptors, HER2 and nectin1/HVEM, exemplified by SK-OV-3 cells. The question was whether one receptor was preferentially used over the other, or each one was used alternatively. In the latter case, we expected that a block in the access to one of the two sets of receptors—e.g. HER2—should result in low extent of inhibition, whereas the simultaneous block to both sets of receptors should result in strong inhibition. The latter was indeed the case. As controls, we included the two retargeted viruses R-LM113 and R-LM249, and wt R-LM5. R-LM113 is detargeted from natural gD receptors [33,43], even though the AA 6–38 deletion in gD removes only some residues implicated in the nectin1-binding site, in addition to the entire HVEM binding site. The nectin1 binding site is widespread in the molecule, and includes the Ig-folded core and portions located between AA 35–38, 199–201, 214–217, 219–221 [12,13,34]. SK-OV-3 cells were infected with the indicated viruses, in the presence of trastuzumab, MAb HD1 to gD, or both. Fig 3A shows that trastuzumab or HD1 exerted almost no inhibition on R-VG803 when given singly, but practically abolished infection when given together. In contrast, R-LM113 and R-LM249 were inhibited by trastuzumab alone. Thus, R-VG803 can use alternatively HER2 or nectin1/HVEM to infect SK-OV-3 cells. Usage of one or the other portals of entry by R-VG803 depends on the spectrum of receptors displayed by the cells.

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