The long-term goal of the laboratory is to understand the molecular events that lead to herpes simplex virus (HSV) entry into the cell. These studies led us to the design of oncolytic herpes simplex viruses retargeted to receptors specifically expressed on cancer cell, and to investigate some aspects of the innate response of the cell to invading HSV, in particular the role of integrins as sensors of pathogens, in synergy with Toll Like Receptors.
Molecular basis of herpes simplex virus (HSV) entry into the cell and exit out of the cell
Viral glycoproteins and cellular receptors. Four viral glycoproteins, gB, gD, gH and gL are essential for HSV entry and spread, a process that additionally requires one of several different receptors. In the late 1990s this and another laboratory independently discovered that nectin1, an immunoglobulin-like intercellular adhesion molecule, serves as a receptor for HSV. An alternative receptor is HVEM (HVEA), a member of the TNF receptor family. Studies on the roles played by each of the glycoproteins showed that gD, in addition to serve as receptor-binding and major determinant of HSV tropism, also signals receptor-recognition to gH/gL and gB, and thus triggers fusion of the virion envelope with cell membranes. Indeed, gD ectodomain is organized in two topologically and functionally distinct regions. The N-terminus (aa 1-260 of mature gD) carries the receptor binding sites. The C-terminus (aa 260-310) carries the pro-fusion domain (PFD) required for the triggering of fusion but not for receptor binding. These studies led to a model of activation of the fusion glycoproteins mediated by the binding of gD to one of its receptors.
Integrins: additional cellular receptors which interact with HSV gH/gL. Is gD the only receptor-binding glycoprotein in HSV? The heterodimer gH/gL, which – together with gB - is part of the fusion apparatus conserved across the Herpesviridae family, physically interacts with αvβ3-integrin as well as with other integrins.
αvβ3-integrin is a determinant of the pathway of HSV entry. HSV may enter cells through multiple alternative pathways: fusion at plasma membrane, endocytosis into neutral or acidic endosomes, followed by fusion with endosomal membrane, and, perhaps, macropynocytosis. The cell, not the virus, determines the choice as to which pathway of entry is taken by HSV. This laboratory has demonstrated that a determining factor is αvβ3-integrin, which relocalizes the nectin1 receptor, hence HSV, to cholesterol-rich microdomains, from where the HSV in endocytosed in a pathway dependent on dynamin2 and acidic endosomes.
Design and preclinical efficacy of oncolytic HSVs retargeted to cancer-specific receptors
Herpes simplex virus 1 (HSV-1) has numerous properties desired for an oncolytic agent. Virtually all HSVs that have been or are in clinical trials to date as oncolytic agents carry mutations that attenuate the virus, and enable its replication in a subset of tumors, deficient in innate response to the virus. The goal of the laboratory is to generate non-attenuated oncolytic HSVs, by reprogramming the entry apparatus of HSV. Essentially, we genetically engineer HSVs and target them to specific receptors present in cancer cells. The retargeted oncolytic HSVs no longer infect normal cells, yet retain the full-blown killing capacity of wt-HSV. Retargeting of HSV to cancer-specific receptors has been achieved by insertion of a novel ligand in a position of gD that leaves the glycoprotein capable of responding to receptor recognition, and to trigger fusion. The target receptor that we have selected is the HER2/neu receptor, a member of the EGFR family overexpressed in 25-30 % of breast and ovarian tumors. Because HER2/neu lacks a natural ligand, we engineered in gD a single chain antibody (scFv) to HER2/neu. Overall, the insertion almost doubled the size of the gD ectodomain, yet left gD functional. In an alternative construct, we deleted the Ig folded core of gD and replaced it with the single chain antibody to HER2. The HER-2-retargeted HSVs have lost the ability to interact with the natural HSV receptors HVEM and nectin1; hence, they exhibit a high safety profile when injected in mice. Importantly, in collaborative studies with the team Lollini-Nanni-De Giovanni (DIMES, University of Bologna), the HER-2-retargeted HSVs was shown to exert anti-tumor activity in nude mice carrying human ovarian or breast cancers. When administered by the intraperitoneal route, they also cured metastatic diffusion of human breast and ovarian cancer cells in the abdomen of laboratory mice.
HER-2, along with other receptors, is expressed also in glioblastoma, a highly malignant form of brain cancer. In collaboration wih dr Paolo Malatesta (Department of Translational Oncology, Genova, Italy), our laboratory has provided evidence that the HER-2-retargeted HSV exerts anticancer activity against glioblastoma in murine models.