A number of sentinels sense incoming herpes simplex virus (HSV) virions and initiate an immediate innate response. The first line of defense at the cell surface is TLR2 (Toll-like receptor 2), whose signature signaling activity leads to activation of the key transcription factor NF-κB. We report that the HSV pathogen-associated molecular patterns for TLR2 are the virion glycoproteins gH/gL and gB, which constitute the conserved fusion core apparatus across the members of the Herpesviridae family. Specifically, virions devoid singly of one of essential fusion glycoproteins (gD, gB, or gH null), able to attach to cells but defective in fusion/entry, were sufficient to elicit the first wave of NF-κB response to HSV. The most effective were the gD-null virions, positive for gH/gL and gB. A soluble form of gB, truncated upstream of the transmembrane sequence (gB 730t-st ), was produced in human cells and purified by means of a Strep tag. gH/gL and gB were each able to physically interact with TLR2 in coimmunoprecipitation assays, one independently of the other, yet gH t-st /gL, but not gB 730t-st , elicited an NF-κB response. Thus, whereas both HSV gH/gL and gB are ligands to TLR2, only gH/gL is sufficient to initiate a signaling cascade which leads to NF-κB activation.

Pathogens are sensed by Toll-like receptors (TLRs) and a growing number of non-TLR receptors. Integrins constitute a family of signaling receptors exploited by viruses and bacteria to access cells. By gain- and loss-of-function approaches we found that αvβ3-integrin is a sensor of and plays a crucial role in the innate defense against herpes simplex virus (HSV). αvβ3-integrin signaled through two pathways. One concurred with TLR2, affected activation/induction of interferons type 1 (IFNs-1), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), and a polarized set of cytokines and receptors. The virion glycoproteins gH/gL sufficed to induce IFN1 and NF-κB via this pathway. The other pathway was TLR2-independent, involved sarcoma (SRC)-spleen tyrosine kinase (SYK)-Caspase recruitment domain-containing protein 9 (CARD9)-TRIF (TIR-domain-containing adapter-inducing interferon-β), and affected interferon regulatory factor 3 and 7 (IRF3-IRF7). The importance of αvβ3-integrin-mediated defense is reflected in the observation that HSV evolved the immediate-early infected cellular protein 0 (ICP0) protein to counteract it. We propose that αvβ3-integrin is considered a class of non-TLR pattern recognition receptors, a role likely exerted toward viruses and bacteria that interact with integrins and mount an innate response.

Oncolytic herpes simplex viruses (oHSVs) showed efficacy in clinical trials and practice. Most of them gain cancer-specificity from deletions/mutations in genes that counteract the host response, and grow selectively in cancer cells defective in anti-viral response. Because of the deletions/mutations, they are frequently attenuated or over-attenuated. We developed next-generation oHSVs, which carry no deletion/mutation, gain cancer-specificity from specific retargeting to tumor cell receptors—e.g. HER2 (human epidermal growth factor receptor 2)—hence are fully-virulent in the targeted cancer cells. The type of immunotherapy they elicit was not predictable, since non-attenuated HSVs induce and then dampen the innate response, whereas deleted/attenuated viruses fail to contrast it, and since the retargeted oHSVs replicate efficiently in tumor cells, but spare other cells in the tumor. We report on the first efficacy study of HER2-retargeted, fully-virulent oHSVs in immunocompetent mice. Their safety profile was very high. Both the unarmed R-LM113 and the IL-12-armed R-115 inhibited the growth of the primary HER2-Lewis lung carcinoma-1 (HER2-LLC1) tumor, R-115 being constantly more efficacious. All the mice that did not die because of the primary treated tumors, were protected from the growth of contralateral untreated tumors. The long-term survivors were protected from a second contralateral tumor, providing additional evidence for an abscopal immunotherapeutic effect. Analysis of the local response highlighted that particularly R-115 unleashed the immunosuppressive tumor microenvironment, i.e. induced immunomodulatory cytokines, including IFNγ, T-bet which promoted Th1 polarization. Some of the tumor infiltrating cells, e.g. CD4+, CD335+ cells were increased in the tumors of all responders mice, irrespective of which virus was employed, whereas CD8+, Foxp3+, CD141+ were increased and CD11b+ cells were decreased preferentially in R-115-treated mice. The durable response included a breakage of tolerance towards both HER2 and the wt tumor cells, and underscored a systemic immunotherapeutic vaccine response.

Fully retargeted oncolytic herpes simplex viruses (o-HSVs) gain cancer-specificity from redirection of tropism to cancer-specific receptors, and are non-attenuated. To overcome the hurdles of systemic delivery, and enable oncolytic viruses (o-viruses) to reach metastatic sites, carrier cells are being exploited. Mesenchymal stromal cells (MSCs) were never tested as carriers of retargeted o-viruses, given their scarse-null expression of the cancer-specific receptors. We report that MSCs from different sources can be forcedly infected with a HER2-retargeted oncolytic HSV. Progeny virus spread from MSCs to cancer cells in vitro and in vivo. We evaluated the organ distribution and therapeutic efficacy in two murine models of metastatic cancers, following a single i.v. injection of infected MSCs. As expected, the highest concentration of carrier-cells and of viral genomes was in the lungs. Viral genomes persisted throughout the body for at least two days. The growth of ovarian cancer lung metastases in nude mice was strongly inhibited, and the majority of treated mice appeared metastasis-free. The treatment significantly inhibited also breast cancer metastases to the brain in NSG mice, and reduced by more than one-half the metastatic burden in the brain.

␣v␤3 integrin represents a novel sensing system which detects herpes simplex virus (HSV) and bacterial constituents. In cooperation with Toll-like receptor 2 (TLR2), it elicits an innate response that leads to activation of type I interferon (IFN), NF-B, and a specific set of cytokines. We report that this defensive branch is functional in cells which represent experimental models of epithelial, including keratinocytic, and neuronal cells. These are the major targets of HSV in vivo. HSV entered the three cell lines via distinct routes. Hence, the defensive response was independent of the route of virus entry. Soluble gH/gL sufficed to elicit type I IFN and NF-B activation and represents the viral pathogen-associated molecular pattern (PAMP) of this defense system. P attern recognition receptors (PRRs) are responsible for sensing pathogens and for initiating signaling cascades which culminate in the innate response, whose hallmarks are the production of interferons (IFNs) and proinflammatory cytokines and the activation of the transcription factor NF-B (1). Based on the nature of the pathogen-associated molecular pattern (PAMP) and the properties of the respective PRR, the innate response to herpesviruses can be divided into three main branches (for an indepth review, see reference 2). Thus, the Toll-like receptors (TLRs) localized at the plasma membranes, e.g., TLR2, recognize proteic or lipidic PAMPs. The endosomal TLR3 and TLR7, as well as the cytoplasmic RIG-I (RNA helicase retinoic acid-inducible gene I), sense single-or double-stranded RNAs. The endosomal TLR9 and a number of cytosolic molecules, such as DAI (DNAdependent activator of interferon-regulatory factor), IFI16 (gamma interferon-inducible protein 16), and AIM2 (absent in melanoma 2), etc., sense double-stranded DNA (2, 3).Until recently, the prevalent paradigm has been that the IFN production occurs in response to activation of the cytosolic sensors, whereas the TLRs located at the plasma membrane elicit a predominantly inflammatory response (4-6). In particular, until recently, TLR2 was known to sense bacteria and was thought not to be involved in recognition of viruses. When a role for TLR2 against herpes simplex virus (HSV) and other herpesviruses was documented, TLR2 was found to be polarized toward an inflammatory response (7,8). Kurt-Jones and coworkers (9) found that encephalitis caused by HSV was less severe in mice lacking TLR2, thus providing evidence for a role of TLR2 in the response to HSV and highlighting an inflammatory effect (10). In contrast with this view, Barbalat et al. (11) demonstrated that TLR2 was capable of inducing a type I IFN in response to viral ligands in inflammatory monocytes; the PAMPs remained elusive. Key to understanding the significance of the multiple, apparently redundant, innate defense branches against herpesviruses is the fact that they have been often demonstrated in highly specific cell types. For example, the TLR9 response was observed in plasmacytoid cells, the TLR9 and TLR2 responses were observed i...