Human herpesvirus 6: An emerging pathogen.

Infections with human herpesvirus 6 (HHV-6), a beta-herpesvirus of which two variant groups (A and B) are recognized, is very common, approaching 100% in seroprevalence. Primary infection with HHV-6B causes roseola infantum or exanthem subitum, a common childhood disease that resolves spontaneously. After primary infection, the virus replicates in the salivary glands and is shed in saliva, the recognized route of transmission for variant B strains; it remains latent in lymphocytes and monocytes and persists at low levels in cells and tissues. Not usually associated with disease in the immunocompetent, HHV-6 infection is a major cause of opportunistic viral infections in the immunosuppressed, typically AIDS patients and transplant recipients, in whom HHV-6 infection/reactivation may culminate in rejection of transplanted organs and death. Other opportunistic viruses, human cytomegalovirus and HHV-7, also infect or reactivate in persons at risk. Another disease whose pathogenesis may be correlated with HHV-6 is multiple sclerosis. Data in favor of and against the correlation are discussed

Critical Residues in the CC′ Ridge of the Human Nectin1 Receptor V Domain Enable Herpes Simplex Virus Entry into the Cell and Act Synergistically with the Downstream Region

AbstractThe site on nectin1 receptor required for herpes simplex virus (HSV) entry into the cell was previously mapped to the 64–94 region, encompassing the predicted CC′C" region of the immunoglobulin V domain. Within it lies a minimal HSV entry site (residues 77–94). Here we transferred the 65–76 region (C strand and CC′ loop) and portions, or single amino acids, thereof to nectin2, a homolog nonfunctional for wt HSV-1 entry. Replacement of the seven- or of three-amino-acid-long stretches from nectin1 to nectin2 (amino acids 69–75, 69–71, or 72–75) transferred wt HSV-1 and BHV-1 entry activity and enhanced HSV-2, PrV, and HSV-HSV(U21) entry to levels observed with nectin1. Thus, the CC′ ridge is sufficient to mediate wt HSV entry at a reduced level and responsible for the wide virus range of the receptor. Altogether the HSV entry site appears to be composed of contiguous synergistic regions, 64–76 and 77–94, each independently capable of mediating virus entry at reduced efficiency

The simultaneous insertion of two ligands in gD for the cultivation of oncolytic HSVs in non-cancer cells and the retargeting to cancer receptors

Insertion of a single chain antibody (scFv) to HER2 (human epidermal growth factor receptor 2) in gD, gH, or gB gives rise to herpes simplex viruses (HSVs) specifically retargeted to HER2-positive cancer cells, hence in highly specific non-attenuated oncolytic agents. Clinical grade virus production can not rely on cancer cells. Recently, we developed a double retargeting strategy whereby gH carries the GCN4 peptide for retargeting to the non-cancer producer Vero-GCN4R cell line, and gD carries the scFv to HER2 for cancer retargeting. Here, we engineered double retargeted recombinants, which carry both the GCN4 peptide and the scFv to HER2 in gD. Novel, more advantageous detargeting strategies were devised, so as to optimize the cultivation of the double-retargeted recombinants. Nectin1 detargeting was achieved by deletion of aa 35-39, 214-223, or 219-223, and replacement of the deleted sequences with one of the two ligands. The latter two deletions were not attempted before. All recombinants exhibited the double retargeting to HER2 and to the Vero-GCN4R cells, as well as detargeting from the natural receptors HVEM and nectin1. Of note, some recombinants grew to higher yields than others. The best performing recombinants carried a gD deletion as small as 5 amino acids, and grew to titers similar to those exhibited by the singly retargeted R-LM113, and by the non-retargeted R-LM5. This study shows that double retargeting through insertion of two ligands in gD is feasible and, when combined with appropriate detargeting modifications, can result in recombinants highly effectivein vitroandin vivo.IMPORTANCEThere is increasing interest in oncolytic viruses, following FDA and EMA approval of the oncolytic HSV OncovexGM-CSF, and, mainly, because they greatly boost the immune response to the tumor and can be combined with immunotherapeutic agents, particularly immune checkpoint inhibitors. A strategy to gain high cancer specificity and avoid virus attenuation is to retarget the virus tropism to cancer-specific receptors of choice. However, cultivation of retargeted oncolytics in cells expressing the cancer receptor may not be approvable by regulatory agencies. We devised a strategy for their cultivation in non-cancer cells. Here, we describe a double retargeting strategy, based on the simultaneous insertion of two ligands in gD, one for retargeting to a producer, universal Vero cell derivative, one for retargeting to the HER2 cancer receptor. These insertions were combined with novel, minimally-disadvantageous detargeting modifications. The current and accompanying studies teach how to best achieve the clinical-grade cultivation of retargeted oncolytics

Prominent role of the Ig-like V domain in trans-interactions of nectins. Nectin3 and nectin 4 bind to the predicted C-C'-C"-D beta-strands of the nectin1 V domain.

Nectins form a family of integral molecules that belong to the immunoglobulin superfamily. Their ectodomain is made of three Ig-like domains (V, C, C). This family comprises at least five members, namely nectin1, -2, -3, -4, and poliovirus receptor (PVR), that are involved in different physiological and pathological processes. (i) Nectins are adhesion molecules localized at adherens junctions in epithelial cells. (ii) Some nectins act as poliovirus or alpha-herpesvirus receptors (nectin1). (iii) Nectin1 mutations are involved in orofacial developmental abnormalities in humans. Adhesion properties of nectins are mediated by Ca(2+)-independent homophilic and heterophilic processes through ectodomain trans-interactions. We have described a nectin trans-hetero-interaction network: nectin3 binds to nectin1, nectin2, and PVR; nectin1 also binds to nectin4. In the present study we compared the affinities of the different trans-interactions mediated by nectin1. We found that the K(D) of nectin1/nectin3 and nectin1/nectin4 interactions is 1 and 100 nm, respectively, whereas the K(D) of the nectin1-mediated homophilic interaction is 1 microm. We show that nectin1/nectin3 and nectin1/nectin4 trans-hetero-interactions were mediated through trans V to V domain interactions, whereas C domains contributed to increase the affinity of the interaction. Nectin3 and nectin4 share a common binding region in the nectin1 V domain: (i) nectin3 strongly competed with nectin4 binding, (ii) nectin3 and nectin4 binding to nectin1 was reduced by a number of monoclonal antibodies directed against the nectin1 V domain, and (iii) the glycoprotein D of herpes simplex virus-1 that binds to the V domain of nectin1 reduced nectin3 and nectin4 binding. Finally, using chimeric nectin1/PVR receptors where PVR V domain beta-strands were substituted with the corresponding regions of nectin1, the nectin3 and nectin4 minimal binding region on nectin1 V domain was mapped to the C-C'-C"-D beta-strands

Persistence of Human Herpesvirus 7 in Normal Tissues Detected by Expression of a Structural Antigen

Human herpesvirus 7 (HHV-7) infection in histologically normal human tissues was investigated by immunohistochemical detection of the 85-kDa tegument phosphoprotein (pp85) encoded by the U14 gene. So far, two cell types were recognized as sites of HHV-7 infection in vivo: CD4+ T lymphocytes, believed to be the site of latent infection, and epithelial cells of salivary glands, the site of productive infection and viral shedding. Unexpectedly, cells expressing the HHV-7 structural antigen were detectable in lungs, skin, and mammary glands. Morphologically and phenotypically, they were distinct from lymphocytes. Liver, kidney, and tonsils were positive, although the number of HHV-7-positive cells was low. Large intestine, spleen, and brain were negative. Different from the current notion of the state of HHV-7 in humans, the results show that a variety of tissues harbor cells at a late stage of infection and suggest that HHV-7 causes a persistent rather than a true latent infectio

Immunotherapeutic efficacy of retargeted ohsvs designed for propagation in an ad hoc cell line

Our laboratory has pursued the generation of cancer‐specific oncolytic herpes simplex viruses (oHSVs) which ensure high efficacy while maintaining a high safety profile. Their blueprint included retargeting to a Tumor‐Associated Antigen, e.g., HER2, coupled to detargeting from natural receptors to avoid off‐target and off‐tumor infections and preservation of the full complement of unmodified viral genes. These oHSVs are “fully virulent in their target cancer cells”. The 3rd generation retargeted oHSVs carry two distinct retargeting moieties, which enable infection of a producer cell line and of the target cancer cells, respectively. They can be propagated in an ad hoc Vero cell derivative at about tenfold higher yields than 1st generation recombinants, and more effectively replicate in human cancer cell lines. The R‐335 and R‐337 prototypes were armed with murine IL‐12. Intratumorally‐administered R‐337 conferred almost complete protection from LLC‐ 1‐HER2 primary tumors, unleashed the tumor microenvironment immunosuppression, synergized with the checkpoint blockade and conferred long‐term vaccination against distant challenge tumors. In summary, the problem intrinsic to the propagation of retargeted oHSVs—which strictly require cells positive for targeted receptors—was solved in 3rd generation viruses. They are effective as immunotherapeutic agents against primary tumors and as antigen‐agnostic vaccines

Genotype of Immunologically Hot or Cold Tumors Determines the Antitumor Immune Response and Efficacy by Fully Virulent Retargeted oHSV

We report on the efficacy of the non-attenuated HER2-retargeted oHSV named R-337 against the immunologically hot CT26-HER2 tumor, and an insight into the basis of the immune protection. Preliminarily, we conducted an RNA immune profiling and immune cell content characterization of CT26-HER2 tumor in comparison to the immunologically cold LLC1-HER2 tumor. CT26-HER2 tumor was implanted into HER2-transgenic BALB/c mice. Hallmarks of R-337 effects were the protection from primary tumor, long-term adaptive vaccination directed to both HER2 and CT26-wt cell neoantigens. The latter effect differentiated R-337 from OncoVEXGM-CSF. As to the basis of the immune protection, R-337 orchestrated several changes to the tumor immune profile, which cumulatively reversed the immunosuppression typical of this tumor (graphical abstract). Thus, Ido1 (inhibitor of T cell anticancer immunity) levels and T regulatory cell infiltration were decreased; Cd40 and Cd27 co-immunostimulatory markers were increased; the IFNγ cascade was activated. Of note was the dampening of IFN-I response, which we attribute to the fact that R-337 is fully equipped with genes that contrast the host innate response. The IFN-I shut-down likely favored viral replication and the expression of the mIL-12 payload, which, in turn, boosted the antitumor response. The results call for a characterization of tumor immune markers to employ oncolytic herpesviruses more precisely

Efficacy of Systemically Administered Retargeted Oncolytic Herpes Simplex Viruses—Clearance and Biodistribution in Naïve and HSV-Preimmune Mice

We investigated the anticancer efficacy, blood clearance, and tissue biodistribution of systemically administered retargeted oncolytic herpes simplex viruses (ReHVs) in HSV-naive and HSV-preimmunized (HSV-IMM) mice. Efficacy was tested against lung tumors formed upon intravenous administration of cancer cells, a model of metastatic disease, and against subcutaneous distant tumors. In naive mice, HER2- and hPSMA-retargeted viruses, both armed with mIL-12, were highly effective, even when administered to mice with well-developed tumors. Efficacy was higher for combination regimens with immune checkpoint inhibitors. A significant amount of infectious virus persisted in the blood for at least 1 h. Viral genomes, or fragments thereof, persisted in the blood and tissues for days. Remarkably, the only sites of viral replication were the lungs of tumor-positive mice and the subcutaneous tumors. No replication was detected in other tissues, strengthening the evidence of the high cancer specificity of ReHVs, a property that renders ReHVs suitable for systemic administration. In HSV-IMM mice, ReHVs administered at late times failed to exert anticancer efficacy, and the circulating virus was rapidly inactivated. Serum stability and in vivo whole blood stability assays highlighted neutralizing antibodies as the main factor in virus inactivation. Efforts to deplete mice of the neutralizing antibodies are ongoing

oHSV Genome Editing by Means of galK Recombineering

open8noThis work was supported by European Research Council (ERC) Advanced Grant number 340060, VII framework program to G. C.-F., by RFO (University of Bologna) to L.M. and T.G, and by Fondi Pallotti to T.G.Since the cloning of the herpes simplex virus (HSV) genome as BAC (bacterial artificial chromosome), the genetic engineering of the viral genome has become readily feasible. The advantage is that the modification of the animal virus genome is carried out in bacteria, with no replication or production of viral progeny, and is separated from the reconstitution or regeneration of the recombinant virus in mammalian cells. This allows an easy engineering of essential genes, as well. Many technologies have been developed for herpesvirus BAC engineering. In our hands the most powerful is galK recombineering that exploits a single marker (galK) for positive and negative selection and PCR amplicons for seamless modification in the desired genome locus. Here we describe the engineering of the HSV recombinant BAC 115 by the insertion of a heterologous cassette for the expression of murine interleukin 12 (mIL12) in the intergenic sequence between US1 and US2 ORFs.embargoed_20201017Laura Menotti, Valerio Leoni, Valentina Gatta, Biljana Petrovic, Andrea Vannini, Simona Pepe, Tatiana Gianni, Gabriella Campadelli-FiumeLaura Menotti, Valerio Leoni, Valentina Gatta, Biljana Petrovic, Andrea Vannini, Simona Pepe, Tatiana Gianni, Gabriella Campadelli-Fium

The structure of Herpesvirus Fusion Glycoprotein B-Bilayer Complex reveals the protein-membrane and lateral protein-protein interaction

Glycoprotein B (gB) is a key component of the complex herpesvirus fusion machinery. We studied membrane interaction of two gB ectodomain forms and present an electron cryotomography structure of the gB-bilayer complex. The two forms differed in presence or absence of the membrane proximal region (MPR) but showed an overall similar trimeric shape. The presence of the MPR impeded interaction with liposomes. In contrast, the MPR-lacking form interacted efficiently with liposomes. Lateral interaction resulted in coat formation on the membranes. The structure revealed that interaction of gB with membranes was mediated by the fusion loops and limited to the outer membrane leaflet. The observed intrinsic propensity of gB to cluster on membranes indicates an additional role of gB in driving the fusion process forward beyond the transient fusion pore opening and subsequently leading to fusion pore expansion