HSV Entry Research Articles

Aescin Inhibits Herpes simplex Virus Type 1 Induced Membrane Fusion.

Infections with Herpes simplex virus can cause severe ocular diseases and encephalitis. The present study aimed to investigate potential inhibitors of fusion between HSV-1 and the cellular membrane of the host cell. Fusion and entry of HSV-1 into the host cell is mimicked by a virus-free eukaryotic cell culture system by co-expression of the HSV-1 glycoproteins gD, gH, gL, and gB in presence of a gD receptor, resulting in excessive membrane fusion and polykaryocyte formation. A microscopic read-out was used for the screening of potential inhibitors, whereas luminometric quantification of cell-cell fusion was used in a reporter fusion assay. HSV-1 gB was tagged at its C-terminus with mCherry to express mCherry-gB in both assay systems for the visualization of the polykaryocyte formation. Reporter protein expression of SEAP was regulated by a Tet-On 3 G system. The saponin mixture aescin was identified as the specific inhibitor (IC50 7.4 µM, CC50 24.3 µM, SI 3.3) of membrane fusion. A plaque reduction assay on Vero cells reduced HSV-1 entry into cells and HSV-1 cell-to-cell spread significantly; 15 µM aescin decreased relative plaque counts to 41%, and 10 µM aescin resulted in a residual plaque size of 11% (HSV-1 17 syn+) and 2% (HSV-1 ANG path). Release of the HSV-1 progeny virus was reduced by one log step in the presence of 15 µM aescin. Virus particle integrity was mainly unaffected. Analytical investigation of aescin by UHPLC-MS revealed aescin IA and -IB and isoaescin IA and -IB as the main compounds with different functional activities. Aescin IA had the lowest IC50, the highest CC50, and an SI of > 4.6.

Herpes simplex virus spreads rapidly in human foreskin, partly driven by chemokine-induced redistribution of Nectin-1 on keratinocytes.

HSV infects keratinocytes in the epidermis of skin via nectin-1. We established a human foreskin explant infection model to investigate HSV entry and spread. HSV1 entry could only be achieved by the topical application of virus via high density microarray projections (HD-MAPs) to the epidermis, which penetrated beyond one third of its thickness, simulating in vivo microtrauma. Rapid lateral spread of HSV1 to a mean of 13 keratinocytes wide occurred after 24 hours and free virus particles were observed between keratinocytes, consistent with an intercellular route of spread. Nectin-1 staining was markedly decreased in foci of infection in the epidermis and in the human keratinocyte HaCaT cell line. Nectin-1 was redistributed, at the protein level, in adjacent uninfected cells surrounding infection, inducible by CCL3, IL-8 (or CXCL8), and possibly CXCL10 and IL-6, thus facilitating spread. These findings provide the first insights into HSV1 entry and spread in human inner foreskin in situ.

Development of a novel, high-efficacy oncolytic herpes simplex virus type 1 platform equipped with two distinct retargeting modalities

To retarget oncolytic herpes simplex virus (oHSV) to cancer-specific antigens, we designed a novel, double-retargeted oHSV platform that uses single-chain antibodies (scFv) incorporated into both glycoprotein H (gH) and a bispecific adapter expressed from the viral genome to mediate infection predominantly via tumor-associated antigens. Successful retargeting was achieved using a nectin-1-detargeted HSV that remains capable of interacting with HVEM, the second canonical HSV entry receptor, and is therefore recognized by the adapter consisting of the virus-binding N-terminal 82 residues of HVEM fused to the target-specific scFv. We tested both an EpCAM- and a HER2-specific scFv separately and together to target cells expressing one, the other, or both receptors. Our results show not only dose-dependent, target receptor-specific infection in vitro, but also enhanced virus spread compared to single-retargeted virus. In addition, we observed effective infection and spreading of the EpCAM double-retargeted virus in vivo. Remarkably, a single intravenous dose of the EpCAM-specific virus eliminated all detectable tumors in a subcutaneous xenograft model, and the same intravenous dose appeared harmless in immunocompetent FVB/N mice. Our findings suggest that our double-retargeted oHSV platform can provide a potent and versatile, systemically deliverable class of anti-cancer therapeutics that specifically target cancer cells while ensuring safety.

Herpes Simplex Virus 1 Glycoprotein B from a Hyperfusogenic Virus Mediates Enhanced Cell-Cell Fusion.

Herpes simplex virus 1 (HSV-1) causes significant morbidity and death in humans worldwide. Herpes simplex virus 1 has a complex fusion mechanism that is incompletely understood. The HSV-1 strain ANG has notable fusion and entry activities that distinguish it from wild type. HSV-1 ANG virions fused with the Vero cell surface at 4 °C and also entered cells more efficiently at 15 °C, relative to wild type HSV-1 strain KOS virions, consistent with a hyperfusogenic phenotype. Understanding the molecular basis for the unique entry and fusion activities of HSV-1 strain ANG will help decipher the HSV fusion reaction and entry process. Sequencing of HSV-1 ANG genes revealed multiple changes in gB, gC, gD, gH, and gL proteins relative to wild type HSV-1 strains. The ANG UL45 gene sequence, which codes for a non-essential envelope protein, was identical to wild type KOS. HSV-1 ANG gB, gD, and gH/gL were necessary and sufficient to mediate cell-cell fusion in a virus-free reporter assay. ANG gB, when expressed with wild type KOS gD and gH/gL, increased membrane fusion, suggesting that ANG gB has hyperfusogenic cell-cell fusion activity. Replacing the KOS gD, gH, or gL with the corresponding ANG alleles did not enhance cell-cell fusion. The novel mutations in the ANG fusion and entry glycoproteins provide a platform for dissecting the cascade of interactions that culminate in HSV fusion and entry.

Engineered CHO cells as a novel AAV production platform for gene therapy delivery

The Herpes simplex virus (HSV)-based platform for production of recombinant adeno-associated viral vectors (rAAVs) yields higher titers and increased percentage of full capsids when compared to the triple transient transfection (TTT) method. However, this platform currently faces two major challenges. The first challenge is the reliance on commercial media, sometimes supplemented with serum, leading to costly manufacturing and a high risk for introduction of adventitious agents. The second challenge is that the production of HSV-1 relies on adherent complementing Vero cells (V27), making it difficult to scale up. We engineered serum-free-adapted CHO cells expressing key HSV-1 entry receptors, HVEM and/or Nectin-1 to address the first challenge. Using high-throughput cloning methods, we successfully selected a HVEM receptor-expressing clone (CHO–HV–C1) that yields 1.62 × 109, 2.51 × 109, and 4.07 × 109 viral genome copies/mL with rAAV6.2-GFP, rAAV8-GFP, and rAAV9-GFP vectors respectively, within 24 h post rHSV-1 co-infection. Moreover, CHO–HV–C1-derived rAAVs had comparable in vitro transduction, infectivity, and biodistribution titers to those produced by TTT. The second challenge was addressed via engineering CHO–HV–C1 cells to express HSV-1 CP27. These cells successfully produced rHSV-1 vectors, but with significantly lower titers than V27 cells. Taken together, the CHO/HSV system provides a novel, scalable, reduced cost, serum-free AAV manufacturing platform.

Epigallocatechin Gallate-Modified Silver Nanoparticles Show Antiviral Activity against Herpes Simplex Type 1 and 2.

(1) Background: Epigallocatechin gallate (EGCG) has been recognized as a flavonoid showing antiviral activity against various types of DNA and RNA viruses. In this work, we tested if EGCG-modified silver nanoparticles (EGCG-AgNPs) can become novel microbicides with additional adjuvant properties to treat herpes infections. (2) Methods: The anti-HSV and cytotoxic activities of EGCG-AgNPs were tested in human HaCaT and VK-2-E6/E7 keratinocytes. HSV-1/2 titers and immune responses after treatment with EGCG-AgNPs were tested in murine models of intranasal HSV-1 infection and genital HSV-2 infection. (3) Results: EGCG-AgNPs inhibited attachment and entry of HSV-1 and HSV-2 in human HaCaT and VK-2-E6/E7 keratinocytes much better than EGCG at the same concentration. Infected mice treated intranasally (HSV-1) or intravaginally (HSV-2) with EGCG-AgNPs showed lower virus titers in comparison to treatment with EGCG alone. After EGCG-AgNPs treatment, mucosal tissues showed a significant infiltration in dendritic cells and monocytes in comparison to NaCl-treated group, followed by significantly better infiltration of CD8+ T cells, NK cells and increased expression of IFN-α, IFN-γ, CXCL9 and CXCL10. (4) Conclusions: Our findings show that EGCG-AgNPs can become an effective novel antiviral microbicide with adjuvant properties to be applied upon the mucosal tissues.

Method for the growth of Herpes Simplex Virus (HSV-1 and HSV-2) in an oxygen-free environment

Vaginal herpes infections occur under low oxygen conditions, including anoxia. Using an anaerobic (0% O2) cell system, the infection rate, early protein expression, and progeny viability were determined. Anaerobic HeLa 229 cells (0% O2; anaerobic DMEM; 24h) were infected with MOI 1 of HSV1/2, then incubated under aerobic (control) and anaerobic conditions. Infectivity (6h post-infection (pi)) was assessed by ONPG activity and enumerating X-gal-stained cells. HSV1/2 viral immediate-early entry, early entry, and late entry protein expression were determined by fluorescent antibody staining followed by HSV1/2 glycoprotein B mouse monoclonal antibody and DAPI. Statistical analysis was performed using t-test and data were considered significant when p ≤ 0.05 (GraphPad Prism). HSV1/2 infection under standard aerobic conditions was approximately twice that measured for anaerobically infected cells. In addition, the level of HSV-2 infectivity was significantly (p < 0.05) higher (~8-fold) than that of HSV-1, regardless of oxygen level. HSV entry stage protein expression (ICP8, ICP27, VP5) was detected for anaerobic HSV1/2. A similar pattern was observed for anaerobically grown progeny of HSV1/2, i.e., the number of progeny was higher when anaerobic progenies were grown under aerobic conditions (anaerobic to aerobic) as compared to those grown under anaerobic conditions (anaerobic to anaerobic). In addition, the progeny levels grown solely under anaerobic conditions trended higher for HSV-1 than HSV-2 progeny but were not significantly different. These findings show that HSV1/2 can infect and replicate in anaerobically growing cells and that oxygen is not essential for productive replication.

Host Cell Neddylation Facilitates Alphaherpesvirus Entry in a Virus-Specific and Cell-Dependent Manner.

ABSTRACTHerpes simplex virus 1 (HSV-1) commandeers the host cell proteasome at several steps of its replication cycle, including entry. Here we demonstrate that HSV-2, pseudorabies virus (PRV), and bovine herpesvirus 1 (BoHV-1) entry are blocked by bortezomib, a proteasome inhibitor that is an FDA-approved cancer drug. Proteasome-dependent entry of HSV-1 is thought to be ubiquitin-independent. To interrogate further the proteasomal mechanism of entry, we determined the involvement of the ubiquitin-like molecule NEDD8 and the neddylation cascade in alphaherpesvirus entry and infection. MLN4924 is a small-molecule inhibitor of neddylation that binds directly to the NEDD8-activating enzyme. Cell treatment with MLN4924 inhibited plaque formation and infectivity by HSV-1, PRV, and BoHV-1 at noncytotoxic concentrations. Thus, the neddylation pathway is broadly important for alphaherpesvirus infection. However, the neddylation inhibitor had little effect on entry of the veterinary viruses but had a significant inhibitory effect on entry of HSV-1 and HSV-2 into seven different cell types. Washout experiments indicated that MLN4924’s effect on viral entry was reversible. A time-of-addition assay suggested that the drug was acting on an early step in the entry process. Small interfering RNA (siRNA) knockdown of NEDD8 significantly inhibited HSV entry. In probing the neddylation-dependent step in entry, we found that MLN4924 dramatically blocked endocytic uptake of HSV from the plasma membrane by >90%. In contrast, the rate of HSV entry into cells that support direct fusion of HSV with the cell surface was unaffected by MLN4924. Interestingly, proteasome activity was less important for the endocytic internalization of HSV from the cell surface. The results suggest that the NEDD8 cascade is critical for the internalization step of HSV entry.IMPORTANCE Alphaherpesviruses are ubiquitous pathogens of humans and veterinary species that cause lifelong latent infections and significant morbidity and mortality. Host cell neddylation is important for cell homeostasis and for the infection of many viruses, including HSV-1, HSV-2, PRV, and BoHV-1. Inhibition of neddylation by a pharmacologic inhibitor or siRNA blocked HSV infection at the entry step. Specifically, the NEDD8 pathway was critically important for HSV-1 internalization from the cell surface by an endocytosis mechanism. The results expand our limited understanding of cellular processes that mediate HSV internalization. To our knowledge, this is the first demonstration of a function for the neddylation cascade in virus entry.

Conformational Changes in Herpes Simplex Virus Glycoprotein C.

Low endosomal pH facilitates herpesvirus entry in a cell-specific manner. Herpes simplex virus 1 (HSV-1) causes significant morbidity and death in humans worldwide. HSV-1 enters cells by low-pH and neutral-pH pathways. Low-pH-induced conformational changes in the HSV envelope glycoprotein B (gB) may mediate membrane fusion during viral entry. HSV-1 gC, a 511-amino acid, type I integral membrane glycoprotein, mediates HSV-1 attachment to host cell surface glycosaminoglycans, but this interaction is not essential for viral entry. We previously demonstrated that gC regulates low-pH viral entry independent of its known role in cell attachment. Low-pH-triggered conformational changes in gB occur at a lower pH when gC is absent, suggesting that gC positively regulates gB conformational changes. Here, we demonstrate that mildly acidic pH triggers conformational changes in gC itself. Low-pH treatment of virions induced antigenic changes in distinct gC epitopes, and those changes were reversible. One of these gC epitopes is recognized by a monoclonal antibody that binds to a linear sequence that includes residues within gC amino acids 33 to 123. This antibody inhibited low-pH entry of HSV, suggesting that its gC N-terminal epitope is particularly important. We propose that gC plays a critical role in HSV entry through a low-pH endocytosis pathway, which is a major entry route in human epithelial cells. IMPORTANCE Herpesviruses are ubiquitous pathogens that cause lifelong latent infections and are characterized by multiple entry pathways. The HSV envelope gC regulates HSV entry by a low-pH entry route. The fusion protein gB undergoes pH-triggered conformational changes that are facilitated by gC. Here, we report that gC itself undergoes a conformational change at low pH. A monoclonal antibody to gC that binds to a region that undergoes pH-induced changes also selectively inhibits HSV low-pH entry, corroborating the importance of gC in the low-pH entry pathway. This study illustrates the complex role of endosomal pH during HSV entry and provides novel insights into the functions of gC.

CGRP inhibits human Langerhans cells infection with HSV by differentially modulating specific HSV-1 and HSV-2 entry mechanisms.

Herpes simplex virus (HSV) is widespread globally, with both HSV-1 and HSV-2 responsible for genital herpes. During sexual transmission, HSV targets epithelial cells, sensory peripheral pain neurons secreting the mucosal neuropeptide calcitonin gene-related peptide (CGRP), and mucosal immune cells including Langerhans cells (LCs). We previously described a neuro-immune crosstalk, whereby CGRP inhibits LCs-mediated human immunodeficiency virus type 1 (HIV-1) transmission. Herein, to further explore CGRP-mediated anti-viral function, we investigated whether CGRP affects LCs infection with HSV. We found that both HSV-1 and HSV-2 primary isolates productively infect monocyte-derived LCs (MDLCs) and inner foreskin LCs. Moreover, CGRP significantly inhibits infection with both HSV subtypes of MDLCs and langerinhigh, but not langerinlow, inner foreskin LCs. For HSV-1, infection is mediated via the HSV-1-specific entry receptor 3-O sulfated heparan sulfate (3-OS HS) in a pH-depended manner, and CGRP down-regulates 3-OS HS surface expression, as well as abrogates pH dependency. For HSV-2, infection involves langerin-mediated endocytosis in a pH-independent manner, and CGRP up-regulates surface expression of atypical langerin double-trimer oligomers. Our results show that CGRP inhibits mucosal HSV infection by differentially modulating subtype-specific entry receptors and mechanisms in human LCs. CGRP could turn out useful for prevention of LCs-mediated HSV infection and HSV/HIV-1 co-infection.

Actin Polymerization Is Required for Filopodia Formation Supporting HSV-1 Entry into Activated T Cells.

Enhanced HSV-1 production is found in activated T-lymphocytes, but the mechanism is still unknown. In this paper, the HSV-1 entry step in CD3+CD4-CD8-Jurkat T lymphocytes was investigated. Observation under electron microscopy revealed the level of filopodia formation on the surface of activated Jurkat cells was significantly higher than that of non-activated Jurkat cells especially after adding HSV-1 for 15min. A significant increase of actin protein was demonstrated in HSV-1 infected, activated Jurkat cells compared to HSV-1 infected, non-activated Jurkat cells. After the cells were treated with 2.5 and 5µg/mL cytochalasin D, an inhibitor of actin polymerization that causes depolymerization of actin's filamentous form, the actin protein was decreased significantly, resulting in an absence of filopodia formation. In summary, this is the first study revealing that HSV-1 induced filopodia formation through actin polymerization in activated T cells similar to epithelial, mucosal and neuronal cells. This phenomenon supported the virus entry resulting to increased yield of HSV-1 production.

Newly Developed Oncolytic Herpes Simplex Viruses Expressing Multiple Immunomodulatory Transgenes Effectively Target AML Cells

Acute myeloid leukemia (AML) is the most common human leukemia and is a major area of unmet medical need among hematologic malignancies. Progress has been made in identifying therapeutic targets and several approved therapies, but resistance to frontline chemotherapy remains a major cause of treatment failure, highlighting the need for new therapies. Oncolytic viruses (OV) are a promising new class of therapeutics that rely on tumor specific oncolysis and the generation of a potent adaptive anti-tumor immune response for efficacy. To investigate if our newly developed oncolytic herpes simplex viruses (oHSVs), designed to potentiate anti-leukemia immunity, effectively target primitive AML cells, we evaluated oHSV-VG161, which is engineered to express IL-12, IL-15 and the IL-15 receptor alpha subunit, along with a peptide fusion protein capable of disrupting PD-1/PD-L1 interaction. After screening several AML cell lines that expressed relatively high levels of a HSV entry receptor (HVEM), we demonstrated that VG161-infected OCIAML3 and MOLM13 cells significantly enhanced cell killing (IC 50: 0.4 & 1.8 multiplicity of infection (MOI) as compared to MV4-11 and U973 cells (IC 50: 3.0 & 9.5 MOI). These effects were 2-3 folds lower in control VG160-infected cells. We also observed that VG161-infected AML cells induced apoptosis in a dose-dependent manner (~50%) after 48 hours and cleaved PARP, Caspase-3 and Caspase-8 were increased in these cells, and to a lesser extent in control VG160-infected cells. Both VG160 and VG161 viruses replicated efficiently in OCIAML3 and MOLM13 cells in a timely, dose-dependent manner, evidenced by qPCR detection of HSV-1 ICP27 DNA copy numbers (>500-fold increase) over 48 hours of treatment. This result was supported by detection of protein expression of HSV-1 glycoprotein D in VG160 and VG161-infected cells (up to 40% of protein detected) by FACS analysis. Interestingly, IL-12 but not IL-15 protein expression was found in intracellular-stained VG161-infected OCIAML3 and MOLM13 cells in a dose-dependent manner (up to 13% of protein detected, P<0.01) but not in VG160-infected cells, as assessed by FACS analysis. Production of IL-12 was also detected in cultured media obtained from VG161-infected AML cells (up to 150 pg/mL) by ELISA. To investigate potential molecular mechanisms of VG161-mediated anti-leukemia response and specific signalling pathways, we have screened several potential candidates and found immune regulating genes, such as IRF3, IRF7, IRF9, NFkB and ISGs, as well as type I IFN to be highly increased in VG161-infected cells as compared to VG160-infected cells (2-4-folds, P<0.001) in a dose dependent manner over 48 hours of treatment, assayed by qRT-PCR. Western blot analysis demonstrated increased phosphorylation of p-STAT1 and its protein expression in VG161-infected cells compared to VG160 control cells (~2-fold). These results suggest that VG161 viruses expressing several engineered immunomodulatory transgenes, particularly IL-12, contribute to anti-leukemia responses by activating specific immune regulating pathways such as the JAK/STAT pathway. In addition, we detected an increase in both RNA and protein levels of PD-L1 in VG161-infected AML cells, suggesting the necessity of PD-L1 blocking peptide in the viral construct. To further investigate VG161's role in regulating innate and adaptive immune responses, we have examined the biological effects of VG160/VG161 in the presence of healthy peripheral blood mononuclear cells (PBMC) in both AML cell lines and primary AML patient cells in vitro. Most interestingly, VG160 or VG161-infected OCIAML3 and MOLM13 cells show enhanced cell killing when co-cultured with PBMC and this cell killing effect was greatly enhanced in VG161-infected cells as compared to VG160-infected cells, especially in the MOLM13 cell line (up to 90% killing). This observation was further supported when primitive AML patient cells were co-cultured with VG161 and PBMC as compared to VG160 control cells. Moreover, PD-L1 expression was highly increased in AML patient cells when cultured with VG161 as compared to VG160 (2.7-fold) and this was further enhanced when co-cultured with VG161 and PBMC. Thus, we have demonstrated that newly developed oHSVs engineered with several immunomodulatory transgenes effectively target primitive AML cells, suggesting a potential treatment strategy for AML. DisclosuresNo relevant conflicts of interest to declare.

Preclinical evaluation of an engineered oncolytic herpes simplex virus for pediatric osteosarcoma.

10040 Background: Osteosarcoma is the most common primary bone tumor in children. For those with relapsed or metastatic disease, the five-year survival rate is approximately 20%, and survivors often suffer from long-term disability from current therapies. The high morbidity and mortality for these patients highlight a great need for improved therapies. One such novel therapeutic approach is oncolytic herpes simplex virus (oHSV) immunovirotherapy. We previously demonstrated that M002, an engineered oHSV that contains deletions of the neurovirulence gene preventing infection of normal cells, effectively infects and kills neuroblastoma and rhabdomyosarcoma. Currently, similar oHSVs are being evaluated in early phase clinical trials for children and adults with relapsed or refractory brain tumors. To date, there has been limited investigation of oncolytic virotherapy in osteosarcoma. Thus, we sought to examine the ability of oHSV, M002, to infect and kill osteosarcoma cells in vitro. Methods: We evaluated two long-term passaged human osteosarcoma cell lines, U2-OS and MG-63. Flow cytometry was used to assess baseline expression of oHSV viral entry-mediated receptors (CD111, CD112, syndecan, HVEM). Single and multi-step viral recovery experiments measured virus infectivity and replication. Cells were infected with increasing multiplicity of infection (MOI) of M002, and cell viability was measured 72 hours post-infection via alamarBlue assay. Results: Both MG-63 and U2-OS cells expressed HSV entry molecules (Table) including high levels of the primary HSV entry molecule CD111. Single step virus recovery experiments in MG-63 cells infected at a MOI of 10 plaque-forming units (PFU)/cell demonstrated a 3 log-fold increase in virus titer from 12 to 24 hours post-infection. For multi-step experiments, MG-63 cells were infected with a MOI of 0.1 PFU/cell; viral replication significantly increased from 1.1x103 PFU at 6 hours post-infection to 3.8x1010 PFU at 72 hours post-infection. M002 successfully decreased osteosarcoma viability with a lethal dose in 50% of cells (LD50)of 2.82 and0.67 PFU/cell for MG-63 and U2-OS cells, respectively. Notably, at a virus MOI of 5 PFU/cell, viability was decreased by 64% ± 0.1% (p&lt;0.001 vs control) in MG-63 cells and 96% ± 0.1% (p&lt;0.001 vs control) in U2-OS cells. Conclusions: MG-63 and U2-OS osteosarcoma cells express high levels of HSV entry receptors. Virus recovery experiments demonstrated the ability of M002 to infect cells and replicate over time. The viability of osteosarcoma cells significantly decreased following infection with M002. These data suggest M002 may be a promising novel therapeutic option for patients with osteosarcoma and warrant further investigation for translation to the clinical setting.[Table: see text]

Halting the Spread of Herpes Simplex Virus-1: The Discovery of an Effective Dual αvβ6/αvβ8 Integrin Ligand.

Over recent years, αvβ6 and αvβ8 Arg-Gly-Asp (RGD) integrins have risen to prominence as interchangeable co-receptors for the cellular entry of herpes simplex virus-1 (HSV-1). In fact, the employment of subtype-specific integrin-neutralizing antibodies or gene-silencing siRNAs has emerged as a valuable strategy for impairing HSV infectivity. Here, we shift the focus to a more affordable pharmaceutical approach based on small RGD-containing cyclic pentapeptides. Starting from our recently developed αvβ6-preferential peptide [RGD-Chg-E]-CONH2 (1), a small library of N-methylated derivatives (2–6) was indeed synthesized in the attempt to increase its affinity toward αvβ8. Among the novel compounds, [RGD-Chg-(NMe)E]-CONH2 (6) turned out to be a potent αvβ6/αvβ8 binder and a promising inhibitor of HSV entry through an integrin-dependent mechanism. Furthermore, the renewed selectivity profile of 6 was fully rationalized by a NMR/molecular modeling combined approach, providing novel valuable hints for the design of RGD integrin ligands with the desired specificity profile.

Herpes Simplex Virus type 1 infects Langerhans cells and the novel epidermal dendritic cell, Epi-cDC2s, via different entry pathways

Skin mononuclear phagocytes (MNPs) provide the first interactions of invading viruses with the immune system. In addition to Langerhans cells (LCs), we recently described a second epidermal MNP population, Epi-cDC2s, in human anogenital epidermis that is closely related to dermal conventional dendritic cells type 2 (cDC2) and can be preferentially infected by HIV. Here we show that in epidermal explants topically infected with herpes simplex virus (HSV-1), both LCs and Epi-cDC2s interact with HSV-1 particles and infected keratinocytes. Isolated Epi-cDC2s support higher levels of infection than LCs in vitro, inhibited by acyclovir, but both MNP subtypes express similar levels of the HSV entry receptors nectin-1 and HVEM, and show similar levels of initial uptake. Using inhibitors of endosomal acidification, actin and cholesterol, we found that HSV-1 utilises different entry pathways in each cell type. HSV-1 predominantly infects LCs, and monocyte-derived MNPs, via a pH-dependent pathway. In contrast, Epi-cDC2s are mainly infected via a pH-independent pathway which may contribute to the enhanced infection of Epi-cDC2s. Both cells underwent apoptosis suggesting that Epi-cDC2s may follow the same dermal migration and uptake by dermal MNPs that we have previously shown for LCs. Thus, we hypothesize that the uptake of HSV and infection of Epi-cDC2s will stimulate immune responses via a different pathway to LCs, which in future may help guide HSV vaccine development and adjuvant targeting.

Antibody Screening System Using a Herpes Simplex Virus (HSV)-Based Probe To Identify a Novel Target for Receptor-Retargeted Oncolytic HSVs.

Herpes simplex virus (HSV) is a promising tool for developing oncolytic virotherapy. We recently reported a platform for receptor-retargeted oncolytic HSVs that incorporates single-chain antibodies (scFvs) into envelope glycoprotein D (gD) to mediate virus entry via tumor-associated antigens. Therefore, it would be useful to develop an efficient system that can screen antibodies that might mediate HSV entry when they are incorporated as scFvs into gD. We created an HSV-based screening probe by the genetic fusion of a gD mutant with ablated binding capability to the authentic HSV entry receptors and the antibody-binding C domain of streptococcal protein G. This engineered virus failed to enter cells through authentic receptors. In contrast, when this virus was conjugated with an antibody specific to an antigen on the cell membrane, it specifically entered cells expressing the cognate antigen. This virus was used as a probe to identify antibodies that mediate virus entry via recognition of certain molecules on the cell membrane other than authentic receptors. Using this method, we identified an antibody specific to epiregulin (EREG), which has been investigated mainly as a secreted growth factor and not necessarily for its precursor that is expressed in a transmembrane form. We constructed an scFv from the anti-EREG antibody for insertion into the retargeted HSV platform and found that the recombinant virus entered cells specifically through EREG expressed by the cells. This novel antibody-screening system may contribute to the discovery of unique and unexpected molecules that might be used for the entry of receptor-retargeted oncolytic HSVs.IMPORTANCE The tropism of the cellular entry of HSV is dependent on the binding of the envelope gD to one of its authentic receptors. This can be fully retargeted to other receptors by inserting scFvs into gD with appropriate modifications. In theory, upon binding to the engineered gD, receptors other than authentic receptors should induce a conformational change in the gD, which activates downstream mechanisms required for viral entry. However, prerequisite factors for receptors to be used as targets of a retargeted virus remain poorly understood, and it is difficult to predict which molecules might be suitable for our retargeted HSV construct. Our HSV-based probe will allow unbiased screening of antibody-antigen pairs that mediate virus entry and might be a useful tool to identify suitable pairs for our construct and to enhance our understanding of virus-cell interactions during infection by HSV and possibly other viruses.

Herpes Simplex Virus Entry by a Nonconventional Endocytic Pathway.

Herpes simplex virus 1 (HSV-1) causes significant morbidity and mortality in humans worldwide. HSV-1 enters epithelial cells via an endocytosis mechanism that is low-pH dependent. However, the precise intracellular pathway has not been identified, including the compartment where fusion occurs. In this study, we utilized a combination of molecular and pharmacological approaches to better characterize HSV entry by endocytosis. HSV-1 entry was unaltered in both cells treated with small interfering RNA (siRNA) to Rab5 or Rab7 and cells expressing dominant negative forms of these GTPases, suggesting entry is independent of the conventional endo-lysosomal network. The fungal metabolite brefeldin A (BFA) and the quinoline compound Golgicide A (GCA) inhibited HSV-1 entry via beta-galactosidase reporter assay and impaired incoming virus transport to the nuclear periphery, suggesting a role for trans-Golgi network (TGN) functions and retrograde transport in HSV entry. Silencing of Rab9 or Rab11 GTPases, which are involved in the retrograde transport pathway, resulted in only a slight reduction in HSV infection. Together, these results suggest that HSV enters host cells by an intracellular route independent of the lysosome-terminal endocytic pathway.IMPORTANCE Herpes simplex virus 1 (HSV-1), the prototype alphaherpesvirus, is ubiquitous in the human population and causes lifelong infection that can be fatal in neonatal and immunocompromised individuals. HSV enters many cell types by endocytosis, including epithelial cells, the site of primary infection in the host. The intracellular itinerary for HSV entry remains unclear. We probed the potential involvement of several Rab GTPases in HSV-1 entry and suggest that endocytic entry of HSV-1 is independent of the canonical lysosome-terminal pathway. A nontraditional endocytic route may be employed, such as one that intersects with the trans-Golgi network (TGN). These results may lead to novel targets for intervention.

Hsv-1 Endocytic Entry into a Human Oligodendrocytic Cell Line is Mediated by Clathrin and Dynamin but Not Caveolin.

Endocytosis is a pathway used by viruses to enter cells that can be classified based on the proteins involved, such as dynamin, clathrin or caveolin. Although the entry of herpes simplex type 1 (HSV-1) by endocytosis has been documented in different cell types, its dependence on clathrin has not been described whereas its dependence on dynamin has been shown according to the cell line used. The present work shows how clathrin-mediated endocytosis (CME) is one way that HSV-1 infects the human oligodendroglial (HOG) cell line. Partial dynamin inhibition using dynasore revealed a relationship between decrease of infection and dynamin inhibition, measured by viral titration and immunoblot. Co-localization between dynamin and HSV-1 was verified by immunofluorescence at the moment of viral entry into the cell. Inhibition by chlorpromazine revealed that viral progeny also decreased when clathrin was partially inhibited in our cell line. RT-qPCR of immediately early viral genes, specific entry assays and electron microscopy all confirmed clathrin’s participation in HSV-1 entry into HOG cells. In contrast, caveolin entry assays showed no effect on the entry of this virus. Therefore, our results suggest the participation of dynamin and clathrin during endocytosis of HSV-1 in HOG cells.

Cetylpyridinium chloride blocks herpes simplex virus replication in gingival fibroblasts

Infections with herpes simplex viruses are lifelong and highly prevalent worldwide. Individuals with clinical symptoms elicited by HSVs may suffer from occasional or recurrent herpetic lesions in the orofacial and genital areas. Despite the existence of nucleoside analogues that interfere with HSV replication, such as acyclovir, these drugs are somewhat ineffective in treating skin lesions as topical formulations only reduce in one or few days the duration of the herpetic ulcers. Cetylpyridinium chloride (CPC) is a quaternary ammonium compound present in numerous hygiene products, such as mouthwashes, deodorants, aphtae-treating formulations and oral tablets as an anti-septic to limit bacterial growth. Some reports indicate that CPC can also modulate host signaling pathways, namely NF-κB signaling. Because HSV infection is modulated by NF-κB, we sought to assess whether CPC has antiviral effects against HSVs. Using wild-type HSV-1 and HSV-2, as well as viruses that are acyclovir-resistant or encode GFP reporter genes, we assessed the antiviral capacity of CPC in epithelial cells and human gingival fibroblasts expanded from the oral cavity and its mechanism of action. We found that a short, 10-min exposure to CPC added after HSV entry into the cells, significantly limited viral replication in both cell types by impairing viral gene expression. Interestingly, our results suggest that CPC blocks HSV replication by interfering with the translocation of NF-κB into the nucleus of HSV-infected cells. Taken together, these findings suggest that formulations containing CPC may help limit HSV replication in infected tissues and consequently reduce viral shedding.

Saliva enhances infection of gingival fibroblasts by herpes simplex virus 1.

Oral herpes is a highly prevalent infection caused by herpes simplex virus 1 (HSV-1). After an initial infection of the oral cavity, HSV-1 remains latent in sensory neurons of the trigeminal ganglia. Episodic reactivation of the virus leads to the formation of mucocutaneous lesions (cold sores), but asymptomatic reactivation accompanied by viral shedding is more frequent and allows virus spread to new hosts. HSV-1 DNA has been detected in many oral tissues. In particular, HSV-1 can be found in periodontal lesions and several studies associated its presence with more severe periodontitis pathologies. Since gingival fibroblasts may become exposed to salivary components in periodontitis lesions, we analyzed the effect of saliva on HSV-1 and -2 infection of these cells. We observed that human gingival fibroblasts can be infected by HSV-1. However, pre-treatment of these cells with saliva extracts from some but not all individuals led to an increased susceptibility to infection. Furthermore, the active saliva could expand HSV-1 tropism to cells that are normally resistant to infection due to the absence of HSV entry receptors. The active factor in saliva was partially purified and comprised high molecular weight complexes of glycoproteins that included secretory Immunoglobulin A. Interestingly, we observed a broad variation in the activity of saliva between donors suggesting that this activity is selectively present in the population. The active saliva factor, has not been isolated, but may lead to the identification of a relevant biomarker for susceptibility to oral herpes. The presence of a salivary factor that enhances HSV-1 infection may influence the risk of oral herpes and/or the severity of associated oral pathologies.