TMEFF1 is a neuron-specific restriction factor for herpes simplex virus.

The brain is highly sensitive to damage caused by infection and inflammation1,2. Herpes simplex virus 1 (HSV-1) is a neurotropic virus and the cause of herpes simplex encephalitis3. It is unknown whether neuron-specific antiviral factors control virus replication to prevent infection and excessive inflammatory responses, hence protecting the brain. Here we identify TMEFF1 as an HSV-1 restriction factor using genome-wide CRISPR screening. TMEFF1 is expressed specifically in neurons of the central nervous system and is not regulated by type I interferon, the best-known innate antiviral system controlling virus infections. Depletion of TMEFF1 in stem-cell-derived human neurons led to elevated viral replication and neuronal death following HSV-1 infection. TMEFF1 blocked the HSV-1 replication cycle at the level of viral entry through interactions with nectin-1 and non-muscle myosin heavy chains IIA and IIB, which are core proteins in virus-cell binding and virus-cell fusion, respectively4-6. Notably, Tmeff1-/- mice exhibited increased susceptibility to HSV-1 infection in the brain but not in the periphery. Within the brain, elevated viral load was observed specifically in neurons. Our study identifies TMEFF1 as a neuron-specific restriction factor essential for prevention of HSV-1 replication in the central nervous system.

Development of an HSV-1 production process involving serum-reduced culturing and bead-to-bead transfer.

Herpes simplex virus type 1 (HSV-1) plays an important role in the field of gene therapy and viral vaccines, especially as an oncolytic virus. However, the mass production of HSV-1 viral vectors remains a challenge in the industry. In this study, a microcarrier-mediated serum-reduced medium culture was used to improve the bioprocess of HSV-1 production and increase HSV-1 yields. The composition of the culture media, which included a basal medium, serum concentration, and glutamine additive, was optimized. The process was successfully conducted in a 1 L bioreactor, and virus production was threefold greater than that of conventional processes with a 10% serum medium. The bead-to-bead transfer process was also developed to further increase scalability. In spinner flasks, the detachment rate increased from 49.4 to 80.6% when combined agitation was performed during digestion; the overall recovery proportion increased from 37.9 to 71.1% after the operational steps were optimized. Specifically, microcarrier loss was reduced during aspiration and transfer, and microcarriers and detached cells were separated with filters. Comparable cell growth was achieved with the baseline process using 2D culture as the inoculum by exchanging the subculture medium. To increase virus production after bead-to-bead transfer, critical parameters, including shear stress during digestion, TrypLE and EDTA concentrations in the subculture, and the CCI, were identified from 47 parameters via correlation analysis and principal component analysis. The optimized bead-to-bead transfer process achieved an average of 90.4% overall recovery and comparable virus production compared to that of the baseline process. This study is the first to report the optimization of HSV-1 production in Vero cells cultured on microcarriers in serum-reduced medium after bead-to-bead transfer. KEY POINTS: • An HSV-1 production process was developed that involves culturing in serum-reduced medium, and this process achieved threefold greater virus production than that of traditional processes. • An indirect bead-to-bead transfer process was developed with over 90% recovery yield in bioreactors. • HSV-1 production after bead-to-bead transfer was optimized and was comparable to that achieved with 2D culture as inoculum.

Histone H2A variant H2A.B is enriched in transcriptionally active and replicating HSV-1 lytic chromatin.

Herpes simplex virus 1 (HSV-1) transcription is restricted in latently infected neurons and the genomes are in mostly silenced chromatin, whereas all viral genes are transcribed in lytically infected cells, in which the genomes are dynamically chromatinized. Epigenetic regulation modulates HSV-1 transcription during lytic, latent, and reactivating infections but the precise mechanisms are not fully defined. Nucleosomes are dynamic: they slide, breathe, assemble, and disassemble. We and others have proposed that the most dynamic HSV-1 chromatin is transcriptionally competent, whereas the least dynamic is silenced. However, the mechanisms yielding the unusually dynamic viral chromatin remain unknown. Histone variants affect nucleosome dynamics. The dynamics of H2A, H2A.X, and macroH2A were enhanced in infected cells, whereas those of H2A.B were uniquely decreased. We constructed stably transduced cells expressing tagged histone H2A, H2A.B, macroH2A, or H2B, which assembles the H2A/H2B nucleosome dimers with all H2A variants. All H2A variants, as well as ectopic and endogenous H2B were assembled into HSV-1 chromatin evenly throughout the genome but canonical H2A was relatively depleted whereas H2A.B was enriched, particularly in the most dynamic viral chromatin. When viral transcription and DNA replication were restricted, H2A.B became as depleted from the viral chromatin through the entire genome as H2A. We propose that lytic HSV-1 nucleosomes are enriched in the dynamic variant H2A.B/H2B dimers to promote HSV-1 chromatin dynamics and transcriptional competency and conclude that the dynamics of HSV-1 chromatin are determined in part by the H2A variants. IMPORTANCE: Herpes simplex virus 1 (HSV-1) transcription is epigenetically regulated during latent and lytic infections, and epigenetic inhibitors have been proposed as potential antiviral drugs to modulate latency and reactivation. However, the detailed epigenetic mechanisms of regulation of HSV-1 transcription have not been fully characterized and may differ from those regulating cellular transcription. Whereas lytic HSV-1 chromatin is unusually dynamic, latent silenced HSV-1 chromatin is not. The mechanisms resulting in the unique dynamics of the lytic chromatin remain unknown. Here we identify the enrichment of the highly dynamic histone 2A variant H2A in the most dynamic viral chromatin, which provides a mechanistic understanding of its unique dynamics. Future work to identify the mechanisms of enrichment in H2A.B on the viral chromatin may identify novel druggable epigenetic regulators that modulate HSV-1 latency and reactivation.

Shared sequence characteristics identified in non-canonical rearrangements of HSV-1 genomes.

IMPORTANCE: Mutations and genetic rearrangements are the primary driving forces of evolution. Viruses provide valuable model systems for investigating these mechanisms due to their rapid evolutionary rates and vast genetic variability. To investigate genetic rearrangements in the double-stranded DNA genome of herpes simplex virus type 1, the viral population was serially passaged in various cell types. The serial passaging led to formation of defective genomes, resulted from cell-specific non-canonical rearrangements (NCRs). Interestingly, we discovered shared sequence characteristics underlying the formation of these NCRs across all cell types. Moreover, most NCRs identified in clinical samples shared these characteristics. Based on our findings, we propose a model elucidating the formation of NCRs during viral replication within the nucleus of eukaryotic cells.

H84T BanLec has broad spectrum antiviral activity against human herpesviruses in cells, skin, and mice.

H84T BanLec is a molecularly engineered lectin cloned from bananas with broad-spectrum antiviral activity against several RNA viruses. H84T BanLec dimers bind glycoproteins containing high-mannose N-glycans on the virion envelope, blocking attachment, entry, uncoating, and spread. It was unknown whether H84T BanLec is effective against human herpesviruses varicella-zoster virus (VZV), human cytomegalovirus (HCMV), and herpes simplex virus 1 (HSV-1), which express high-mannose N-linked glycoproteins on their envelopes. We evaluated H84T BanLec against VZV-ORF57-Luc, TB40/E HCMV-fLuc-eGFP, and HSV-1 R8411 in cells, skin organ culture, and mice. The H84T BanLec EC50 was 0.025 µM for VZV (SI50 = 4000) in human foreskin fibroblasts (HFFs), 0.23 µM for HCMV (SI50 = 441) in HFFs, and 0.33 µM for HSV-1 (SI50 = 308) in Vero cells. Human skin was obtained from reduction mammoplasties and prepared for culture. Skin was infected and cultured up to 14 days. H84T BanLec prevented VZV, HCMV and HSV-1 spread in skin at 10 µM in the culture medium, and also exhibited dose-dependent antiviral effects. Additionally, H84T BanLec arrested virus spread when treatment was delayed. Histopathology of HCMV-infected skin showed no overt toxicity when H84T BanLec was present in the media. In athymic nude mice with human skin xenografts (NuSkin mice), H84T BanLec reduced VZV spread when administered subcutaneously prior to intraxenograft virus inoculation. This is the first demonstration of H84T BanLec effectiveness against DNA viruses. H84T BanLec may have additional unexplored activity against other, clinically relevant, glycosylated viruses.

Microglia Reduce Herpes Simplex Virus 1 Lethality of Mice with Decreased T Cell and Interferon Responses in Brains.

Herpes simplex virus 1 (HSV-1) infects the majority of the human population and can induce encephalitis, which is the most common cause of sporadic, fatal encephalitis. An increase of microglia is detected in the brains of encephalitis patients. The issues regarding whether and how microglia protect the host and neurons from HSV-1 infection remain elusive. Using a murine infection model, we showed that HSV-1 infection on corneas increased the number of microglia to outnumber those of infiltrating leukocytes (macrophages, neutrophils, and T cells) and enhanced microglia activation in brains. HSV-1 antigens were detected in brain neurons, which were surrounded by microglia. Microglia depletion increased HSV-1 lethality of mice with elevated brain levels of viral loads, infected neurons, neuron loss, CD4 T cells, CD8 T cells, neutrophils, interferon (IFN)-ß, and IFN-γ. In vitro studies demonstrated that microglia from infected mice reduced virus infectivity. Moreover, microglia induced IFN-ß and the signaling pathway of signal transducer and activator of transcription (STAT) 1 to inhibit viral replication and damage of neurons. Our study reveals how microglia protect the host and neurons from HSV-1 infection.

Strong alkaline electrolyzed water efficiently inactivates SARS-CoV-2, other viruses, and Gram-negative bacteria.

Air spaces and material surfaces in a pathogen-contaminated environment can often be a source of infection to humans, and disinfection has become a common intervention focused on reducing the contamination levels. In this study, we examined the efficacy of SAIW, a unique electrolyzed water with chlorine-free, high pH, high concentration of dissolved hydrogen, and low oxygen reduction potential, for the inactivation of several viruses and bacteria. Infectivity assays revealed that initial viral titers of enveloped and non-enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, herpes simplex virus type 1, human coronavirus, feline calicivirus, and canine parvovirus, were reduced by 2.9- to 5.5-log10 within 30 s of SAIW exposure. Similarly, the culturability of three Gram-negative bacteria (Escherichia coli, Salmonella, and Legionella) dropped down by 1.9- to 4.9-log10 within 30 s of SAIW treatment. Mechanistically, treatment with SAIW was found to significantly decrease the binding and subsequent entry efficiencies of SARS-CoV-2 on Vero cells. Finally, we showed that this chlorine-free electrolytic ion water had no acute inhalation toxicity in mice, demonstrating that SAIW holds promise for a safer antiviral and antibacterial disinfectant.

Antimicrobial Terpenoids from South China Sea Soft Coral Lemnalia sp.

Chemical investigation of the South China Sea soft coral Lemnalia sp. afforded 13 structurally diverse terpenoids, including three new neolemnane sesquiterpene lineolemnene, E-G (1-3); a new aristolane-type sesquiterpenoid, 2-acetoxy-aristolane (4); four new decalin-type bicyclic diterpenes, named biofloranate A-D (5-8); a new serrulatane, named euplexaurene D (9); and a new aromadendrane-type diterpenoid cneorubin K (10), together with three known related compounds (11-13). The structures of the new compounds were elucidated by NMR spectroscopy, the Mosher's method, and ECD analysis. Compounds 1-13 were tested in a wide panel of biological assays. Lineolemnene J (3) showed weak cytotoxicity against the CCRF-CEM cancer cell line. The isolated new diterpenes, except euplexaurene D (9), demonstrated moderate antimicrobial activity against Bacillus subtilis and Staphylococcus aureus with a MIC of 4-64 µg/mL. Compound 2 exhibited a mild inhibitory effect against influenza A H1N1 virus (IC50 = 5.9 µM).

Untreated herpes simplex virus encephalitis without a fatal outcome.

Herpes simplex virus encephalitis (HSE) is the most common sporadic fatal encephalitis. Although timely administered acyclovir treatment decreases mortality, neuropsychiatric sequelae is still common among survivors. Magnetic resonance imaging is frequently utilized for the diagnosis of HSE, which typically involves temporal lobe(s) and can be mixed with brain tumors involving the same area. Here, we report a case of HSE, who received acyclovir with a delay of 90 days because of presumptive tumor diagnosis and survived with minimal sequelae.

The 3-O-sulfation of heparan sulfate modulates protein binding and lyase degradation.

Humans express seven heparan sulfate (HS) 3-O-sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3-O-sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3-O-sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.

Antiviral Potential of Sea Urchin Aminated Spinochromes against Herpes Simplex Virus Type 1.

Herpes simplex virus type 1 (HSV-1) is one of the most prevalent pathogens worldwide requiring the search for new candidates for the creation of antiherpetic drugs. The ability of sea urchin spinochromes-echinochrome A (EchA) and its aminated analogues, echinamines A (EamA) and B (EamB)-to inhibit different stages of HSV-1 infection in Vero cells and to reduce the virus-induced production of reactive oxygen species (ROS) was studied. We found that spinochromes exhibited maximum antiviral activity when HSV-1 was pretreated with these compounds, which indicated the direct effect of spinochromes on HSV-1 particles. EamB and EamA both showed the highest virucidal activity by inhibiting the HSV-1 plaque formation, with a selectivity index (SI) of 80.6 and 50.3, respectively, and a reduction in HSV-1 attachment to cells (SI of 8.5 and 5.8, respectively). EamA and EamB considerably suppressed the early induction of ROS due to the virus infection. The ability of the tested compounds to directly bind to the surface glycoprotein, gD, of HSV-1 was established in silico. The dock score of EchA, EamA, and EamB was -4.75, -5.09, and -5.19 kcal/mol, respectively, which correlated with the SI of the virucidal action of these compounds and explained their ability to suppress the attachment and penetration of the virus into the cells.

A review: Natural polysaccharides from medicinal plants and microorganisms and their anti-herpetic mechanism.

The infections caused by Herpes simplex viruses (HSV-1 and -2) are seriously endangering the health of all human beings. Once infected with these two viruses, it will cause life-long latency in the host, and the continuous recurrence of the infection will seriously affect the quality of life. Moreover, infections with HSV-1 and HSV-2 have been reported to make the body susceptible to other diseases, such as Alzheimer's disease and HIV. Thus, more attention should be paid to the development of novel anti-HSV drugs. Polysaccharides obtained from medicinal plants and microorganism (both land and sea) are reported to be promising anti-herpes substances. However, their antiviral mechanisms are complex and diverse, which includes direct inhibition of virus life cycle (Adsorption, penetration, genetic material and protein synthesis) and indirectly through improving the body's immunity. And each step of the research processes from extraction to structural analysis contributes to the result in terms of antiviral activity. Therefore, The complex mechanisms involved in the treatment of Herpes simplex infections makes development of new antiviral compounds is difficult. In this paper, the mechanisms of polysaccharides in the treatment of Herpes simplex infections, the research processes of polysaccharides and their potential clinical applications were reviewed.

Characterization of a Novel Alphaherpesvirus Isolated from the Fruit Bat Pteropus lylei in Vietnam.

Herpesviruses exist in nature within each host animal. Ten herpesviruses have been isolated from bats and their biological properties reported. A novel bat alphaherpesvirus, which we propose to name "Pteropus lylei-associated alphaherpesvirus (PLAHV)," was isolated from urine of the fruit bat Pteropus lylei in Vietnam and characterized. The entire genome sequence was determined to be 144,008 bp in length and predicted to include 72 genes. PLAHV was assigned to genus Simplexvirus with other bat alphaherpesviruses isolated from pteropodid bats in Southeast Asia and Africa. The replication capacity of PLAHV in several cells was evaluated in comparison with that of herpes simplex virus 1 (HSV-1). PLAHV replicated better in the bat-originated cell line and less in human embryonic lung fibroblasts than HSV-1 did. PLAHV was serologically related to another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from a Pteropus hypomelanus-related bat captured in Indonesia, but not with HSV-1. PLAHV caused lethal infection in mice. PLAHV was as susceptible to acyclovir as HSV-1 was. Characterization of this new member of bat alphaherpesviruses, PLAHV, expands the knowledge on bat-associated alphaherpesvirology.IMPORTANCE A novel bat alphaherpesvirus, Pteropus lylei-associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Vietnam. The whole-genome sequence was determined and was predicted to include 72 open reading frames in the 144,008-bp genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei, in Asia. This study expands the knowledge on bat-associated alphaherpesvirology.

The intracellular DNA sensors cGAS and IFI16 do not mediate effective antiviral immune responses to HSV-1 in human microglial cells.

Glia play a key role in immunosurveillance within the central nervous system (CNS) and can recognize a wide range of pathogen-associated molecular patterns (PAMPS) via members of multiple pattern recognition receptor (PRR) families. Of these, the expression of cytosolic/nuclear RNA and DNA sensors by glial cells is of particular interest as their ability to interact with intracellular nucleic acids suggests a critical role in the detection of viral pathogens. The recently discovered DNA sensors cyclic GMP-AMP synthase (cGAS) and interferon gamma-inducible protein 16 (IFI16) have been reported to be important for the recognition of DNA pathogens such as herpes simplex virus-1 (HSV-1) in peripheral human cell types, and we have recently demonstrated that human glia express cGAS and its downstream adaptor molecule stimulator of interferon genes (STING). Here, we have demonstrated that human microglial cells functionally express cGAS and exhibit robust constitutive IFI16 expression. While cGAS serves as a significant component in IRF3 activation and IFN-ß production by human microglial cells in response to foreign intracellular DNA, IFI16 is not required for such responses. Surprisingly, neither of these sensors mediate effective antiviral responses to HSV-1 in microglia, and this may be due, at least in part, to viral suppression of cGAS and/or IFI16 expression. As such, this ability may represent an important HSV immune evasion strategy in glial cells, and approaches that mitigate such suppression might represent a novel strategy to limit HSV-1-associated neuropathology.

Experimental Data Based Machine Learning Classification Models with Predictive Ability to Select in Vitro Active Antiviral and Non-Toxic Essential Oils.

In the last decade essential oils have attracted scientists with a constant increase rate of more than 7% as witnessed by almost 5000 articles. Among the prominent studies essential oils are investigated as antibacterial agents alone or in combination with known drugs. Minor studies involved essential oil inspection as potential anticancer and antiviral natural remedies. In line with the authors previous reports the investigation of an in-house library of extracted essential oils as a potential blocker of HSV-1 infection is reported herein. A subset of essential oils was experimentally tested in an in vitro model of HSV-1 infection and the determined IC50s and CC50s values were used in conjunction with the results obtained by gas-chromatography/mass spectrometry chemical analysis to derive machine learning based classification models trained with the partial least square discriminant analysis algorithm. The internally validated models were thus applied on untested essential oils to assess their effective predictive ability in selecting both active and low toxic samples. Five essential oils were selected among a list of 52 and readily assayed for IC50 and CC50 determination. Interestingly, four out of the five selected samples, compared with the potencies of the training set, returned to be highly active and endowed with low toxicity. In particular, sample CJM1 from Calaminta nepeta was the most potent tested essential oil with the highest selectivity index (IC50 = 0.063 mg/mL, SI > 47.5). In conclusion, it was herein demonstrated how multidisciplinary applications involving machine learning could represent a valuable tool in predicting the bioactivity of complex mixtures and in the near future to enable the design of blended essential oil possibly endowed with higher potency and lower toxicity.

Potential antiviral agents of Rosmarinus officinalis extract against herpes viruses 1 and 2.

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) belong to the herpesviridae family and cause neurological disorders by infecting the nervous system. The present study aimed to investigate the effects of Rosmarinus officinalis L. (rosemary) extract against HSV-1 and HSV-2 in vitro. The antioxidant activity of this extract was investigated by superoxide anion and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical assays. Rosemary extract was evaluated by an HSV-1 antiviral assay, in which viral replication in Vero cells was determined and quantified using a cytopathic effect assay. The present study showed that rosemary extract at 30 µg/ml caused 55% inhibition of HSV-1 plaques, whereas 40 µg/ml rosemary extract caused 65% inhibition of HSV-2 plaques. The extracts completely inhibited HSV-1 and HSV-2 plaque formation at 50 µg/ml. Scavenging activity of the superoxide anion radical was observed at 65.74 mg/ml, whereas 50% scavenging activity of the DPPH radical was observed at 67.34 mg/ml. These data suggest that rosemary extract may be suitable as a topical prophylactic or therapeutic agent for herpes viral infections. However, further research is required to elucidate the plant's active constituents, which may be useful in drug development.

HSV-1 infection and pathogenesis in the tree shrew eye following corneal inoculation.

Herpes simplex virus type I (HSV-1) infection causes inflammation in the cornea known as herpes simplex virus keratitis (HSK), a common but serious corneal disease. It is not entirely clear whether the virus during recurring infection comes from the trigeminal ganglia or the eye tissue, including the retina and ciliary ganglion. Because the tree shrew is closely related to primates and tree shrew eye anatomic structures are similar to humans, we studied HSV-1 corneal infection in the tree shrew. We found that HSK symptoms closely mimic those found in human HSK showing typical punctiform and dendritic viral keratitis during the acute infection period. Following the HSV-specific lesions, complications such as stromal scarring, corneal thickening (primary infection), opacity, and neovascularization were observed. In the tree shrew model, following ocular inoculation, the cornea becomes infected, and viral protein can be detected using anti-HSV-1 antibodies in the epithelial layer and retina neuronal ganglion cells. The HSV-1 transcripts, ICP0, ICP4, and LAT can be detected at 3 days post-infection (dpi), peaking at 5 dpi. After 2 weeks, ICP4 and ICP0 transcripts are reduced to a basal level, but the Latency Associated Transcripts (LATs) continue to accumulate. Interestingly, after the acute infection, we still detected abundant active HSV-1 in tree shrew eyes. Further, we found HSV-1 persistent in the ciliary ganglion and cornea. These findings are discussed in support of the tree shrew as a non-human primate HSK model, which could be useful for mechanistic studies of HSK.

Herpes Simplex Virus Growth, Preparation, and Assay.

The human herpesvirus family members, in particular herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2), are abundant and extremely contagious viruses with a high seroprevalence in the human population emphasizing the importance of studying their biology. Hence, the propagation and purification of virus stocks constitute a key element in laboratory work.

Engineering HSV-1 Vectors for Gene Therapy.

Virus vectors have been employed as gene transfer vehicles for various preclinical and clinical gene therapy applications and with the approval of Glybera (Alipogene tiparvovec) as the first gene therapy product as a standard medical treatment (Yla-Herttuala, Mol Ther 20:1831-1832, 2013), gene therapy has reached the status of being a part of standard patient care. Replication-competent herpes simplex virus (HSV) vectors that replicate specifically in actively dividing tumor cells have been used in Phase I-III human trials in patients with glioblastoma multiforme (GBM), a fatal form of brain cancer, and in malignant melanoma. In fact, Imlygic® (T-VEC, Talimogene laherparepvec, formerly known as OncoVex GM-CSF), displayed efficacy in a recent Phase-III trial when compared to standard GM-CSF treatment alone (Andtbacka et al., J Clin Oncol 31:sLBA9008, 2013), and has since become the first FDA-approved viral gene therapy product used in standard patient care (October 2015) (Pol et al., Oncoimmunology 5:e1115641, 2016). Moreover, increased efficacy was observed when Imlygic® was combined with checkpoint inhibitory antibodies as a frontline therapy for malignant melanoma (Ribas et al., Cell 170:1109-1119.e1110, 2017; Dummer et al., Cancer Immunol Immunother 66:683-695, 2017). In addition to the replication-competent oncolytic HSV vectors like T-VEC, replication-defective HSV vectors have been employed in Phase I-II human trials and have been explored as delivery vehicles for disorders such as pain, neuropathy and other neurodegenerative conditions. Research during the last decade on the development of HSV vectors has resulted in the engineering of recombinant vectors that are completely replication defective, nontoxic, and capable of long-term transgene expression in neurons. This chapter describes methods for the construction of recombinant genomic HSV vectors based on the HSV-1 replication-defective vector backbones, steps in their purification, and their small-scale production for use in cell culture experiments as well as preclinical animal studies.

Preparation of Herpes Simplex Virus Type 1 (HSV-1)-Based Amplicon Vectors.

Amplicon vectors, or amplicons, are defective, helper-dependent, herpes simplex virus type 1 (HSV-1)-based vectors. The main interest of amplicons as gene transfer tools stems from the fact that the genomes of these vectors do not carry protein-encoding viral sequences. Consequently, they are completely safe for the host and nontoxic for the infected cells. Moreover, the complete absence of virus genes provides a genomic space authorizing a very large payload, enough to accommodate foreign DNA sequences up to almost 150-kbp, the size of the HSV-1 genome. This transgene capacity can be used to deliver complete gene loci, including introns and exons, as well as long regulatory sequences conferring tissue-specific expression or stable maintenance of the transgene in proliferating cells. For many years the development of these vectors and their application in gene transfer experiments was hindered by the presence of contaminating toxic helper virus particles in the vector stocks. In recent years, however, two different methodologies have been developed that allow generating amplicon stocks either completely free of helper particles or only faintly contaminated with fully defective helper particles. This chapter describes these two methodologies.