Oncolytic HSV Vectors and Anti-Tumor Immunity.

The therapeutic promise of oncolytic viruses (OVs) rests on their ability to both selectively kill tumor cells and induce anti-tumor immunity. The potential of tumors to be recognized and eliminated by an effective anti-tumor immune response has been spurred on by the discovery that immune checkpoint inhibition can overcome tumor-specific cytotoxic T cell (CTL) exhaustion and provide durable responses in multiple tumor indications. OV-mediated tumor destruction is now recognized as a powerful means to assist in the development of anti-tumor immunity for two important reasons: (i) OVs, through the elicitation of an anti-viral response and the production of type I interferon, are potent stimulators of inflammation and can be armed with transgenes to further enhance anti-tumor immune responses; and (ii) lytic activity can promote the release of tumor-associated antigens (TAAs) and tumor neoantigens that function as in situ tumor-specific vaccines to elicit adaptive immunity. Oncolytic herpes simplex viruses (oHSVs) are among the most widely studied OVs for the treatment of solid malignancies, and Amgen's oHSV Imlygic® for the treatment of melanoma is the only OV approved in major markets. Here we describe important biological features of HSV that make it an attractive OV, clinical experience with HSV-based vectors, and strategies to increase applicability to cancer treatment.

Generation of an Oncolytic Herpes Simplex Viral Vector Completely Retargeted to the GDNF Receptor GFRα1 for Specific Infection of Breast Cancer Cells.

Oncolytic herpes simplex viruses (oHSV) are under development for the treatment of a variety of human cancers, including breast cancer, a leading cause of cancer mortality among women worldwide. Here we report the design of a fully retargeted oHSV for preferential infection of breast cancer cells through virus recognition of GFRα1, the cellular receptor for glial cell-derived neurotrophic factor (GDNF). GFRα1 displays a limited expression profile in normal adult tissue, but is upregulated in a subset of breast cancers. We generated a recombinant HSV expressing a completely retargeted glycoprotein D (gD), the viral attachment/entry protein, that incorporates pre-pro-GDNF in place of the signal peptide and HVEM binding domain of gD and contains a deletion of amino acid 38 to eliminate nectin-1 binding. We show that GFRα1 is necessary and sufficient for infection by the purified recombinant virus. Moreover, this virus enters and spreads in GFRα1-positive breast cancer cells in vitro and caused tumor regression upon intratumoral injection in vivo. Given the heterogeneity observed between and within individual breast cancers at the molecular level, these results expand our ability to deliver oHSV to specific tumors and suggest opportunities to enhance drug or viral treatments aimed at other receptors.

Cellular Antisilencing Elements Support Transgene Expression from Herpes Simplex Virus Vectors in the Absence of Immediate Early Gene Expression.

Inactivation of all herpes simplex virus (HSV) immediate early (IE) genes to eliminate vector cytotoxicity results in rapid silencing of the viral genome, similar to the establishment of HSV latency. We recently reported that silencing of a nonviral reporter cassette could be overcome in nonneuronal cells by positioning the cassette in the viral latency (LAT) locus between resident chromatin boundary elements. Here, we tested the abilities of the chicken hypersensitive site 4 insulator and the human ubiquitous chromatin opening element A2UCOE to promote transgene expression from an IE-gene-inactivated HSV vector. We found that A2UCOE was particularly active in nonneuronal cells and reduced reporter promoter occupancy by a repressive histone mark. We determined whether multiple transgenes could be expressed under the control of different promoters from different loci of the same virus. The results showed abundant coexpression of LAT-embedded and A2UCOE-flanked genes in nonneuronal cells. In addition, a third reporter gene without known protective elements was active in cultured rat sensory neurons. These findings indicate that cellular antisilencing sequences can contribute to the expression of multiple genes from separate promoters in fully IE gene-disabled HSV vectors, providing an opportunity for therapeutic applications requiring mutually independent expression of different gene products from a single vector.IMPORTANCE Gene therapy has now entered a phase of development in which a growing number of recessive single gene defects can be successfully treated by vector-mediated introduction of a wild-type copy of the gene into the appropriate tissue. However, many disease conditions, such as neurodegeneration, cancer, and inflammatory processes, are more complex, requiring either multiple gene corrections or provision of coordinated gene activities to achieve a therapeutic outcome. Although herpes simplex virus (HSV) vectors have the capacity to meet this need, the challenge has been to genetically engineer the HSV genome in a manner to prevent expression of any viral genes while retaining the ability to express multiple therapeutic transgenes under independent transcriptional control. Here, we show that non-HSV insulator elements can be applied to retain at least transient transgene activity from multiple viral loci, thereby opening the door for more complex gene therapy applications in the future.

Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity.

The design of highly defective herpes simplex virus (HSV) vectors for transgene expression in nonneuronal cells in the absence of toxic viral-gene activity has been elusive. Here, we report that elements of the latency locus protect a nonviral promoter against silencing in primary human cells in the absence of any viral-gene expression. We identified a CTCF motif cluster 5' to the latency promoter and a known long-term regulatory region as important elements for vigorous transgene expression from a vector that is functionally deleted for all five immediate-early genes and the 15-kb internal repeat region. We inserted a 16.5-kb expression cassette for full-length mouse dystrophin and report robust and durable expression in dystrophin-deficient muscle cells in vitro. Given the broad cell tropism of HSV, our design provides a nontoxic vector that can accommodate large transgene constructs for transduction of a wide variety of cells without vector integration, thereby filling an important void in the current arsenal of gene-therapy vectors.

Syncytial Mutations Do Not Impair the Specificity of Entry and Spread of a Glycoprotein D Receptor-Retargeted Herpes Simplex Virus.

Membrane fusion, which is the key process for both initial cell entry and subsequent lateral spread of herpes simplex virus (HSV), requires the four envelope glycoproteins gB, gD, gH, and gL. Syncytial mutations, predominantly mapped to the gB and gK genes, confer hyperfusogenicity on HSV and cause multinucleated giant cells, termed syncytia. Here we asked whether interaction of gD with a cognate entry receptor remains indispensable for initiating membrane fusion of syncytial strains. To address this question, we took advantage of mutant viruses whose viral entry into cells relies on the uniquely specific interaction of an engineered gD with epidermal growth factor receptor (EGFR). We introduced selected syncytial mutations into gB and/or gK of the EGFR-retargeted HSV and found that these mutations, especially when combined, enabled formation of extensive syncytia by human cancer cell lines that express the target receptor; these syncytia were substantially larger than the plaques formed by the parental retargeted HSV strain. We assessed the EGFR dependence of entry and spread separately by using direct entry and infectious center assays, respectively, and we found that the syncytial mutations did not override the receptor specificity of the retargeted viruses at either stage. We discuss the implications of these results for the development of more effective targeted oncolytic HSV vectors. IMPORTANCE: Herpes simplex virus (HSV) is investigated not only as a human pathogen but also as a promising agent for oncolytic virotherapy. We previously showed that both the initial entry and subsequent lateral spread of HSV can be retargeted to cells expressing tumor-associated antigens by single-chain antibodies fused to a receptor-binding-deficient envelope glycoprotein D (gD). Here we introduced syncytial mutations into the gB and/or gK gene of gD-retargeted HSVs to determine whether viral tropism remained dependent on the interaction of gD with the target receptor. Entry and spread profiles of the recombinant viruses indicated that gD retargeting does not abolish the hyperfusogenic activity of syncytial mutations and that these mutations do not eliminate the dependence of HSV entry and spread on a specific gD-receptor interaction. These observations suggest that syncytial mutations may be valuable for increasing the tumor-specific spreading of retargeted oncolytic HSV vectors.

Use of miRNA response sequences to block off-target replication and increase the safety of an unattenuated, glioblastoma-targeted oncolytic HSV.

Glioblastoma multiforme (GBM) is an aggressive brain cancer for which there is no effective treatment. Oncolytic HSV vectors (oHSVs) are attenuated lytic viruses that have shown promise in the treatment of human GBM models in animals, but their efficacy in early phase patient trials has been limited. Instead of attenuating the virus with mutations in virulence genes, we engineered four copies of the recognition sequence for miR-124 into the 3'UTR of the essential ICP4 gene to protect healthy tissue against lytic virus replication; miR-124 is expressed in neurons but not in glioblastoma cells. Following intracranial inoculation into nude mice, the miR-124-sensitive vector failed to replicate or show overt signs of pathogenesis. To address the concern that this safety feature may reduce oncolytic activity, we inserted the miR-124 response elements into an unattenuated, human receptor (EGFR/EGFRvIII)-specific HSV vector. We found that miR-124 sensitivity did not cause a loss of treatment efficiency in an orthotopic model of primary human GBM in nude mice. These results demonstrate that engineered miR-124 responsiveness can eliminate off-target replication by unattenuated oHSV without compromising oncolytic activity, thereby providing increased safety.

Novel mutations in gB and gH circumvent the requirement for known gD Receptors in herpes simplex virus 1 entry and cell-to-cell spread.

Both entry and cell-to-cell spread of herpes simplex virus (HSV) involve a cascade of cooperative interactions among the essential glycoproteins D, B, and H/L (gD, gB, and gH/gL, respectively) initiated by the binding of gD to a cognate HSV entry receptor. We previously reported that a variant (D285N/A549T) of glycoprotein B (gB:NT) enabled primary virus entry into cells that were devoid of typical HSV entry receptors. Here, we compared the activities of the gB:NT variant with those of a newly selected variant of glycoprotein H (gH:KV) and a frequently coselected gB variant (gB:S668N). In combination, gH:KV and gB:S668N enabled primary virus entry into cells that lacked established HSV entry receptors as efficiently as did gB:NT, but separately, each variant enabled only limited entry. Remarkably, gH:KV uniquely facilitated secondary virus spread between cells that lacked canonical entry receptors. Transient expression of the four essential entry glycoproteins revealed that gH:KV, but not gB:NT, induced fusion between cells lacking the standard receptors. Because the involvement of gD remained essential for virus spread and cell fusion, we propose that gH:KV mimics a transition state of gH that responds efficiently to weak signals from gD to reach the active state. Computational modeling of the structures of wild-type gH and gH:KV revealed relatively subtle differences that may have accounted for our experimental findings. Our study shows that (i) the dependence of HSV-1 entry and spread on specific gD receptors can be reduced by sequence changes in the downstream effectors gB and gH, and (ii) the relative roles of gB and gH are different in entry and spread.

Effective treatment of an orthotopic xenograft model of human glioblastoma using an EGFR-retargeted oncolytic herpes simplex virus.

Glioblastoma multiforme (GBM) remains an untreatable human brain malignancy. Despite promising preclinical studies using oncolytic herpes simplex virus (oHSV) vectors, efficacy in patients has been limited by inefficient virus replication in tumor cells. This disappointing outcome can be attributed in part to attenuating mutations engineered into these viruses to prevent replication in normal cells. Alternatively, retargeting of fully replication-competent HSV to tumor-associated receptors has the potential to achieve tumor specificity without impairment of oncolytic activity. Here, we report the establishment of an HSV retargeting system that relies on the combination of two engineered viral glycoproteins, gD and gB, to mediate highly efficient HSV infection exclusively through recognition of the abundantly expressed epidermal growth factor receptor (EGFR) on glioblastoma cells. We demonstrate efficacy in vitro and in a heterotopic tumor model in mice. Evidence for systemically administered virus homing to the tumor mass is presented. Treatment of orthotopic primary human GBM xenografts demonstrated prolonged survival with up to 73% of animals showing a complete response as confirmed by magnetic resonance imaging. Our study describes an approach to HSV retargeting that is effective in a glioma model and may be applicable to the treatment of a broad range of tumor types.

Oncolytic herpes simplex viruses designed for targeted treatment of EGFR-bearing tumors.

Oncolytic herpes simplex viruses (oHSVs) have emerged as leading cancer therapeutic agents. Effective oHSV virotherapy may ultimately require both intratumoral and systemic vector administration to target the primary tumor and distant metastases. An attractive approach to enhancing oHSV tumor specificity is engineering the virus envelope glycoproteins for selective recognition of and infection via tumor-specific cell surface proteins. We previously demonstrated that oHSVs could be retargeted to EGFR-expressing cells by the incorporation of a single-chain antibody (scFv) at the N terminus of glycoprotein D (gD). Here, we compared retargeted oHSVs generated by the insertion of scFv, affibody molecule, or VHH antibody ligands at different positions within the N terminus of gD. When compared to the scFv-directed oHSVs, VHH and affibody molecules mediated enhanced EGFR-specific tumor cell entry, spread and cell killing in vitro, and enabled long-term tumor-specific virus replication following intravenous delivery in vivo. Moreover, oHSVs retargeted via a VHH ligand reduced tumor growth upon intravenous injection and achieved complete tumor destruction after intratumoral injection. Systemic oHSV delivery is important for the treatment of metastatic disease, and our enhancements in targeted oHSV design are a critical step in creating an effective tumor-specific oHSVs for safe administration via the bloodstream.

Novel mutations in UL24 and gH rescue efficient infection of an HSV vector retargeted to TrkA.

Transductional targeting of herpes simplex virus (HSV)-based gene therapy vectors offers the potential for improved tissue-specific delivery and can be achieved by modification of the viral entry machinery to incorporate ligands that bind the desired cell surface proteins. The interaction of nerve growth factor (NGF) with tropomyosin receptor kinase A (TrkA) is essential for survival of sensory neurons during development and is involved in chronic pain signaling. We targeted HSV infection to TrkA-bearing cells by replacing the signal peptide and HVEM binding domain of glycoprotein D (gD) with pre-pro-NGF. This TrkA-targeted virus (KNGF) infected cells via both nectin-1 and TrkA. However, infection through TrkA was inefficient, prompting a genetic search for KNGF mutants showing enhanced infection following repeat passage on TrkA-expressing cells. These studies revealed unique point mutations in envelope glycoprotein gH and in UL24, a factor absent from mature particles. Together these mutations rescued efficient infection of TrkA-expressing cells, including neurons, and facilitated the production of a completely retargeted KNGF derivative. These studies provide insight into HSV vector improvements that will allow production of replication-defective TrkA-targeted HSV for delivery to the peripheral nervous system and may be applied to other retargeted vector studies in the central nervous system.

Gene therapy for the treatment of chronic peripheral nervous system pain.

Chronic pain is a major health concern affecting 80 million Americans at some time in their lives with significant associated morbidity and effects on individual quality of life. Chronic pain can result from a variety of inflammatory and nerve damaging events that include cancer, infectious diseases, autoimmune-related syndromes and surgery. Current pharmacotherapies have not provided an effective long-term solution as they are limited by drug tolerance and potential abuse. These concerns have led to the development and testing of gene therapy approaches to treat chronic pain. The potential efficacy of gene therapy for pain has been reported in numerous pre-clinical studies that demonstrate pain control at the level of the spinal cord. This promise has been recently supported by a Phase-I human trial in which a replication-defective herpes simplex virus (HSV) vector was used to deliver the human pre-proenkephalin (hPPE) gene, encoding the natural opioid peptides met- and leu-enkephalin (ENK), to cancer patients with intractable pain resulting from bone metastases (Fink et al., 2011). The study showed that the therapy was well tolerated and that patients receiving the higher doses of therapeutic vector experienced a substantial reduction in their overall pain scores for up to a month post vector injection. These exciting early clinical results await further patient testing to demonstrate treatment efficacy and will likely pave the way for other gene therapies to treat chronic pain.

Bispecific adapter-mediated retargeting of a receptor-restricted HSV-1 vector to CEA-bearing tumor cells.

The safety and efficacy of viral therapies for solid tumors can be enhanced by redirecting the virus infection to tumor-specific cell-surface markers. Successful retargeting of herpes simplex virus type 1 (HSV-1) has been achieved using vectors that carry a modified envelope glycoprotein D (gD) engineered to interact directly with novel receptors. In addition, soluble bridging molecules (adapters) have been used to link gD indirectly to cell-specific receptors. Here, we describe the development of an adapter connecting gD to the common tumor antigen carcinoembryonic antigen (CEA). The adapter consisted of a CEA-specific single-chain antibody fused to the gD-binding region of the gD receptor, herpes virus entry mediator (HVEM). We used this adapter in combination with a vector that is detargeted for recognition of the widely expressed gD receptor nectin-1, but retains an intact binding region for the less common HVEM. We show that the adapter enabled infection of HSV-resistant Chinese hamster ovary (CHO) cells expressing ectopic CEA and nectin-1/CEA-bearing human gastric carcinoma cells that are resistant to the vector alone. We observed cell-to-cell spread following adapter-mediated infection in vitro and reduced tumor growth in vivo, indicating that this method of vector retargeting may provide a novel strategy for tumor-specific delivery of tumoricidal HSV.

Evaluation of parameters for efficient purification and long-term storage of herpes simplex virus-based vectors.

Replication competent oncolytic herpes simplex virus (HSV) vectors have been used extensively to treat solid tumors with promising results. However, highly defective HSV vectors will be needed for applications that require sustained therapeutic gene expression in the absence of vector-related toxicity or inflammation. These vectors require complementing cell lines for their manufacture, creating significant challenges to achieve high yields of infectious virus particles. We recently described an improved upstream process for the production of a non-cytotoxic HSV vector for gene therapy applications. Here, we sought to optimize the downstream conditions for purification and long-term storage of the same vector, JΔNI5. We compared different methods to remove cellular impurities and concentrate the vector by monitoring both physical and biological titers, resulting in the establishment of optimal conditions for vector production. To optimize the long-term storage parameters for non-cytotoxic HSV vectors, we evaluated vector stability at low temperature and sensitivity to freeze-thaw cycles. We report that suboptimal purification and storage methods resulted in loss of vector viability. Our results describe effective and reproducible protocols for purification and storage of HSV vectors for pre-clinical studies.

A herpes simplex virus vector system for expression of complex cellular cDNA libraries.

Viral vector-based gene expression libraries from normal or diseased tissues offer opportunities to interrogate cellular functions that influence or participate directly in specific biological processes. Here we report the creation and characterization of a herpes simplex virus (HSV)-based expression library consisting of cDNAs derived from PC12 pheochromocytoma cells. A replication-defective HSV vector backbone was engineered to contain both a bacterial artificial chromosome (BAC) and the Invitrogen in vitro Gateway recombination system, creating DBAC-GW. A cDNA library was produced and transferred into the DBAC-GW genome by in vitro recombination and selection in bacteria to produce DBAC-L. DBAC-L contained at least 15,000 unique cDNAs, as shown by DNA array analysis of PCR-amplified cDNA inserts, representing a wide range of cancer- and neuron-related cellular functions. Transfection of the recombinant DBAC-L DNA into complementing animal cells produced more than 1 million DBAC-L virus particles representing the library genes. By microarray analysis of vector-infected cells, we observed that individual members of this vector population expressed unique PC12 cDNA-derived mRNA, demonstrating the power of this system to transfer and express a variety of gene activities. We discuss the potential utility of this and similarly derived expression libraries for genome-wide approaches to identify cellular functions that participate in complex host-pathogen interactions or processes related to disease and to cell growth and development.

A double mutation in glycoprotein gB compensates for ineffective gD-dependent initiation of herpes simplex virus type 1 infection.

Herpes simplex virus (HSV) entry into cells is triggered by the binding of envelope glycoprotein D (gD) to a specific receptor, such as nectin-1 or herpesvirus entry mediator (HVEM), resulting in activation of the fusion effectors gB and gH and virus penetration. Here we report the identification of a hyperactive gB allele, D285N/A549T, selected by repeat passage of a gD mutant virus defective for nectin-1 binding through cells that express a gD-binding-impaired mutant nectin-1. The gB allele in a wild-type virus background enabled the use of other nectins as virus entry receptors. In addition, combination of the mutant allele with an epidermal growth factor receptor (EGFR)-retargeted gD gene yielded dramatically increased EGFR-specific virus entry compared to retargeted virus carrying wild-type gB. Entry of the gB mutant virus into nectin-1-bearing cells was markedly accelerated compared to that of wild-type virus, suggesting that the gB mutations affect a rate-limiting step in entry. Our observations indicate that ineffective gD activation can be complemented by hypersensitization of a downstream component of the entry cascade to gD signaling.

Treatment of glioblastoma with current oHSV variants reveals differences in efficacy and immune cell recruitment.

Oncolytic herpes simplex viruses (oHSVs) have demonstrated efficient lytic replication in human glioblastoma tumors using immunodeficient mouse models, but early-phase clinical trials have reported few complete responses. Potential reasons for the lack of efficacy are limited vector potency and the suppressive glioma tumor microenvironment (TME). Here we compare the oncolytic activity of two HSV-1 vectors, a KOS-strain derivative KG4:T124 and an F-strain derivative rQNestin34.5v.1, in the CT2A and GL261N4 murine syngeneic glioma models. rQNestin34.5v1 generally demonstrated a greater in vivo viral burden compared to KG4:T124. However, both vectors were rapidly cleared from CT2A tumors, while virus remained ensconced in GL261N4 tumors. Immunological evaluation revealed that the two vectors induced similar changes in immune cell recruitment to either tumor type at 2 days after infection. However, at 7 days after infection, the CT2A microenvironment displayed the phenotype of an untreated tumor, while GL261N4 tumors exhibited macrophage and CD4+/CD8+ T cell accumulation. Furthermore, the CT2A model was completely resistant to virus therapy, while in the GL261N4 model rQNestin34.5v1 treatment resulted in enhanced macrophage recruitment, impaired tumor progression, and long-term survival of a few animals. We conclude that prolonged intratumoral viral presence correlates with immune cell recruitment, and both are needed to enhance anti-tumor immunity.

Generation of herpesvirus entry mediator (HVEM)-restricted herpes simplex virus type 1 mutant viruses: resistance of HVEM-expressing cells and identification of mutations that rescue nectin-1 recognition.

Both initial infection and cell-to-cell spread by herpes simplex virus type 1 (HSV-1) require the interaction of the viral glycoprotein D (gD) with an entry receptor on the cell surface. The two major HSV entry receptors, herpesvirus entry mediator (HVEM) and nectin-1, mediate infection independently but are coexpressed on a variety of cells. To determine if both receptors are active in these instances, we have established mutant viruses that are selectively impaired for recognition of one or the other receptor. In plaque assays, these viruses showed approximately 1,000-fold selectivity for the matched receptor over the mismatched receptor. Separate assays showed that each virus is impaired for both infection and spread through the mismatched receptor. We tested several human tumor cell lines for susceptibility to these viruses and observed that HT29 colon carcinoma cells are susceptible to infection by nectin-1-restricted virus but are highly resistant to HVEM-restricted virus infection, despite readily detectable HVEM expression on the cell surface. HVEM cDNA isolated from HT29 cells rendered HSV-resistant cells permissive for infection by the HVEM-restricted virus, suggesting that HT29 cells lack a cofactor for HVEM-mediated infection or express an HVEM-specific inhibitory factor. Passaging of HVEM-restricted virus on nectin-1-expressing cells yielded a set of gD missense mutations that each restored functional recognition of nectin-1. These mutations identify residues that likely play a role in shaping the nectin-1 binding site of gD. Our findings illustrate the utility of these receptor-restricted viruses in studying the early events in HSV infection.

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.

Protocol Optimization for the Production of the Non-Cytotoxic JΔNI5 HSV Vector Deficient in Expression of Immediately Early Genes.

Non-toxic herpes simplex virus (HSV) vectors can be generated by functional deletion of all immediate-early (IE) genes, providing a benign vehicle with potential for gene therapy. However, deletion of multiple IE genes raises manufacturing concerns and thus limits clinical application of these vectors. To address this issue, we previously developed a novel production cell line, called U2OS-ICP4/27, by lentiviral transduction of human osteosarcoma U2OS cells with two essential HSV IE genes, ICP4 and ICP27. To optimize the process of vector manufacturing on this platform, we evaluated several cell culture parameters of U2OS-ICP4/27 for high-titer and -quality production of non-toxic HSV vectors, revealing that the yields and functionality of these vectors can be significantly influenced by culturing conditions. We also found that several chemical compounds can enhance the replication of non-toxic HSV vectors and their release from producer cells into the supernatants. Notably, the vector produced by our optimized protocol displayed a greatly improved vector yield and quality and showed elevated transgene expression in cultures of primary dorsal root ganglion neurons. Taken together, our optimized production approach emerges as a relevant protocol for high-yield and high-quality preparation of non-toxic HSV-based gene therapy vectors.

Point Mutations in Retargeted gD Eliminate the Sensitivity of EGFR/EGFRvIII-Targeted HSV to Key Neutralizing Antibodies.

Effective oncolytic virotherapy may require systemic delivery, tumor targeting, and resistance to virus-neutralizing (VN) antibodies. Since herpes simplex virus (HSV) glycoprotein D (gD) is the viral attachment/entry protein and predominant VN target, we examined the impact of gD retargeting alone and in combination with alterations in dominant VN epitopes on virus susceptibility to VN antibodies. We compared the binding of a panel of anti-gD monoclonal antibodies (mAbs) that mimic antibody specificities in human HSV-immune sera to the purified ectodomains of wild-type and retargeted gD, revealing the retention of two prominent epitopes. Substitution of a key residue in each epitope, separately and together, revealed that both substitutions (1) blocked retargeted gD recognition by mAbs to the respective epitopes, and, in combination, caused a global reduction in mAb binding; (2) protected against fusion inhibition by VN mAbs reactive with each epitope in virus-free cell-cell fusion assays; and (3) increased the resistance of retargeted HSV-1 to these VN mAbs. Although the combined modifications of retargeted gD allowed bona fide retargeting, incorporation into virions was partially compromised. Our results indicate that stacking of epitope mutations can additively block retargeted gD recognition by VN antibodies but also that improvements in gD incorporation into virus particles may be required.