HSV-1 Mutant Research Articles

Antitumor efficacy of CRISPR/Cas9-engineered ICP6 mutant herpes simplex viruses in a mouse xenograft model for lung adenocarcinoma.

Oncolytic viruses (OVs), including oncolytic herpes simplex viruses (oHSVs), are promising therapeutics against cancer. Here, we report two ICP6-mutated HSVs (type I) generated by CRISPR/Cas9, rHSV1/∆RR (with ICP6 ribonucleotide reductase [RR]domain deleted) and rHSV1/∆ICP6 (with a complete deletion of ICP6), exhibiting potent antitumor efficacy against lung adenocarcinoma. Both the mutants showed strong cytotoxicity in vitro, comparable with the control viruses expressing intact ICP6, but in relatively lower titers. Moreover, these mutant viruses exhibited preferential killing ability against lung tumor cells rather than normal lung fibroblast cells. Further, unlike the control HSV-1 causing severe illness or death in the mouse model, the ICP6-mutated viruses did not induce significant pathogenicity but instead effectively reduced tumor burden in vivo and led to 100% survival of the animals, indicating notable antitumor activity and attenuated virulence. In addition, rHSV1/∆RR seemed to have even better antitumor efficacy than rHSV1/∆ICP6, albeit no statistical significance in inhibition of tumor volume. Histopathologically, rHSV1/∆RR induced massive neutrophil infiltration to the tumor microenvironment and consistently, triggered more antitumor immune and neutrophil chemotactic cytokines or higher expression levels of them (indicated by quantitative polymerase chain reaction and transcriptome analyses). These results demonstrate the anti-adenocarcinoma potential of the CRISPR/Cas9-engineered ICP6 mutant HSV1, especially the rHSV1/∆RR, which likely induces stronger innate antitumor immune response. Together, these findings may provide new valuable clues for further development of OV-based therapeutics against lung adenocarcinoma or other types of tumors.

Characterization of the Immunologic Phenotype of Dendritic Cells Infected With Herpes Simplex Virus 1.

Due to viral envelope glycoprotein D binding to cellular membrane HVEM receptor, HSV-1 can infect certain dendritic cells, which becomes an event in the viral strategy to interfere with the host’s immune system. We previously generated the HSV-1 mutant strain M6, which produced an attenuated phenotype in mice and rhesus monkeys. The attenuated M6 strain was used to investigate how HSV-1 infection of dendritic cells interferes with both innate and adaptive immunity. Our study showed that dendritic cells membrane HVEM receptors could mediate infection of the wild-type strain and attenuated M6 strain and that dendritic cells infected by both viruses in local tissues of animals exhibited changes in transcriptional profiles associated with innate immune and inflammatory responses. The infection of pDCs and cDCs by the two strains promoted cell differentiation to the CD103+ phenotype, but varied transcriptional profiles were observed, implying a strategy that the HSV-1 wild-type strain interferes with antiviral immunity, probably due to viral modification of the immunological phenotype of dendritic cells during processing and presentation of antigen to T cells, leading to a series of deviations in immune responses, ultimately generating the deficient immune phenotype observed in infected individuals in the clinical.

Efficacy of an HSV-1 Neuro-Attenuated Vaccine in Mice Is Reduced by Preventing Viral DNA Replication.

We previously isolated an HSV-1 mutant, KOS-NA, that contains two non-synonymous mutations in UL39. One of the mutations, resulting in an R950H amino acid substitution in ICP6, renders KOS-NA severely neuro-attenuated and significantly reduces HSV-1 latency. Vaccination of mice with KOS-NA prior to corneal challenge provides significant protection against HSV-1-mediated eye diseases even at a very low immunizing dose, indicating its utility as a vaccine scaffold. Because KOS-NA contains a neuro-attenuating mutation in a single gene, we sought to improve its safety by deleting a portion of the UL29 gene whose protein product, ICP8, is essential for viral DNA replication. Whereas KOS-NA reduced replication of HSV-1 challenge virus in the corneal epithelium and protected mice against blepharitis and keratitis induced by the challenge virus, KOS-NA/8- and an ICP8- virus were significantly less efficacious except at higher doses. Our results suggest that the capacity to replicate, even at significantly reduced levels compared with wild-type HSV-1, may be an important feature of an effective vaccine. Means to improve safety of attenuated viruses as vaccines without compromising efficacy should be sought.

The HSV1 Tail-Anchored Membrane Protein pUL34 Contains a Basic Motif That Supports Active Transport to the Inner Nuclear Membrane Prior to Formation of the Nuclear Egress Complex

Herpes simplex virus type 1 nucleocapsids are released from the host nucleus by a budding process through the nuclear envelope called nuclear egress. Two viral proteins, the integral membrane proteins pUL34 and pUL31, form the nuclear egress complex at the inner nuclear membrane, which is critical for this process. The nuclear import of both proteins ensues separately from each other: pUL31 is actively imported through the central pore channel, while pUL34 is transported along the peripheral pore membrane. With this study, we identified a functional bipartite NLS between residues 178 and 194 of pUL34. pUL34 lacking its NLS is mislocalized to the TGN but retargeted to the ER upon insertion of the authentic NLS or a mimic NLS, independent of the insertion site. If co-expressed with pUL31, either of the pUL34-NLS variants is efficiently, although not completely, targeted to the nuclear rim where co-localization with pUL31 and membrane budding seem to occur, comparable to the wild-type. The viral mutant HSV1(17+)Lox-UL34-NLS mt is modestly attenuated but viable and associated with localization of pUL34-NLS mt to both the nuclear periphery and cytoplasm. We propose that targeting of pUL34 to the INM is facilitated by, but not dependent on, the presence of an NLS, thereby supporting NEC formation and viral replication.

Characteristics of Helicase-Primase Inhibitor Amenamevir-Resistant Herpes Simplex Virus.

The antiherpetic drug amenamevir (AMNV) inhibits the helicase-primase complex of herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus directly as well as inhibiting the replication of these viruses. Although several mutated HSV viruses resistant to helicase-primase inhibitors have been reported, the mutations contributing to the resistance remain unclear, as recombinant viruses containing a single mutation have not been analyzed. We obtained AMNV-resistant viruses with amino acid substitutions by several passages under AMNV treatment. Twenty HSV-1 and 19 HSV-2 mutants with mutation(s) in UL5 helicase and/or UL52 primase, but not in cofactor UL8, were isolated. The mutations in UL5 were located downstream of motif IV, with UL5 K356N in HSV-1 and K355N in HSV-2, in particular, identified as having the highest frequency, which was 9/20 and 9/19, respectively. We generated recombinant AMNV-resistant HSV-1 with a single amino acid substitution using bacterial artificial chromosome (BAC) mutagenesis. As a result, G352C in UL5 helicase and F360C/V and N902T in UL52 primase were identified as novel mutations. The virus with K356N in UL5 showed 10-fold higher AMNV resistance than did other mutants and showed equivalent viral growth in vitro and virulence in vivo as the parent HSV-1, although other mutants showed attenuated virulence. All recombinant viruses were susceptible to the other antiherpetic drugs, acyclovir and foscarnet. In conclusion, based on BAC mutagenesis, this study identified, for the first time, mutations in UL5 and UL52 that contributed to AMNV resistance and found that a mutant with the most frequent K356N mutation in HSV-1 maintained viral growth and virulence equivalent to the parent virus.

Pathogenesis and chemotherapy of a thymidine kinase deficient mutant strain of HSV‐1 in a murine zosteriform infection model.

Type I and II Herpesvirus simplex virus [HSV] are contagious and cause oral (cold sores) and genital ulcers (genital HSV) before becoming latent in trigeminal ganglia (oral HSV) or sacral ganglia (genital HSV), and can reactivate and cause disease almost at the same spot of the initial virus exposure. Acyclovir is one of the gold standard treatments for labial and genital HSV for decades. Topical Acyclovir and penciclovir and their prodrugs act on thymidine kinase (TK) and are viral DNA chain inhibitor agents and hence inhibit virus replication. In TK deletion mutants, drugs which interact with TK enzyme and inhibit virus replication do not have any inhibitory effect. TK deletion mutants also replicate at slower rates and therefore take longer to make plaques in tissue culture compared to wild type strains. Resistant virus could also arise due to suboptimal treatment regimens although it is not a great problem currently in immunocompetent patients. However resistant HSV could be a major challenge to treat in immunocompromised and immunosuppressed patients as not many options are available to use antiviral drugs whose mode of action is to work on TK inhibition. In this study pathogenesis of TK deletion mutant resistant strains of HSV were used in a immunocompetent neck-ear zosteriform infection mouse model and efficacy of prodrugs on zoster spread was studied. Resistant strains of HSV mutants received were originally raised in vitro and plaques purified. These strains were then grown in tissue culture and working stock was produced. We observed that rate of replication of these mutant viruses was slower as they took longer to make plaque in tissue culture compared to wild type strains. Resistant strain of HSV were used in the immunocompetent murine zosteriform infection model along with the wild strain and efficacy of prodrugs was studied to see if murine immunocompetent natural defense will have any effect on the pathogenicity and if antiviral agents have any effect in vivo. A murine neck ear zosteriform infection model was used and development of zosteriform lesion were noted on the ear pinna along with ear thickness and body weight. Mutant viruses show delayed zosteriform spread. It was noticed that though lesions were not completely inhibited by prodrugs, zosteriform lesion and intensity was lower after antiviral treatment. This was not expected so we tend to believe that either immunocompetent immune defense system of mice were providing extra help to reduce the virus lesion or a possibility of helper virus in the murine model. In order to see the effect of TK activity in resistant mutants original stock was further analyzed by radiolabeled C-14 TK assay to confirm the deletion mutants status. In this communication In vivo results of zoster spread, radiolabeled C-14 TK assays to identify TK deletion mutant, purified cloning, other antiviral which can have an effect on TK deletion resistant HSV in immunocompromised and immunosuppressed patients will be discussed.

Role of the Herpes Simplex Virus CVSC Proteins at the Capsid Portal Vertex.

The packaging of DNA into preformed capsids is a critical step during herpesvirus infection. For herpes simplex virus, this process requires the products of seven viral genes: the terminase proteins pUL15, pUL28, and pUL33; the capsid vertex-specific component (CVSC) proteins pUL17 and pUL25; and the portal proteins pUL6 and pUL32. The pUL6 portal dodecamer is anchored at one vertex of the capsid by interactions with the adjacent triplexes as well as helical density attributed to the pUL17 and pUL25 subunits of the CVSC. To define the roles and structures of the CVSC proteins in virus assembly and DNA packaging, we isolated a number of recombinant viruses expressing pUL25, pUL17, and pUL36 fused with green or red fluorescent proteins as well as viruses with specific deletions in the CVSC genes. Biochemical and structural studies of these mutants demonstrated that (i) four of the helices in the CVSC helix bundle can be attributed to two copies each of pUL36 and pUL25, (ii) pUL17 and pUL6 are required for capsid binding of the terminase complex in the nucleus, (iii) pUL17 is important for determining the site of the first cleavage reaction generating replicated genomes with termini derived from the long-arm component of the herpes simplex virus 1 (HSV-1) genome, (iv) pUL36 serves no direct role in cleavage/packaging, (v) cleavage and stable packaging of the viral genome involve an ordered interaction of the terminase complex and pUL25 with pUL17 at the portal vertex, and (vi) packaging of the viral genome results in a dramatic displacement of the portal.IMPORTANCE Herpes simplex virus 1 (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. A critical step during productive HSV-1 infection is the cleavage and packaging of replicated, concatemeric viral DNA into preformed capsids. A key knowledge gap is how the capsid engages the replicated viral genome and the subsequent packaging of a unit-length HSV genome. Here, biochemical and structural studies focused on the unique portal vertex of wild-type HSV and packaging mutants provide insights into the mechanism of HSV genome packaging. The significance of our research is in identifying the portal proteins pUL6 and pUL17 as key viral factors for engaging the terminase complex with the capsid and the subsequent cleavage, packaging, and stable incorporation of the viral genome in the HSV-1 capsid.

HSV1 VP1-2 deubiquitinates STING to block type I interferon expression and promote brain infection.

Herpes simplex virus (HSV) is the main cause of viral encephalitis in the Western world, and the type I interferon (IFN) system is important for antiviral control in the brain. Here, we have compared Ifnb induction in mixed murine brain cell cultures by a panel of HSV1 mutants, each devoid of one mechanism to counteract the IFN-stimulating cGAS-STING pathway. We found that a mutant lacking the deubiquitinase (DUB) activity of the VP1-2 protein induced particularly strong expression of Ifnb and IFN-stimulated genes. HSV1 ΔDUB also induced elevated IFN expression in murine and human microglia and exhibited reduced viral replication in the brain. This was associated with increased ubiquitination of STING and elevated phosphorylation of STING, TBK1, and IRF3. VP1-2 associated directly with STING, leading to its deubiquitination. Recruitment of VP1-2 to STING was dependent on K150 of STING, which was ubiquitinated by TRIM32. Thus, the DUB activity of HSV1 VP1-2 is a major viral immune-evasion mechanism in the brain.

Characterization of immune response in mice vaccinated with highly efficacious vaccine 0ΔNLS against HSV-1

Abstract Using an experimental mouse model we compared the host immune response of a mutant HSV-1, termed 0ΔNLS (deficient in the nuclear location signal of ICP0), a highly efficacious prophylactic vaccine against ocular HSV-1 infection, to that of the parental HSV-1 virus, termed GFP105 (KOS background). C57BL/6 mice were infected in the footpad (FP) with 1×105 plaque forming units of virus and at times post infection (PI), organized lymphoid tissues were analyzed for changes in the immune profile. 0ΔNLS infected animals were more efficient in clearance of virus from the FP and generated a more robust chemokine responses and myeloid cell infiltration in FP at early time points (day 1–3 PI) compared to GFP105 infected mice. By day 7 PI, there were significantly more GC B cells and isotype switched B cells in the PLN of mice infected with 0ΔNLS in comparison to GFP105 infected animals. In addition, an increased number of follicular helper CD4 T cells along with enhanced IL-21 cytokine production and an increased number of antigen-specific CD8 T cells against major and sub-major HSV-1 epitopes was noted. In contrast, by day 21 PI, no difference in T cell and B cell subset was found in the PLN comparing the two groups of infected mice. Furthermore, mice which were re-exposed to 0ΔNLS 21 days PI showed an increased number of metabolically active plasma cells from the bone marrow and secretion of IgG by memory B cells after pre-activation compared to those re-infected with GFP105. In addition, an increased number of IFN-ɤ secreting, HSV-1-specific CD8 T cells from the re-exposed 0ΔNLS mice was observed as well. Collectively, these results warrant further studies to fully understand the efficacy of the 0ΔNLS virus as an effective vaccine against ocular HSV-1 infection.

HSV-1 triggers paracrine fibroblast growth factor response from cortical brain cells via immediate-early protein ICP0

BackgroundHerpes simplex virus-1 (HSV-1) infections of the central nervous system (CNS) can result in HSV-1 encephalitis (HSE) which is characterized by severe brain damage and long-term disabilities. Different cell types including neurons and astrocytes become infected in the course of an HSE which leads to an activation of glial cells. Activated glial cells change their neurotrophic factor profile and modulate inflammation and repair. The superfamily of fibroblast growth factors (FGFs) is one of the largest family of neurotrophic factors comprising 22 ligands. FGFs induce pro-survival signaling in neurons and an anti-inflammatory answer in glial cells thereby providing a coordinated tissue response which favors repair over inflammation. Here, we hypothesize that FGF expression is altered in HSV-1-infected CNS cells.MethodWe employed primary murine cortical cultures comprising a mixed cell population of astrocytes, neurons, microglia, and oligodendrocytes. Astrocyte reactivity was morphometrically monitored by an automated image analysis algorithm as well as by analyses of A1/A2 marker expression. Altered FGF expression was detected by quantitative real-time PCR and its paracrine FGF activity. In addition, HSV-1 mutants were employed to characterize viral factors important for FGF responses of infected host cells.ResultsAstrocytes in HSV-1-infected cortical cultures were transiently activated and became hypertrophic and expressed both A1- and A2-markers. Consistently, a number of FGFs were transiently upregulated inducing paracrine neurotrophic signaling in neighboring cells. Most prominently, FGF-4, FGF-8, FGF-9, and FGF-15 became upregulated in a switch-on like mechanism. This effect was specific for CNS cells and for a fully functional HSV-1. Moreover, the viral protein ICP0 critically mediated the FGF switch-on mechanism.ConclusionsHSV-1 uses the viral protein ICP0 for the induction of FGF-expression in CNS cells. Thus, we propose that HSV-1 triggers FGF activity in the CNS for a modulation of tissue response upon infection.

ATIM-04. PHASE I IMMUNOVIROTHERAPY TRIAL OF IL-12 EXPRESSING HSV-1 (M032) IN PET DOGS WITH SPONTANEOUS HIGH GRADE GLIOMAS: A PRELIMINARY REPORT

Abstract Malignant gliomas are the most common primary brain tumors in humans, accounting for approximately 30% of all primary central nervous system (CNS) tumors in adults. Incidence and outcomes of gliomas in pet dogs are similar to humans. Previous methods of studying gliomas in rodent models do not adequately represent several features that define human cancers, most notably the sporadic nature of tumor development. Murine models lack intra-tumoral heterogeneity or hosts do not have an intact immune system. To overcome these rodent model deficiencies, many investigators have used canine models to study human diseases. We describe a prospective, phase I clinical trial in canine patients with sporadic high-grade gliomas to evaluate M032, a mutant HSV-1 expressing IL-12, alone or combined with a checkpoint inhibitor (Indoximod) to assess safety, tolerability and efficacy. M032 has been shown to infect and kill glioma cells, producing a virus and tumor cell antigen-rich debris field; will provide an increased adjuvant effect from released viral DNA recognized by TLR-9 receptors; and express IL-12 locally, stimulating activation of TH1 lymphocytes. The intratumoral inflammatory cell infiltration is expected to result in immune-related anti-viral responses with cross-epitope spreading to tumor antigens. Indoximod immunomodulation will be tested in latter cohorts to prolong anti-tumor responses. To date, six canines with high-grade gliomas have received intratumoral M032 without virus-related toxicities. Resected tumor and serial blood samples are being cryopreserved for analysis of lymphoid and monocyte/myeloid subset markers. This same virus is being tested in a phase 1 clinical trial in humans with high-grade malignant gliomas; eight human patients have received treatment at this writing without virus-related toxicities. The concurrent prosecution of both trials allows unprecedented real time comparison of safety and efficacy of immunovirotherapy as a stringent test of suitability of the dog as a valid and informative model of human brain tumors.

1365P - Final results from phase II of combination with canerpaturev (formerly HF10), an oncolytic viral immunotherapy, and ipilimumab in unresectable or metastatic melanoma in second-or later line treatment

1365P - Final results from phase II of combination with canerpaturev (formerly HF10), an oncolytic viral immunotherapy, and ipilimumab in unresectable or metastatic melanoma in second-or later line treatment

Synthesis of 4′-substituted 2′-deoxy-4′-thiocytidines and its evaluation for antineoplastic and antiviral activities

4′-Azido- (7), 4′-C-fluoromethyl- (8) 4′-C-ethynyl- (9) and 4′-C-cyano- (10) 2′-deoxy-4′-thiocytidines have been synthesized. In this study, it was found that the isolated yield of 4′-thiouracil nucleoside 13 in a Lewis acid-promoted Vorbrüggen-type glycosidation utilizing 12 was better than that of the electrophilic glycosidation reaction between silylated uracil and 11. This improved result prompted us to perform the glycosidation utilizing 36 and 43 for the synthesis of 37 and 44. Introduction of the azido group was carried out by nucleophilic substitution in the 4′-benzoyloxy derivative 22a. On the other hand, 9 and 10 were synthesized by way of the chemical manipulation of the hydroxymethyl group at the 4′-position of 46.Evaluation of the antineoplastic activity of 2 and 7–10 against human B-cell (CCRF-SB) and T-cell leukemia (Molt-4) cell lines revealed that 4′-azido- (7) and 4′-C-fluoromethyl- (8) derivatives exhibited cytotoxic activity whereas no cytotoxicity was observed in the 4′-C-ethynyl- (9) and 4′-C-cyano- (10) derivatives as well as the parent compound 2. Compound 7 was also found to possess promising antiviral activity against VZV and HSV-1 without any cytotoxity against HEL host cells. It is noteworthy that 7 exhibited potent inhibitory activities against the thymidine kinase-deficient (TK−) mutant of VZV and HSV-1.

Hidden regulation of herpes simplex virus 1 pre-mRNA splicing and polyadenylation by virally encoded immediate early gene ICP27.

In contrast to human cells, very few HSV-1 genes are known to be spliced, although the same pre-mRNA processing machinery is shared. Here, through global analysis of splice junctions in cells infected with HSV-1 and an HSV-1 mutant virus with deletion of infectious cell culture protein 27 (ICP27), one of two viral immediate early (IE) genes essential for viral replication, we identify hundreds of novel alternative splice junctions mapping to both previously known HSV-1 spliced genes and previously unknown spliced genes, the majority of which alter the coding potential of viral genes. Quantitative and qualitative splicing efficiency analysis of these novel alternatively spliced genes based on RNA-Seq and RT-PCR reveals that splicing at these novel splice sites is efficient only when ICP27 is absent; while in wildtype HSV-1 infected cells, the splicing of these novel splice junctions is largely silenced in a gene/sequence specific manner, suggesting that ICP27 not only promotes accumulation of ICP27 targeted transcripts but also ensures correctness of the functional coding sequences through inhibition of alternative splicing. Furthermore, ICP27 toggles expression of ICP34.5, the major viral neurovirulence factor, through inhibition of splicing and activation of a proximal polyadenylation signal (PAS) in the newly identified intron, revealing a novel regulatory mechanism for expression of a viral gene. Thus, through the viral IE protein ICP27, HSV-1 co-opts both splicing and polyadenylation machinery to achieve optimal viral gene expression during lytic infection. On the other hand, during latent infection when ICP27 is absent, HSV-1 likely takes advantages of host splicing machinery to restrict expression of randomly activated antigenic viral genes to achieve immune evasion.

Synthetic lethality of cytolytic HSV-1 in cancer cells with ATRX and PML deficiency.

ABSTRACTCancers that utilize the alternative lengthening of telomeres (ALT) mechanism for telomere maintenance are often difficult to treat and have a poor prognosis. They are also commonly deficient for expression of ATRX protein, a repressor of ALT activity, and a component of promyelocytic leukemia nuclear bodies (PML NBs) that are required for intrinsic immunity to various viruses. Here, we asked whether ATRX deficiency creates a vulnerability in ALT cancer cells that could be exploited for therapeutic purposes. We showed in a range of cell types that a mutant herpes simplex virus type 1 (HSV-1) lacking ICP0, a protein that degrades PML NB components including ATRX, was ten- to one thousand-fold more effective in infecting ATRX-deficient cells than wild-type ATRX-expressing cells. Infection of co-cultured primary and ATRX-deficient cancer cells revealed that mutant HSV-1 selectively killed ATRX-deficient cells. Sensitivity to mutant HSV-1 infection also correlated inversely with PML protein levels, and we showed that ATRX upregulates PML expression at both the transcriptional and post-transcriptional levels. These data provide a basis for predicting, based on ATRX or PML levels, which tumors will respond to a selective oncolytic herpesvirus.

Role of Herpes Simplex Virus Type 1 (HSV-1) Glycoprotein K (gK) Pathogenic CD8+ T Cells in Exacerbation of Eye Disease.

HSV-1-induced corneal scarring (CS), also broadly referred to as Herpes Stromal Keratitis (HSK), is the leading cause of infectious blindness in developed countries. It is well-established that HSK is in fact an immunopathological disease. The contribution of the potentially harmful T cell effectors that lead to CS remains an area of intense study. Although the HSV-1 gene(s) involved in eye disease is not yet known, we have demonstrated that gK, which is one of the 12 known HSV-1 glycoproteins, has a crucial role in CS. Immunization of HSV-1 infected mice with gK, but not with any other known HSV-1 glycoprotein, significantly exacerbates CS, and dermatitis. The gK-induced eye disease occurs independently of the strain of the virus or mouse. HSV-1 mutants that lack gK are unable to efficiently infect and establish latency in neurons. HSV-1 recombinant viruses expressing two additional copies of the gK (total of three gK genes) exacerbated CS as compared with wild type HSV-1 strain McKrae that contains one copy of gK. Furthermore, we have shown that an 8mer (ITAYGLVL) within the signal sequence of gK enhanced CS in ocularly infected BALB/c mice, C57BL/6 mice, and NZW rabbits. In HSV-infected “humanized” HLA-A*0201 transgenic mice, this gK 8mer induced strong IFN-γ-producing cytotoxic CD8+ T cell responses. gK induced CS is dependent on gK binding to signal peptide peptidase (SPP). gK also binds to HSV-1 UL20, while UL20 binds GODZ (DHHC3) and these quadruple interactions are required for gK induced pathology. Thus, potential therapies might include blocking of gK-SPP, gK-UL20, UL20-GODZ interactions, or a combination of these strategies.

Temperature-sensitive origin-binding protein as a tool for investigations of herpes simplex virus activities in vivo.

While it is fairly clear that herpes simplex virus (HSV) DNA replication requires at least seven virus-encoded proteins in concert with various host cell factors, the mode of this process in infected cells is still poorly understood. Using HSV-1 mutants bearing temperature-sensitive (ts) lesions in the UL9 gene, we previously found that the origin-binding protein (OBP), a product of the UL9 gene, is only needed in the first 6 hours post-infection. As this finding was just a simple support for the hypothesis of a biphasic replication mode, we became convinced through these earlier studies that the mutants tsR and tsS might represent suitable tools for more accurate investigations in vivo. However, prior to engaging in highly sophisticated research projects, knowledge of the biochemical features of the mutated versions of OBP appeared to be essential. The results of our present study demonstrate that (i) tsR is most appropriate for cell biological studies, where only immediate early and early HSV gene products are being expressed without the concomital viral DNA replication, and (ii) tsS is a prime candidate for the analysis of HSV DNA replication processes because of its reversibly thermosensitive OBP-ATPase, which allows one to switch on the initiation of DNA synthesis precisely.

1267P - Topline results from phase II of combination treatment with canerpaturev (HF10), an oncolytic viral immunotherapy, and ipilimumab in patients with unresectable or metastatic melanoma after anti-PD-1 therapy

Background: Canerpaturev (C-REV, formerly HF10) is an oncolytic, spontaneous mutant of HSV-1, and is one of immunotherapies that combine direct tumor cell killing with immune modulation. We report the safety and antitumor activity data of Phase II multicenter trial of C-REV in combination with ipilimumab (ipi) in melanoma. Methods: The Phase II was to determine the efficacy and safety of i.t. C-REV and i.v. ipi. Key entry criteria: age ≥ 20 yrs, ECOG PS ≤ 2, Stage IIIB, IIIC or IV unresectable melanoma, who received prior therapies and had measurable non-visceral lesion(s) suitable for injection. C-REV was injected into each tumor (1 x 107 TCID50/mL/dose, up to 5mL); 4 injections q1wk; then up to 15 injections q3wk. Four ipi infusions (3 mg/kg) were administered at q3wk. AEs were graded per CTCAE 4.0. Tumor responses were assessed per irRC and mWHO at 6, 12, 18 and 24 wks. Primary endpoint was Best Overall Response Rate (BORR) by irRC at 24 wks. Results: Of 28 pts enrolled and treated as of the data cut-off 04 May 2018: 43% men, 31 to 81 yrs, disease stage 7% IIIB, 29% IIIC and 64% IV, and disease type 39% acral lentiginous and 21% mucosal melanoma. All pts were received prior cancer therapies: 89% PD-1 monotherapy, 7% DAVFeron and 11% DTIC. 12% had ≥G3 drug-related AEs; Hepatic function disorder, Malaise, Hyponatraemia, Constipation, Nausea, Toxicoderma, Adrenal insufficiency, and Muscle weakness lower limb. Of 27 efficacy evaluable pts, BORR by irRC at 24 wks was 7% and disease stability rate was 56%. One patient had PR in injected lesions and systemic lesions also showed the response after 6 wks of the combo therapy. Conclusions: The combination of C-REV with ipi did not show the exacerbate ipi toxicity, and had a favorable benefit/risk profile. The encouraging antitumor activity was observed in Japanese pts who had received prior therapies such as PD-1. It is recently well-known that the response to ipi after anti-PD-1 therapy was unsatisfactory and associated with a high frequency of severe irAEs, in particular Asian populations. From this study, C-REV+ipi therapy has potential to become a new 2nd line treatment for melanoma. Clinical trial identification: NCT03153085. Legal entity responsible for the study: Takara Bio Inc. Funding: Takara Bio Inc. Disclosure: T. Isei: Honoraria: Novartis, Toray Industries, GlaxoSmithKline, Daiichi Sankyo, Maruho, Pola Pharma, BMS UK, Ono Pharmaceuticals, MSD China; Consulting or advisory role: Novartis, BMS UK, Ono Pharmaceuticals, Merck Serono Ltd, MSD China; Research funding: Nocartis, Takara Bio, Array BioPharma, Amgenm, BMS UK, Ono Pharmaceuticals, MSD China. K. Yokota, H. Uchi, H. Ihn, T. Inozume, S. Fukushima: Research funding: Takara Bio. H. Uhara: Honoraria: MSD, Chugai Pharma, Novartis, Pola Pharma, Maruho, BMS UK, Ono Pharmaceuticals; Consulting or advisory role: Chugai Pharma, Novartis, MSD, Kyowa Hakko Kirin, BMS UK, Ono Pharmaceuticals; Speakers' bureau: Chugai Pharma, Novartis, MSD, Kyowa Hakko Kirin, Mitsubisi Tanabe Pharma, Pola Pharma, BMS UK, Ono Pharmaceuticals, Janssen Inc. Pharmaceuticals; Research funding: Kyowa Hakko Kirin, Mitsubishi Tanabe Pharma, Eisai, Abbvie, Maruho, Daiichi Sankyo, Tsumura&Co., Pola Pharma, Mochida Pharmaceutical Co. Ltd., Nihonlayaku, Torii Pharmaceutical, Kaken Pharmaceutical, Takara Bio, Ono Pharmaceuticals, Taiho Pharmaceutical. Y. Fujisawa: Consulting or advisory role: Novartis; Research funding: Maruho, Eisai, Chugai Pharma, Novartis, Toray Industries, Takara Bio, BMS UK, Ono Pharmaceuticals; Patients, Royalities, Other intellectural property: Novartis. T. Takenouchi: Speakers' bureau: MSD, Chugai Pharma, Novartis, BMS UK, Ono Pharmaceuticals; Research Funding: Takara Bio. Y. Kiyohara: Honoraria: MSD, Novartis, Chugai Pharma, Toray Industries, Takara Bio, Amgen, BMS UK, Ono Pharmaceuticals, Boehringer Ingelheim International GmbH, Merck Serono Ltd; Research funding: MSD, Takara Bio, BMS UK, ONO Pharmaceuticals, Merck Serono Ltd; Travel, accommodations, expenses: Ono Pharmaceuticals. H. Saruta: Speakers' bureau: Kyowa Hakko Kirin, Ono Pharmaceuticals; Research funding: Takara Bio, MSD; Travel, accommodations, expenses: Novartis, Minophargen Pharmaceuticals. D. Watanabe: Consulting or advisory role: Maruho, Japan Vaccine; Speakers' Bureau: Maruho; Research funding: Maruho, Sanofi, Nihon Zouki, Daiichi Sankyo, Takara Bio. A. Takahashi: Honoraria: Novartis, BMS UK, Ono Pharmaceuticals; Research funding: Novartis, Takara Bio, BMS UK, Ono Pharmaceuticals. M. Tanaka: Employee: Takara Bio. N. Yamazaki: Honoraria: Novartis, MSD, BMS UK, Ono Pharmaceuticals; Consulting or advisory role: Novartis, MSD, BMS UK, Ono Pharmaceuticals; Research funding: Novartis, MSD, Takara Bio, BMS UK, Ono Pharmaceuticals.

A VP26-mNeonGreen Capsid Fusion HSV-2 Mutant Reactivates from Viral Latency in the Guinea Pig Genital Model with Normal Kinetics.

Fluorescent herpes simplex viruses (HSV) are invaluable tools for localizing virus in cells, permitting visualization of capsid trafficking and enhancing neuroanatomical research. Fluorescent viruses can also be used to study virus kinetics and reactivation in vivo. Such studies would be facilitated by fluorescent herpes simplex virus recombinants that exhibit wild-type kinetics of replication and reactivation and that are genetically stable. We engineered an HSV-2 strain expressing the fluorescent mNeonGreen protein as a fusion with the VP26 capsid protein. This virus has normal replication and in vivo recurrence phenotypes, providing an essential improved tool for further study of HSV-2 infection.

Immune mechanisms induced by an HSV-1 mutant strain: Discrepancy analysis of the immune system gene profile in comparison with a wild-type strain

Herpes simplex virus is a prevalent pathogen of humans of various age groups. The fact that no prophylactic or therapeutic vaccine is currently available suggests a significant need to further investigate the immune mechanisms induced by the virus and various vaccine candidates. We previously generated an HSV-1 mutant strain, M3, with partial deletions in ul7, ul41 and LAT that produced an attenuated phenotype in mice. In the present study, we performed a comparative analysis to characterize the immune responses induced by M3 versus wild-type HSV-1 in a mouse model. Infection with wild-type HSV-1 triggered an inflammatory-dominated response and adaptive immunity suppression and was accompanied by severe pathological damage. In contrast, infection with M3 induced a systematic immune response involving full activation of both innate and adaptive immunity and was accompanied by no obvious pathological changes. Furthermore, the immune response induced by M3 protected mice from lethal challenge with wild-type strains of HSV-1 and restrained virus proliferation and impaired latency. These data are useful for further HSV-1 vaccine development using a mutant strain construction strategy.