Virus Thymidine Kinase Research Articles

Exploring the Effectiveness of Acyclovir against Ranaviral Thymidine Kinases: Molecular Docking and Experimental Validation in a Fish Cell Line.

The effectiveness of acyclovir, a selective anti-herpesvirus agent, was tested both in silico and in vitro against two ranaviruses, namely the European catfish virus (ECV) and Frog virus 3 (FV3). ECV can cause significant losses in catfish aquaculture, while FV3 poses a risk to vulnerable amphibian populations. The genome of ranaviruses encodes thymidine kinases (TKs) similar to those of herpesviruses. Molecular docking simulations demonstrated that the acyclovir molecule can bind to the active sites of both investigated viral TKs in an orientation conducive to phosphorylation. Subsequently, the antiviral effect of acyclovir was tested in vitro in Epithelioma Papulosum Cyprini (EPC) cells with endpoint titration and qPCR. Acyclovir was used at a concentration of 800 µM, which significantly reduced the viral loads and titers of the ranaviruses. A similar reduction rate was observed with Ictalurid herpesvirus 2, which was used as a positive control virus. These promising results indicate that acyclovir might have a wider range of uses; besides its effectiveness against herpesviruses, it could also be used against ranavirus infections.

CRISPR/Cas9-mediated genome editing of the thymidine kinase gene in a clinical HSV-1 isolate identifies F289S as novel acyclovir-resistant mutation

Herpes simplex virus type 1 (HSV-1) is a neurotropic alphaherpesvirus that establishes a lifelong infection in sensory neurons of infected individuals, accompanied with intermittent reactivation of latent virus causing (a)symptomatic virus shedding. Whereas acyclovir (ACV) is a safe and highly effective antiviral to treat HSV-1 infections, long-term usage can lead to emergence of ACV resistant (ACVR) HSV-1 and subsequently ACV refractory disease. Here, we isolated an HSV-1 strain from a patient with reactivated herpetic eye disease that did not respond to ACV treatment. The isolate carried a novel non-synonymous F289S mutation in the viral UL23 gene encoding the thymidine kinase (TK) protein. Because ACV needs conversion by viral TK and subsequently cellular kinases to inhibit HSV-1 replication, the UL23 gene is commonly mutated in ACVR HSV-1 strains. The potential role of the F289S mutation causing ACVR was investigated using CRISPR/Cas9-mediated HSV-1 genome editing. Reverting the F289S mutation in the original clinical isolate to the wild-type sequence S289F resulted in an ACV-sensitive (ACVS) phenotype, and introduction of the F289S substitution in an ACVS HSV-1 reference strain led to an ACVR phenotype. In summary, we identified a new HSV-1 TK mutation in the eye of a patient with ACV refractory herpetic eye disease, which was identified as the causative ACVR mutation with the aid of CRISPR/Cas9-mediated genome engineering technology. Direct editing of clinical HSV-1 isolates by CRISPR/Cas9 is a powerful strategy to assess whether single residue substitutions are causative to a clinical ACVR phenotype.

Abstract LB441: An intravenous compatible oncolytic vaccinia virus treated hepatic metastasis of colon cancer through extensive cancer cell killing and activation of cytotoxic effector differentiation

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide and nearly 50% of CRC patients develop liver metastasis during the course of their disease. Treatment of colorectal cancer liver metastasis (CRLM) has been particularly challenging due to tolerogenic nature of liver immune microenvironment as well as an eventual development of therapy resistance to standard chemotherapy regimen. One promising area of cancer immunotherapy is oncolytic virotherapy that can selectively destroy tumor cells and stimulate anti-tumor immune response. In this study, we systemically administered mSJ-650, an intravenous compatible oncolytic Wyeth strain vaccinia virus that expresses human complement regulatory protein CD55 and murine GM-CSF in place of viral thymidine kinase gene and has a deletion of the viral K2L gene, and evaluated its anti-tumor efficacy in CRLM. Mouse CRLM was induced by intrasplenic injection of 5×105 MC38-OVA cancer cells. Following tumor nodules formation in the liver, low dose (1×106 pfu) or high dose (3×106 pfu) of mSJ-650 was administered multiple times via tail vein and liver was harvested for evaluation of anti-tumor efficacy. Flow cytometry analysis was performed to investigate changes in hepatic and tumor immune microenvironment upon mSJ-650 treatment. Liver and tumor tissue were stained for immunofluorescence imaging of viral replication within the tumor cells and subsequent oncolysis by the virus. Systemic administration of mSJ-650 showed potent anti-tumor efficacy and led to significant reduction in metastatic nodule formation in liver even at low dose. The anti-tumor efficacy was mediated by tumor cell-specific viral distribution and replication, followed by lytic egress of mSJ-650 from infected tumor cells. Anti-tumor efficacy of mSJ-650 also accompanied a dramatic shift in immunologically tolerant hepatic microenvironment to immune-stimulatory one by decreasing proportion of myeloid-derived suppressor cells, Kupffer cells, and monocyte-derived macrophages (MDMs) in the liver while increasing CD8+ T cell infiltration by 2~3 folds. Moreover, mSJ-650 treatment was associated with phenotypic change in hepatic immune cells, as evidenced by Kupffer cell and MDM polarization to M1 phenotype and elevated expression of activation markers and cytotoxic molecules in CD8+ T cells. Changes in hepatic immune microenvironment were also observed in tumor nodules, especially in those completely surrounded by adjacent liver tissue, suggesting that anti-tumor immune response in tumor bed is potentiated by reprogramming of tumor-adjacent hepatic immune microenvironment. Systemic administration of mSJ-650 demonstrated superior anti-tumor efficacy in CRLM which was mediated by direct tumor cell killing and tumor-adjacent hepatic immune microenvironment reprogramming by the virus. Citation Format: Eunhye Kim, Yeonsoo Yang, Solchan Won, Namhee Lee, Songyi Lee, Mi-Ju Park, Byung-Jin Jung, Yun-Kyoung Hong, Hang-Rae Kim, Dong-Sup Lee, Keunhee Oh. An intravenous compatible oncolytic vaccinia virus treated hepatic metastasis of colon cancer through extensive cancer cell killing and activation of cytotoxic effector differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB441.

Analysis of antiviral drug properties of thymidine kinase of herpes B virus using recombinant herpes simplex virus 1.

Zoonotic infection of humans with herpes B virus (BV) causes severe neurological diseases. Acyclovir (ACV) and ganciclovir (GCV), most frequently used as anti-herpes drugs, are recommended for prophylaxis and therapy in human BV infection. In this study, we examined the property of BV thymidine kinase (TK) against anti-herpes drugs using a recombinant herpes simplex virus type 1 (HSV-1) carrying BV TK gene. We found that HSV-1 carrying BV TK was similarly sensitive to GCV as HSV-1 carrying varicella zoster virus TK. In addition, we demonstrated that BV TK was not mutated in the GCV- and ACV-resistant HSV-1 carrying BV TK, suggesting that ACV- or GCV-resistant BV might be rare during treatment with these antiviral drugs. These data can provide a new insight into the properties of BV TK in terms of the development of drug resistance.

A Computational Study of Famciclovir Derivatives Against Thymidine Kinase as a Molecular Target for the Development of Novel Anticancer Drugs via Suicide Gene Therapy Concepts.

The viral thymidine kinase (TK) phosphorylates the antiviral medication famciclovir (FCV), which treats herpes simplex virus (HSV-TK). The phosphorylated FCV destroys the infected cells by preventing cellular DNA synthesis. We hypothesize that FCV impurity, which is a related substance to FCV, should be efficient in killing cells independent of HSV-TK and is currently the most widely used suicide agent for gene therapy of cancer. This study proposes the binding affinity of these derivatives for the active site of TK through molecular docking to a protein (PDB ID: 1W4R). The derivatives' reliability was ensured through the in-silico preliminary drug designing model by screening their Lipinski rule of five violations, if any, ADMET prediction for their profile using online tools. Using MOE 2009.10 computational software, we performed molecular docking of approximately 22 famciclovir derivatives alongside the famciclovir drug. Our results suggest that these derivatives are indicative of possible chemical stability irrespective of all the parameters used to evaluate the selected derivatives as a possible drug candidates for their cytotoxicity. FC20 (i.e., 2-(2-(2-((1-(9-(4-Acetoxy-3-(acetoxymethyl)butyl)-2-amino-9Hpurin- 8-yl)ethyl)amino)-9H-purin-9-yl)ethyl)propane-1,3-diyl diacetate) and FC21 (i.e., 2-Amino-1,9- dihydro-9-(4-hydroxybutyl)-6H-purin-6-one), showed maximum and minimum scores of -26.95 and - 7.21 kcal/mol, respectively when compared to famciclovir (-15.4122 kcal/mol). Considering that there might be a cytotoxicity effect due to competition between protein TK and the suicidal gene of famciclovir derivatives. The outcome of the study proved that the FCV impurity could successfully modify an HSV-TK-dependent antiviral drug into an anti-tumor drug. Further, it can be used for the design and development of novel compounds of FCV impurity that could be cytotoxic agents if properly delivered to cancer cells.

IRF9 inhibits CyHV-3 replication by regulating the PI3K-AKT signalling pathway in common carp (Cyprinus carpio) epithelial cells

Interferon regulatory factor 9 (IRF9) is an important transcriptional regulator involved in innate and adaptive immunity. Cyprinid herpesvirus-3 (CyHV-3) is a virus causing widespread death and great economic loss in farmed common carp (Cyprinus carpio). However, the effect of IRF9 on CyHV-3 infection in common carp has not been reported. In this study, during CyHV-3 infection, IRF9 overexpression in common carp fin epithelial (CCF) cells significantly reduced the expression of viral factor thymidine kinase (TK) and open reading frame 72 (ORF72), and knockdown of IRF9 produced the opposite results (p < 0.05). In CCF cells. The IRF9 protein was expression in the nucleus and was rapidly induced in CCF cells by CyHV-3 infection. In addition, several genes associated with virus infection, including type I interferon (IFNI), IFN-stimulated gene 15 (ISG15), myxovirus resistance 1 (Mx1) and Viperin were induced in CCF cells overexpressing IRF9 upon CyHV-3 infection. IRF9 overexpression induced by CyHV-3 infection significantly increased the gene expression of Mx1 and phosphoinositide 3-kinase (PI3K) and the protein expression of protein kinase B (AKT) (p < 0.01). Interestingly, IRF9 did not significantly affect Mx1 gene expression when AKT protein levels remained unchanged during CyHV-3 infection of CCF cells. Furthermore, a significant resistance-related locus was found in the IRF9 sequence in “Longke-11” mirror carp (M11) and Yellow River carp (p < 0.05). These results indicated that IRF9 inhibited viral replication by upregulating the expression of Mx1 via the PI3K-AKT signalling pathway during CyHV-3 infection in CCF cells and provide some basis for the study of the antiviral molecular mechanisms of common carp.

Acyclovir-resistant HSV-1 isolates among immunocompromised patients in southern Taiwan: Low prevalence and novel mutations.

Herpes simplex virus type 1 (HSV-1) can establish latency in humans and easily relapse in immunocompromised patients, with significant mortality. Treatment with acyclovir (ACV) can result in the emergence of HSV resistance. A total of 440 frozen HSV-1 isolates collected from 318 patients from January 2014 to July 2019 were obtained from National Cheng Kung University Hospital in southern Taiwan. These 440 isolates were subjected to phenotypic studies for ACV-resistance by initial screening with the plaque reduction assay (PRA) and further validation by the DNA reduction assay (DRA). The ACV-resistant strains were further investigated by Sanger sequencing for the full-length UL23 and UL30 genes, which encode thymidine kinase and DNA polymerase, respectively. Hematological malignancies or hematopoietic stem-cell transplantation patients accounted for 56.9% (124/218) among the immunocompromised patients (218/318) in this study. Repeated sampling for HSV testing was 50% (109/218) in immunocompromised patients. Only 1.38% (3/218) of immunocompromised patients and 0.9% (3/318) of all patients developed ACV-resistant HSV-1 as measured by phenotypic screening assays. It is noteworthy that a novel Y248D mutation in the UL23 gene from an immunocompromised patient was found by both PRA and DRA. In 3D protein predicting analysis, uncharged Y248 was located at an alpha-helix and substituted by negative-charged D248, which may alter the function of viral thymidine kinase. Besides, three unreported mutations related to natural polymorphism were found in virus isolates from two immunocompetent patients, including 683-688 deletion, R227H, and A351D in the UL30 gene. These data show that the prevalence of ACV-resistant HSV-1 among immunocompromised patients in southern Taiwan is low. These results will be helpful for the clinical management and treatment of HSV infections.

Radiovirotherapy at twenty

Radiovirotherapy at twenty

A Herpes Simplex Virus 1 DNA Polymerase Multidrug Resistance Mutation Identified in a Patient With Immunodeficiency and Confirmed by Gene Editing.

Herpes simplex virus 1 can cause severe infections in individuals who are immunocompromised. In these patients, emergence of drug resistance mutations causes difficulties in infection management. Seventeen herpes simplex virus 1 isolates were obtained from orofacial/anogenital lesions in a patient with leaky severe combined immunodeficiency over 7 years, before and after stem cell transplantation. Spatial/temporal evolution of drug resistance was characterized genotypically-with Sanger and next-generation sequencing of viral thymidine kinase (TK) and DNA polymerase (DP)-and phenotypically. CRISPR/Cas9 was used to introduce the novel DP Q727R mutation, and dual infection-competition assays were performed to assess viral fitness. Isolates had identical genetic backgrounds, suggesting that orofacial/anogenital infections derived from the same virus lineage. Eleven isolates proved heterogeneous TK virus populations by next-generation sequencing, undetectable by Sanger sequencing. Thirteen isolates were acyclovir resistant due to TK mutations, and the Q727R isolate additionally exhibited foscarnet/adefovir resistance. Recombinant Q727R mutant virus showed multidrug resistance and increased fitness under antiviral pressure. Long-term follow-up of a patient with severe combined immunodeficiency revealed virus evolution and frequent reactivation of wild-type and TK mutant strains, mostly as heterogeneous populations. The DP Q727R resistance phenotype was confirmed with CRISPR/Cas9, a useful tool to validate novel drug resistance mutations.

Kaposi’s Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of Kaposi's sarcoma, Castleman's disease, and primary effusion lymphoma. Although the functions of the viral thymidine kinases (vTK) of herpes simplex virus-1/2 are well understood, that of KSHV ORF21 (an ortholog of vTK) is largely unknown. Here, we investigated the role of ORF21 in lytic replication and infection by generating two ORF21-mutated KSHV BAC clones: ORF21-kinase activity deficient KSHV (21KD) and stop codon-induced ORF21-deleted KSHV (21del). The results showed that both ORF21 mutations did not affect viral genome replication, lytic gene transcription, or the production of viral genome-encapsidated particles. The ORF21 molecule-dependent function, other than the kinase function of ORF21, was involved in the infectivity of the progeny virus. ORF21 was expressed 36 h after the induction of lytic replication, and endogenously expressed ORF21 was localized in the whole cytoplasm. Moreover, ORF21 upregulated the MEK phosphorylation and anchorage-independent cell growth. The inhibition of MEK signaling by U0126 in recipient target cells suppressed the number of progeny virus-infected cells. These suggest that ORF21 transmitted as a tegument protein in the progeny virus enhances the new infection through MEK up-regulation in the recipient cell. Our findings indicate that ORF21 plays key roles in the infection of KSHV through the manipulation of the cellular function.

Generation of novel oncolytic vaccinia virus with improved intravenous efficacy through protection against complement-mediated lysis and evasion of neutralization by vaccinia virus-specific antibodies

BackgroundOncolytic virus immunotherapy has revolutionized cancer immunotherapy by efficiently inducing both oncolysis and systemic immune activation. Locoregional administration has been used for oncolytic virus therapy, but its applications to deep-seated...

Neomorphic DNA-binding enables tumor-specific therapeutic gene expression in fusion-addicted childhood sarcoma

Chimeric fusion transcription factors are oncogenic hallmarks of several devastating cancer entities including pediatric sarcomas, such as Ewing sarcoma (EwS) and alveolar rhabdomyosarcoma (ARMS). Despite their exquisite specificity, these driver oncogenes have been considered largely undruggable due to their lack of enzymatic activity.Here, we show in the EwS model that – capitalizing on neomorphic DNA-binding preferences – the addiction to the respective fusion transcription factor EWSR1-FLI1 can be leveraged to express therapeutic genes.We genetically engineered a de novo enhancer-based, synthetic and highly potent expression cassette that can elicit EWSR1-FLI1-dependent expression of a therapeutic payload as evidenced by episomal and CRISPR-edited genomic reporter assays. Combining in silico screens and immunohistochemistry, we identified GPR64 as a highly specific cell surface antigen for targeted transduction strategies in EwS. Functional experiments demonstrated that anti-GPR64-pseudotyped lentivirus harboring our expression cassette can specifically transduce EwS cells to promote the expression of viral thymidine kinase sensitizing EwS for treatment to otherwise relatively non-toxic (Val)ganciclovir and leading to strong anti-tumorigenic, but no adverse effects in vivo. Further, we prove that similar vector designs can be applied in PAX3-FOXO1-driven ARMS, and to express immunomodulatory cytokines, such as IL-15 and XCL1, in tumor entities typically considered to be immunologically ‘cold’.Collectively, these results generated in pediatric sarcomas indicate that exploiting, rather than suppressing, the neomorphic functions of chimeric transcription factors may open inroads to innovative and personalized therapies, and that our highly versatile approach may be translatable to other cancers addicted to oncogenic transcription factors with unique DNA-binding properties.

Heterogeneity and viral replication fitness of HSV-1 clinical isolates with mutations in the thymidine kinase and DNA polymerase.

Prolonged antiviral therapy in immunocompromised individuals can result in the emergence of (multi)drug-resistant herpes simplex virus 1 (HSV-1) infections, forming a therapeutic challenge. To evaluate spatial and temporal differences in drug resistance of HSV-1 samples from a HSCT recipient and to determine the effect of resistance mutations on viral replication fitness. Five HSV-1 isolates were recovered from a HSCT recipient who suffered from persistent HSV-1 lesions, consecutively treated with aciclovir, foscarnet, cidofovir and a combination of ganciclovir and cidofovir. Spatial and temporal differences in HSV-1 drug resistance were evaluated genotypically [Sanger sequencing and next-generation sequencing (NGS) of the viral thymidine kinase (TK) and DNA polymerase (DP)] and phenotypically (plaque reduction assay). Viral replication fitness was determined by dual infection competition assays. Rapid evolution to aciclovir and foscarnet resistance was observed due to acquisition of TK (A189V and R222H) and DP (L778M and L802F) mutations. Virus isolates showed heterogeneous populations, spatial virus compartmentalization and minor viral variants in three out of five isolates (detectable by NGS but not by Sanger sequencing). Mutations in the TK and DP genes did not alter replication fitness without drug pressure. TK and/or DP mutants influenced replication fitness under antiviral pressure and showed increased fitness under pressure of the drug they showed resistance to. The use of NGS and dual infection competition assays revealed rapid evolution of HSV-1 drug resistance in a HSCT recipient with spatial and temporal compartmentalization of viral variants that had altered replication fitness under antiviral pressure.

Abstract 5617: A novel genetically modified oncolytic vaccinia virus selectively replicates in tumors to activate an inflammatory TME and induce tumor regression

Abstract Oncolytic viruses (OVs) have been widely investigated as cancer immunotherapy platforms and are represented by a diverse group of DNA and RNA viruses. We are currently investigating a genetically modified oncolytic vaccinia virus as a best-in-class tumor-selective investigational product for the treatment of solid tumors. Specifically, the viral thymidine kinase (TK) has been deleted to enhance safety by greatly limiting replication in healthy tissues while taking advantage of relatively higher endogenous TK levels in tumors. In addition to tumor-selective replication, the oncolytic vaccinia virus encodes an IL-2 variant (IL-2v) protein, which diminishes preferential activation of immunosuppressive Treg cells while preserving activation of CD8+ and NK cells and avoiding toxicities associated with wild type IL-2. Using a murine surrogate of the oncolytic vaccinia virus, we assessed preclinical efficacy in multiple murine syngeneic tumor models demonstrating selective tumor localized replication resulting in tumor growth inhibition. Consistent with tumor selective virus replication, viral copy number and IL-2v transgene levels were increased and had prolonged expression in the tumors compared to the periphery. Intratumoral colocalization of the virus and IL-2v was confirmed by IHC and ISH, respectively. Tumor growth inhibition was associated with increases in tumor infiltrating CD8 and NK effector cells. Additionally, efficacy of the mouse surrogate virus was associated with systemic increases in circulating total CD8 and NK cells as well as pSTAT5+ CD8 and NK cells in the blood. In summary, we have demonstrated that arming a TK-deficient oncolytic vaccinia virus with an IL-2v transgene can effectively couple short-term destruction of tumor cells through oncolysis with the activation of anti-tumor immune response in the tumor microenvironment. These data support the study of this therapy in Phase 1 clinical trials. Citation Format: Anne-Laure Goenaga, I-Ming Wang, Bryan S. Clay, Susanne Lang, Annabel Wang, Peter Weady, Smitha P.S. Pillai, Sripad Ram, Leah Mitchell, Michael Eisenbraun, Joseph Binder, Liliana Maruri Avidal, David Kirn, Robert Hollingsworth, Clare Lees. A novel genetically modified oncolytic vaccinia virus selectively replicates in tumors to activate an inflammatory TME and induce tumor regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5617.

Abstract 3561: Pan-cancer-specific engineered oncolytic vaccinia virus

Abstract Here, we developed a novel oncolytic vaccinia virus (NOV) with the dual advantages of cancer selectivity and normal vessel reconstructive activity by replacing the viral thymidine kinase (vTk) and vaccinia growth factor (VGF) genes with genes encoding TNF-related apoptosis-inducing ligand (TRAIL) and angiopoietin 1 (Ang1), respectively. The pan-cancer-specific oncolytic potency of NOV was confirmed in various human and mouse cancer cell lines (colon, lung, liver, pancreas, cholangiocarcinoma, cervical cancer, osteosarcoma, and melanoma). Vaccinia virus (VV) treatment directly induced early apoptosis in tumors within 24 h, and this effect was enhanced with further engineering; VGF and Tk deletion with Ang1 and TRAIL insertion. Meanwhile, treatment with the conventional anti-cancer drug cisplatin did not induce apoptosis. A virus-treated mouse colon/lung cancer syngeneic model showed attenuated tumor growth, which was in accordance with the results of percent survival measurement, CD8 expression analysis, and TUNEL staining with advanced genetic engineering. Taken together, our results indicate that NOV induces cancer tissue apoptosis and anti-tumor immunity and may constitute a highly advantageous therapeutic agent for next-generation solid tumor virotherapy with pan-cancer-specific oncolytic activity and high biosafety. Citation Format: So Young Yoo. Pan-cancer-specific engineered oncolytic vaccinia virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3561.

Abstract CT176: A phase 1 open label, first in human trial to study safety and tolerability of NK-T cells combined with vvDD: An accelerated dose escalation in multiple tumor types

Abstract Introduction: BioEclipse Therapeutics has developed CRX100 an adoptive NK-T Cells therapy combined with vaccinia vvDD, an oncolytic virus. vvDD is a Western Reserve (WR) strain vaccinia virus (VACV) that has been further attenuated by the removal of the viral thymidine kinase and growth factor genes, and hence its replication and lytic activity is confined to tumor cells. In preclinical studies in immunocompetent mice the combined therapy eradicated tumor cells and initiated an adaptive immune response. Trial Design: Our ongoing phase 1 safety clinical trial (IND 19222 at Clinicaltrials.gov) began in January 2021 and includes planned treatment of approximately 24 patients with the potential for 2 doses each. With the dose of ex vivo activated autologous NK-T cells held constant (2-5 X 109), the dose of vvD is increased at 5 dose levels, 3X107, 1x108, 3x108, 1x109 and 3x109 pfu/dose. The first five doses are accelerated in cohorts of one patient and followed by a non-accelerated phase with a cohort of two patients and then 3 cohorts of three patients to determine the MTD, with an additional 8 patients then treated at the MTD. Dose limiting toxicities evaluated by a BOIN design will assist in the dose decision for phase 2. Inclusion and exclusion criteria: Patients will have tumor progression and have failed standard of care in the indications, triple negative breast cancer, gastric cancer, colorectal carcinoma, hepatocellular carcinoma, ovarian cancer or osteosarcoma. Results: To date, 4 patients have been treated, including 2 patients with ovarian cancer and 2 with CRC at 4 dose levels. No dose limiting toxicities have been observed. The most common AE is mild fever at 6-12 hours after infusion. Two CRC patients treated at a vvDD dose of 3x107 and 1x109 experienced disease progression. Patient 101-005 with ovarian cancer received two separate infusions 3 months apart each at a dose of 1x108 PFU vvDD combined with 2-5 X 109 ex-vivo activated autologous NKT. Tumor assessment after the first dose showed stable disease (+17.2%) by iRECIST. The CA125 serum biomarker increased temporarily about 3 weeks after infusion. The second ovarian patient (101-007) treated with 3x108 vvDD combined with 2-5 X 109 ex-vivo activated autologous NKT had unconfirmed progression (+21%). There was a slight increase and then a decrease of the CA-125 from 303 to 268. The patient developed new ascites 4 weeks after infusion and required a paracentesis. The ascites did not recur after the paracentesis. Conclusion: Thus far in escalation, all doses of the therapy have been safe and well-tolerated. Two patients with ovarian cancer treated with the adoptive NKT cell therapy combined with an oncolytic virus demonstrated changes in CA-125 potentially consistent with immune inflammation. Peripheral blood obtained at 3 and 6 month will be analyzed to assess specific immune responses. Dose escalation and enrollment continue for this study. Citation Format: Oliver Dorigo, Pamela Reilly Contag, Justin Moser, Sandip Pravin Patel, Mark W. Frohlich. A phase 1 open label, first in human trial to study safety and tolerability of NK-T cells combined with vvDD: An accelerated dose escalation in multiple tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT176.

Abstract 3300: Glycoimmune checkpoint and tumor stroma-targeted oncolytic vaccinia virus for the treatment of solid tumors

Abstract Oncolytic virotherapy represents a promising therapeutic strategy for cancers that do not respond well to immune checkpoint inhibitors. Oncolytic viruses (OV) preferentially replicate in and lyse tumor cells, leading to systemic immune stimulation. Studies have shown that hypersialylation promotes tumor growth and metastasis, as well as the suppression of immune cells. Cancer-associated fibroblasts (CAFs) are known to facilitate tumor invasion and angiogenesis and maintain immunosuppressive microenvironment in solid tumors. We are developing vvDD-Sial-FAP/CD3, an oncolytic vaccinia virus expressing a membrane-bound sialidase, to remove sialic acids from the cell surface glycans, and fibroblast activation protein (FAP)-targeted T cell engager, to eliminate glycoimmune checkpoint and tumor stroma, respectively. vvDD-Sial-FAP/CD3 is an engineered vaccinia virus of Western Reserve (WR) strain with: (1) an insertional disruption of the viral thymidine kinase (TK) gene with the sialidase and FAP/CD3 transgenes. TK is an essential enzyme for the pyrimidine synthesis pathway; viral TK gene deletion thus results in selective replication of virus in rapidly dividing cancerous cells with high intracellular nucleotide pools, and (2) a deletion of the vaccinia growth factor (VGF) genes for greater dependence on the cell cycling status of the cancer cells. Our results showed that sialidase expressed from vvDD-Sial-FAP/CD3 efficiently cleaves the sialic acids from the cell surface and the Fc fused to the sialidase induced antibody-dependent cell-mediated cytotoxicity (ADCC) using an ADCC reporter assay. In vitro efficacy studies were conducted using HCT-116 human colon cancer cells mixed with FAP-expressing normal human dermal fibroblasts or FAP-positive HCC1143 human breast cancer cells in the presence of human peripheral blood mononuclear cells. In both tumor models, vvDD-Sial-FAP/CD3 induced activation of both CD4+ and CD8+ T cells, as measured by the upregulation of CD69 and CD25 markers and increased granzyme B release, which resulted in enhanced cell killing. Using A549 co-cultured with CAFs microtissues, we showed that vvDD-Sial-FAP/CD3 spread efficiently within the tumor spheroids. In addition, vvDD-Sial-FAP/CD3 increased tumor-infiltrated lymphocytes, leading to enhanced cell killing. Taken together, our results have demonstrated potent anti-tumor and -stroma effects induced by vvDD-Sial-FAP/CD3 in multiple cancer models. In vivo efficacy of vvDD-Sial-FAP/CD3, either alone or in combination with checkpoint inhibitor or NK cell therapy, is being evaluated. Citation Format: Winnie M. Chan, Mariya Viskovska, Micaela McArthur, Xue Mary Yu, Wei Liang, Zane Norman, Xiaotong Song, Nancy T. Chang, Haiyan Jiang. Glycoimmune checkpoint and tumor stroma-targeted oncolytic vaccinia virus for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3300.

Phenotypic and genotypic acyclovir resistance surveillance of genital herpes simplex virus 2 in South Africa

Acyclovir (ACV) is currently included in the syndromic management algorithm for genital ulcer disease in South Africa, and is the recommended first-line treatment for herpes simplex virus 2 (HSV-2). In the majority of cases, HSV-2 resistance to ACV is due to amino acid changes within the viral thymidine kinase (TK). Phenotypic and genotypic ACV resistance surveillance of HSV-2 derived from genital ulcer disease swab specimens was conducted at a primary healthcare facility in Johannesburg between 2018 and 2020. The objectives of this surveillance were to identify ACV resistance-associated mutations and polymorphisms in HSV-2 TK, and to determine the phenotypic ACV resistance profiles of the corresponding clinical HSV-2 isolates. Genotypic analysis of TK from 67 HSV-2 positive genital ulcer swabs revealed 48 specimens with TK mutations, conferring 113 nucleotide changes. No resistance-associated mutations were found, however, we identified nine known natural polymorphisms (R26H, A27T, S29A, G39E, N78D, L140F, T159I, R220K and R284S) and five amino acid changes of unknown significance (R18C, G39K, M70R, P75S and L263P). Phenotypic susceptibility testing of 52 cultivable HSV-2 isolates revealed all to be susceptible to ACV with IC50 values of <2 μg/ml. The five amino acid changes of unknown significance identified by genotypic testing were not correlated to phenotypic ACV resistance, and therefore grouped as natural polymorphisms. We did not detect any unknown or resistance-associated mutations in specimens that could not be phenotypically tested for ACV resistance. Our findings will supplement existing databases of HSV antiviral resistance-associated mutations and polymorphisms that could be used for genotypic ACV resistance screening.

Deletion of the Viral Thymidine Kinase in a Meq-Deleted Recombinant Marek's Disease Virus Reduces Lymphoid Atrophy but Is Less Protective.

Marek’s disease (MD) is a ubiquitous disease of domesticated chickens and its etiologic agent is the Gallid alphaherpesvirus 2 (GaHV-2), also known as Marek’s disease virus (MDV). MD is currently controlled by vaccination using live attenuated strains of MDV (e.g., CVI988/Rispens), non-pathogenic serotypes of MDV (GaHV-3), or non-pathogenic strains of the related Melagrid alphaherpesvirus 1 (MeHV-1). One attractive strategy for the production of new vaccine strains is a recombinant MDV attenuated by the deletion of the major viral oncogene meq. However, meq-deleted variants of MDV cause atrophy of the bursa and thymus in maternal antibody-negative chickens, and the resulting immunosuppression makes them unsuitable. Herein we detail our attempt to mitigate the lymphoid atrophy caused by meq-deleted MDV by further attenuation of the virus through ablation of the viral thymidine kinase (tk) gene. We demonstrate that ablation of the viral tk from the meq-deleted virus rMd5B40/Δmeq resulted in a virus attenuated for replication in vitro and which spared chickens from atrophy of the lymphoid organs in vivo. When the rMd5B40/Δmeq/Δtk/GFP was used as a vaccine it was protective against challenge with the vv+MDV strain 686, but the protection was less than that provided by the CVI988/Rispens vaccine.

Partial Ablation of Non-Myogenic Progenitor Cells as a Therapeutic Approach to Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD), caused by the loss of dystrophin, remains incurable. Reduction in muscle regeneration with DMD is associated with the accumulation of fibroadipogenic progenitors (FAPs) differentiating into myofibroblasts and leading to a buildup of the collagenous tissue aggravating DMD pathogenesis. Mesenchymal stromal cells (MSCs) expressing platelet-derived growth factor receptors (PDGFRs) are activated in muscle during DMD progression and give rise to FAPs promoting DMD progression. Here, we hypothesized that muscle dysfunction in DMD could be delayed via genetic or pharmacologic depletion of MSC-derived FAPs. In this paper, we test this hypothesis in dystrophin-deficient mdx mice. To reduce fibro/adipose infiltration and potentiate muscle progenitor cells (MPCs), we used a model for inducible genetic ablation of proliferating MSCs via a suicide transgene, viral thymidine kinase (TK), expressed under the Pdgfrb promoter. We also tested if MSCs from fat tissue, the adipose stromal cells (ASCs), contribute to FAPs and could be targeted in DMD. Pharmacological ablation was performed with a hunter-killer peptide D-CAN targeting ASCs. MSC depletion with these approaches resulted in increased endurance, measured based on treadmill running, as well as grip strength, without significantly affecting fibrosis. Although more research is needed, our results suggest that depletion of pathogenic MSCs mitigates muscle damage and delays the loss of muscle function in mouse models of DMD.