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Oncolytic and local immunotherapy
Original research
Oncolytic varicella-zoster virus engineered with ORF8 deletion and armed with drug-controllable interleukin-12
  1. Haifei Jiang1,
  2. Rebecca Nace1,
  3. Talia Fernandez Carrasco1,
  4. Lianwen Zhang1,
  5. Kah Whye Peng1 and
  6. Stephen J Russell2
  1. 1Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, USA
  2. 2Vyriad Inc, Rochester, Minnesota, USA
  1. Correspondence to Dr Haifei Jiang; Jiang.Haifei{at}mayo.edu; Dr Stephen J Russell; sjrussell{at}vyriad.com

Abstract

Background The varicella-zoster virus (VZV), belonging to the group of human α-herpesviruses, has yet to be developed as a platform for oncolytic virotherapy, despite indications from clinical case reports suggesting a potential association between VZV infection and cancer remission.

Methods Here, we constructed oncolytic VZV candidates based on the vaccine strain vOka and the laboratory strain Ellen. These newly engineered viruses were subsequently assessed for their oncolytic properties in the human MeWo melanoma xenograft model and the mouse B16-F10-nectin1 melanoma syngeneic model.

Results In the MeWo xenograft model, both vOka and Ellen exhibited potent antitumor efficacy. However, it was observed that introducing a hyperfusogenic mutation into glycoprotein B led to a reduction in VZV’s effectiveness. Notably, the deletion of ORF8 (encodes viral deoxyuridine triphosphatase) attenuated the replication of VZV both in vitro and in vivo, but it did not compromise VZV’s oncolytic potency. We further armed the VZV Ellen-ΔORF8 vector with a tet-off controlled mouse single-chain IL12 (scIL12) gene cassette. This augmented virus was validated for its oncolytic activity and triggered systemic antitumor immune responses in the immunocompetent B16-F10-nectin1 model.

Conclusions These findings highlight the potential of using Ellen-ΔORF8-tet-off-scIL12 as a novel VZV-based oncolytic virotherapy.

  • Oncolytic Virotherapy
  • Oncolytic Viruses

Data availability statement

Data are available upon reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

https://doi.org/10.1136/jitc-2023-008307

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    WHAT IS ALREADY KNOWN ON THIS TOPIC

    • Clinical case reports suggest a potential association between natural varicella-zoster virus (VZV) infection and cancer remission.

    WHAT THIS STUDY ADDS

    • This study reported a new oncolytic viral platform based on the VZV laboratory strain Ellen. The ORF8 gene (encodes viral deoxyuridine triphosphatase) was deleted to attenuate the replication of VZV and improve safety. In addition, a tet-off controlled single-chain IL12 (scIL12) gene cassette was inserted into the VZV genome to enhance virus-induced systemic antitumor immune responses.

    HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE, OR POLICY

    • This study highlights the potential of using Ellen-ΔORF8-tet-off-scIL12 as a novel VZV-based oncolytic virotherapy.

    Introduction

    Oncolytic viruses are engineered to selectively destroy cancer cells, while simultaneously promoting the immune system to recognize tumor antigens and initiate systemic antitumor immune responses.1 The human α-herpesviruses have long double-stranded DNA (dsDNA) genomes (~124–152 kb) and are a group of epithelial and nerve tropic viruses with a short 8–12 hours replication cycle.2 3 They infect the skin or mucosal surfaces during primary infection and subsequently establish lifelong latency in sensory ganglions.4 Among the three human α-herpesviruses, herpes simplex virus (HSV) type 1 and type 2 have been extensively studied as anticancer agents.5 Presently, two HSV-based therapies have received clinical approval: T-VEC, approved in the USA and Europe for the treatment of recurrent melanoma, and DELYTACT, authorized in Japan for glioblastoma therapy.5

    Unlike HSVs, varicella-zoster virus (VZV) has not been developed as an oncolytic platform. VZV induces robust immune responses during primary infection and virus reactivation,6 7 and clinical case reports revealed a potential association between VZV infection/reactivation and tumor remission in many cancer types.8–11 VZV infection showed durable bone marrow stimulating activity in patients with hematologic disorders,12 and its reactivation after autologous stem cell transplants was found associated with favorable outcome according to a study involving 191 consecutive patients with melanoma (VZV reactivation occurred in 57 patients).10 Besides, VZV exposure has been reported an inverse correlation with glioma risk.11 13 According to the Glioma International Case‐Control Study data that included thousands of glioma and control cases, a positive chickenpox history may lower glioma risk by 21% after the adjustment for age and sex, and the protective effect may be stronger for high‐grade gliomas.11 Simultaneously, VZV-specific T cells were found to cross-reacting with glioma cells ex vivo.13 Another notable case involved a patient with advanced maxillary sinus squamous cell cancer who experienced an exceptional complete response to treatment with pembrolizumab in the course of a natural VZV reactivation.8 Novel VZV-based virotherapies need to be developed and evaluated in preclinical tumor models.

    VZV virions exhibit pleomorphism, displaying diameters ranging from 150 nm to 200 nm. These virions possess an outer lipid envelope that incorporates viral glycoproteins and encompasses an icosahedral nucleocapsid composed of 162 capsomeres. Positioned between the envelope layer and the nucleocapsid is a protein tegument.14 Packaged within the nucleocapsid is a 124 kb linear dsDNA genome that encodes at least 70 open reading frames (ORFs). The VZV genome comprises a unique long fragment flanked by terminal long and internal long repeats, as well as a unique short fragment flanked by internal short and terminal short repeats. All VZV genes except six have homologs in HSV.15 Conversely, VZV lacks homologs for nine HSV genes, including the multifunctional ICP34.5 and the transporter associated with antigen processing inhibitor ICP47, which have been deleted in many oncolytic HSV vectors.15

    The primary infection with VZV, typically acquired through direct contact with skin lesions or exposure to respiratory aerosols, results in the development of chickenpox (varicella). During the lytic phase of infection, which primarily occurs in epithelial and skin cells,16 VZV has the intriguing ability to attract lymphocytes to the infection site and uses them as carriers to facilitate its own systemic spreading.17 The process is partially mediated by the virus-encoded chemokine binding protein glycoprotein C, which binds to more than 20 CXC and CC chemokines and potentiates their ability to recruit lymphocytes (ie, T cells).17 18 During systemic infection, the virus can enter the sensory neurons of trigeminal or dorsal root ganglia and establish life-long latency. In the event of host immunosuppression, VZV may reactivate and lead to the manifestation of shingles (zoster). When this occurs, the virus spreads from sensory ganglion to innervated skin.14 When antiviral drugs and VZV vaccines are available for the treatment and prevention of varicella and singles,16 19 the annual incidence of shingles is still 5–6.5 per 1000 individuals at age 60 and is 8–11 per 1000 at age 70.14 In general, VZV infections are highly prevalent worldwide, with approximately 90% of the US population acquiring VZV infection before the age of 15.20 21 Moreover, a retrospective study on hematopoietic stem cell transplantation patients supports that the pre-existing antiviral antibody does not prevent VZV reinfection but may contribute to decreased systemic viral transmission.22

    In the current study, oncolytic VZVs were constructed based on the vaccine Oka strain and the laboratory strain Ellen using the bacterial artificial chromosome (BAC)-based reverse genetics system. These newly constructed viruses were tested in both a human melanoma xenograft model and a syngeneic melanoma mouse model. Recombinant viruses with or without a hyperfusogenic glycoprotein B (gB) mutation were compared for antitumor potency. A tet-off-controlled interleukin (IL)-12 cassette was inserted into the VZV genome as therapeutic transgene, and ORF8 and ORF65 were deleted to attenuate the virulence. In addition, VZV-induced systemic antitumor immune responses were evaluated in a syngeneic melanoma mouse model.

    Results

    Construction of oncolytic VZV vectors

    The BAC vector-based Lambda Red recombineering was used in this study to manipulate the 125 kb dsDNA genome of VZV.23 To achieve this, a BAC vector PSG7 encompassing two homology arms (ORF52 and ORF53-54) was constructed. The homology arms drove the site-specific integration of BAC vector into the VZV genome between ORF52 and ORF53 within the arising retinal pigment epithelia-19 (ARPE-19) cells (figure 1A). The recombinant VZV-BAC episome was extracted from infected ARPE-19 cells and then transferred into SW102 bacterial cells, which express the Lambda Red recombineering system (exo, bet, and gam).24 Genetic manipulations of VZV were completed within SW102 cells via homologous recombination, and resulting viral constructs were transfected back into ARPE-19 cells for virus reconstitution. Using this reverse genetics platform, VZV-BAC vectors were constructed based on the vaccine strain vOka and the laboratory strain Ellen.

    Figure 1
    Figure 1

    Construction of oncolytic VZV vectors. (A) Construction of VZV-BAC vector. The BAC vector pSG7 encompassing VZV ORF52 and ORF53-54 as homology arms were linearized and transfected into APRE-19 cells on day 1. VZV was inoculated on day 3 to enable the site-specific integration of BAC vector into VZV genome within ARPE-19 cells. The green plaques of recombinant VZV-BAC virus were enriched by G418 treatment, and circular VZV-BAC episomes were extracted from infected ARPE-19 cells and transformed into DH10B bacterial cells. The BAC plasmids containing full-length VZV genome were further transferred to the SW102 recombineering bacterial cells for VZV genome reconstruction. Recombinant VZV-BAC constructs were transfected into ARPE-19 or ARPE-19-cre cells for virus reconstitution. (B) VZV vOka-BAC-bHCG and vOka-BAC-bHCG-gBsyn. The bHCG was inserted between ORF60 and ORF61 of the VZV vaccine Oka strain as a secreted reporter gene, and the ORF31 (gB) A2453T point mutation was introduced to generate the hyperfusogenic vOka-BAC-bHCG-gBsyn vector. (C) Plaques of vOka-BAC-bHCG and vOka-BAC-bHCG-gBsyn (MOI=0.001, 2 dpi). Scale bars=200 µm. (D) gB A2453T mutation increases virus syncytium area. vOka-BAC-bHCG and vOka-BAC-bHCG-gBsyn plasmids were transfected into 293T cells (2 µg per 1×106 cells), and on 3 days post-transfection virus syncytium area was measured and compared (unpaired t-test, n=5). (E) Growth curves of recombinant vOka viruses. The APRE-19 cells cultured in six-well plates were infected with viruses on day 0 (1000 PFUs per well), and virus titers on days 1, 3, 5, and 7 were determined (n=3). Virus titers of vOka-BAC-bHCG and vOka-BAC-bHCG-gBsyn on day 3 and day 5 were compared (unpaired t-test). ns, not significant, *p<0.05, **p<0.01. BAC, bacterial artificial chromosome; bHCG, beta-human chorionic gonadotropin; IRS, internal short; MOI, multiplicity of infection; PFU, plaque-forming unit; TRS, terminal short; UL, unique long; US, unique short; VZV, varicella-zoster virus.

    Initially, we focused on the vaccine-based vOka vector. The human chorionic gonadotropin beta-subunit (bHCG) gene was inserted between ORF60 and ORF61 as a secreted reporter to monitor viral infection (figure 1B). A hyperfusogenic mutation of gB (Y881F) was further introduced to the vOka genome for enhancing cell-to-cell fusion.25 26 Increased syncytium area formed by vOka-BAC-bHCG-gBsyn (gBY881F) was observed compared with the control virus vOka-BAC-bHCG (figure 1C,D) but the growth kinetics of the hyperfusogenic mutant virus was impaired (figure 1E).

    VZV demonstrates potent antitumor efficacy in human melanoma xenograft model

    Following the removal of the LoxP-flanked BAC sequence by cultivating the vOka-bHCG and vOka-bHCG-gBsyn viruses in ARPE-19 cells expressing cre recombinase, their potential for oncolytic activity was evaluated using the MeWo human melanoma xenograft model (figure 2A). The viruses were administered intratumorally (IT), and the attenuated replication of vOka-bHCG-gBsyn was verified via the monitoring of blood bHCG reporter protein levels (figure 2B). In comparison to the hyperfusogenic virus, the vOka-bHCG virus exhibited superior antitumor efficacy in terms of both inhibiting tumor growth and prolonging the survival of animals (figure 2C,D). The gBY881F mutation has been previously demonstrated to interfere with both virus replication and the transcription of host genes.27

    Figure 2
    Figure 2

    VZV vOka vaccine strain demonstrates potent antitumor activity in human melanoma xenograft model. (A) Schematic of experimental design. Human MeWo melanoma cells were implanted subcutaneously on day 0. Viruses were IT delivered on days 14, 16, and 18 (5×104 PFUs per injection). (B) Blood bHCG levels. Blood samples were collected on day 5 post-treatment, and plasma bHCG levels were compared between treatment groups (n=4, unpaired t-test). (C) Tumor growth curves (n=10, two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test). (D) Animal survival curves (n=10, Log-rank (Mantel-Cox) test). ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. bHCG, beta-human chorionic gonadotropin; IT, intratumorally; PFU, plaque-forming unit; VZV, varicella-zoster virus.

    Our efforts progressed to the incorporation of additional/alternative transgenes into the vOka genome. This encompassed the introduction of the human sodium iodide symporter (NIS) gene as an additional reporter gene, as well as the therapeutic transgene single-chain IL12 (scIL12) (online supplemental figure 1A).28 On attempting to reconstitute the recombinant viruses within ARPE-19 cells, it was observed that the incorporation of either NIS or scIL12 transgenes hindered the rescue of vOka constructs. In contrast, the Ellen-BAC-NIS and Ellen-BAC-bHCG-scIL12 viruses were successfully rescued (online supplemental figure 1B). These results imply that the Ellen virus holds an advantage in scenarios where therapeutic transgenes need to be introduced.

    Supplemental material

    Deletion of ORF8 attenuates VZV replication while not reducing virus antitumor potency

    In pursuit of enhancing the safety profile of viruses derived from the Ellen strain, we opted to delete ORF8 and ORF65, two non-essential genes of VZV. Deletion of these genes was chosen due to their non-compromising impact on virus growth in cancer cells while holding the potential to reduce neurovirulence.29 Specifically, ORF8 encodes the viral deoxyuridine triphosphatase, and ORF65 belongs to the evolutionarily conserved α-herpesvirus US9 family, implicated in anterograde axonal transport of the virus.30 ORF8 and ORF65 were removed from Ellen backbone using the galactokinase-based positive/negative selection system (figure 3A). Subsequent assessment of growth curves for the two mutant viruses, Ellen-ΔORF8 and Ellen-ΔORF65, indicated a significant reduction of growth kinetics for Ellen-ΔORF8 in both ARPE epithelial cells and differentiated human neuronal dSH-SY5Y cells but not in MeWo melanoma cells (figure 3B and online supplemental figure 2).31 32 On the other hand, the attenuation observed with ΔORF65 was in dSH-SY5Y neuronal cells but not in ARPE-19 cells or MeWo cells (figure 3B and online supplemental figure 2).

    Figure 3
    Figure 3

    Deletion of ORF8 attenuates VZV replication while not reducing virus antitumor potency. (A) Construction of VZV Ellen-BAC-ΔORF8 and Ellen-BAC-ΔORF65. (B) Growth curves of Ellen-ΔORF8 and Ellen-ΔORF65. The ARPE-19 cells, dSH-SY5Y neuronal cells, or MeWo cells cultured in six-well plates were infected with viruses on day 0 (1000 PFUs per well), and virus titers on days 3, 6, and 9 were detected (n=3). Virus growth curves were compared between the wild-type backbone virus and mutant viruses (two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test (for ARPE-19 cells and MeWo cells) or Tukey’s multiple comparisons test (for dSH-SY5Y cells)). (C) Schematic of experimental design. Human MeWo melanoma cells were implanted subcutaneously on day 0 (n=8). Viruses were IT delivered on days 20, 22, and 24 (5×104 PFUs per injection). (D) Intratumoral virus replication. Tumor slices from different therapy groups (collected on day 5 post-treatment) were stained with anti-VZV ORF68 (gE) antibody (Alexa Flour 594). Representative images were displayed. Scale bars=100 µm. (E) Efficacy of VZV Ellen-ΔORF8 and Ellen-ΔORF65. Tumor growth curves were displayed, and animal survival curves were compared with the Gehan-Breslow-Wilcoxon test. ns, not significant, *p<0.05, **p<0.01, ***p<0.001. BAC, bacterial artificial chromosome; IT, intratumorally; PFU, plaque-forming unit; VZV, varicella-zoster virus.

    The oncolytic characteristics of the two mutant viruses were subsequently assessed using the MeWo melanoma xenograft model (figure 3C). On day 5 postadministration, intratumoral virus replication was observed for both mutant viruses (figure 3D). Analysis of tumor growth curves and animal survival indicated that neither ΔORF8 nor ΔORF65 reduced the antitumor efficacy of VZV in the MeWo model (figure 3E). Treatment-related mortality occurred in one of the eight mice from the wild-type Ellen group (on day 34) but not in the other two groups (figure 3E). We continued to characterize the potential reduced virulence linked to the deletion of ORF8 in vivo. The EF1a-HTLV chimeric promoter-driven scIL12 was inserted between ORF60 and ORF61 in the Ellen-BAC, Ellen-BAC-ΔORF8 and Ellen-BAC-ΔORF65 backbones (figure 4A). After excising the BAC sequence in ARPE-19-cre cells, these three viruses were administered to MeWo tumors to assess both their effectiveness and toxicity (figure 4B). Measurements of IL12 levels in the blood revealed that both ΔORF8 and ΔORF65 variants resulted in a decrease in virus replication on day 5 post-treatment, with the ΔORF8 variant showing a more pronounced attenuation (figure 4C). Importantly, treatment-related animal deaths were recorded in all the nine mice who received Ellen-scIL12 treatment. However, these numbers decreased to two and six in the Ellen-ΔORF8-scIL12 and Ellen-ΔORF65-scIL12 treatment groups, respectively (figure 4D). Compared with the control and the Ellen-scIL12 groups, the Ellen-ΔORF8-scIL12 group exhibited a significant improvement in overall animal survival (figure 4E,F). Collectively, these findings suggest that Ellen-ΔORF8 holds promise as an oncolytic VZV vector, possessing attenuated virulence while retaining its antitumor potency in vivo.

    Figure 4
    Figure 4

    Deletion of ORF8 from VZV improves safety in human MeWo melanoma xenograft model. (A) Insertion of scIL12 into VZV Ellen genome. The elongation factor-1α/ human T-cell leukemia virus (EF1/HTLV) composite promoter-driven scIL12 was inserted between ORF60 and ORF61. (B) In vivo testing of Ellen-scIL12, Ellen-ΔORF8-scIL12, and Ellen-ΔORF65-scIL12. Human MeWo melanoma cells were implanted subcutaneously on day 0 (n=9). Viruses were IT delivered on days 20, 22, and 24 (5×104 PFUs per injection). Tumor growth curves were displayed. (C) Blood IL12 concentration. Plasma samples were collected on day 5 post-treatment, and IL12 p70 concentrations were determined by ELISA and compared with one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. (D) Deletion of ORF8 led to a decrease in treatment-related mortality. Treatment or tumor growth-related animal deaths were recorded for each group. ND, not detected. (E) Animal survival curves (Log-rank (Mantel-Cox) test). (F) Average body weight. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. IL12, interleukin-12; IT, intratumorally; PFU, plaque-forming unit; scIL12, single-chain IL12; VZV, varicella-zoster virus.

    Arming VZV with drug-controllable scIL12

    IL12 is a potent proinflammatory type 1 cytokine that promotes cell-mediated immunity. Even though early clinical trials of systemically administered IL12 revealed dose-limiting toxicities, encouraging preliminary results of both effectiveness and safety have emerged in phase I/II trials when replication-competent viral vectors encoding IL12 were IT delivered.33 34 To combine VZV virotherapy with IL12 immunotherapy and to add a switch for controlling IL12 expression in case of systemic IL12 toxicity, we replaced the ORF8 coding sequence in Ellen genome with the tet-off controllable scIL12, resulting in the generation of the Ellen-ΔORF8-tet-off-scIL12 virus (figure 5A). The scIL12 encodes IL12 p40 and p35 subunits as a fusion protein, and its efficacy has been shown to be comparable to that of the native IL12 p70 complex (online supplemental figure 3). Subsequent experimentation revealed that the presence of doxycycline effectively inhibited the expression of scIL12 in infected MeWo cells, while virus replication remained unaffected (figure 5B,C).

    Figure 5
    Figure 5

    VZV armed with drug-controllable scIL12. (A) Insertion of tet-off controllable scIL12 into VZV Ellen genome. The ORF8 coding sequence was replaced by tet-off-scIL12 cassette. tTA, tetracycline transactivator protein. The pTight promoter contains a modified tet response element (TRE). Without doxycycline, the tTA protein binds to TRE and activates the expression of scIL12. The introduction of doxycycline leads to its binding with tTA, thus silencing the transactivation of genes controlled by TRE. (B, C) Drug controllable expression of IL12 in Ellen-ΔORF8-tet-off-scIL12. The MeWo cells were seeded in a 24-well plate (2×105 cells/well) on day 0 and were cultured overnight. On day 1, cells were infected with Ellen-ΔORF8-tet-off-scIL12 (200 PFUs per well), and doxycycline was added to the infected cells at different concentrations (0, 1, or 5 µg/mL). IL12 p70 concentration in the supernatant and virus titers on day 4 were detected (n=3). For each virus, IL12 concentrations and virus titers in the wells with or without doxycycline treatment were compared (one-way ANOVA with Tukey’s multiple comparisons test). ns, not significant,****p<0.0001. IL12, interleukin-12; PFU, plaque-forming unit; scIL12, single-chain IL12; VZV, varicella-zoster virus.

    IL12-armed VZV induces systemic antitumor immune responses in the B16-F10-nectin1 syngeneic mouse melanoma model

    As previous studies have shown that IL12 efficacy is greatly reduced in nude mice,35 we sought to evaluate the efficacy of the IL12-armed Ellen virus in an immune-competent setting. We found that the B16-F10-nectin1 mouse melanoma cells were permissive to the Ellen-ΔORF8-tet-off-scIL12 virus in terms of entry, genome replication, and gene expression. However, it was impaired in producing infectious virions when compared with the production in human MeWo melanoma cells (online supplemental figure 4).36 37 In the bilateral B16-F10-nectin1 model (figure 6A), intratumoral administration of the Ellen-ΔORF8-tet-off-scIL12 virus demonstrated superior efficacy in inhibiting tumor growth of both injected and uninjected tumors, thereby extending animal survival in comparison to the Ellen-ΔORF8 virus (figure 6C,D). Additionally, intraperitoneal administration of doxycycline significantly reduced the expression of scIL12, subsequently diminishing the antitumor effectiveness of Ellen-ΔORF8-tet-off-scIL12 (figure 6B–D). The experiment included the HSV-1 KOS-ΔICP34.5ΔICP47-gBsyn-scIL12 virus, characterized by its hyperfusogenic gB and scIL12 expression along with the absence of both ICP34.5 and ICP47, as a comparative control.38 When given in equal doses (1×105 plaque-forming units (PFUs) per injection), the Ellen-ΔORF8-tet-off-scIL12 virus exhibited better overall antitumor efficacy in comparison to the KOS-ΔICP34.5ΔICP47-gBsyn-scIL12 virus (figure 6C,D). After being injected, the oncolytic HSV derived from KOS may cause the lysis of target cells and was then rapidly cleared by the immune system.38 No treatment-related toxicity was observed across all treatment groups (figure 6D,E).

    Figure 6
    Figure 6

    In vivo evaluation of Ellen-ΔORF8-tet-off-scIL12 in the bilateral B16-F10-nectin1 melanoma syngeneic model. (A) Schematic of experimental design. B16-F10-nectin1 cells were implanted subcutaneously on both flanks of C57BL/6J mice on day 0 (n=10 per group). Viruses were IT delivered (1×105 PFUs per injection) into tumors on the right flank on days 11, 13, and 15. Each mouse received daily intraperitoneal administration of doxycycline (2.5 mg/kg) or vehicle control (phosphate-buffered saline (PBS)) from day 11 to day 17. The KOS-ΔICP34.5ΔICP47-gBsyn-scIL12 virus was HSV-1 KOS strain derived. (B) Blood IL12 concentration. Plasma samples were collected on day 5 post-treatment (n=5), and IL12 p70 concentrations were compared with one-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test. (C, D) Efficacy of Ellen-ΔORF8-tet-off-scIL12. Tumor growth curves were displayed, and animal survival curves were compared with the Log-rank (Mantel-Cox) test. (E) Average body weight. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. HSV-1, herpes simplex virus type 1; IL12, interleukin-12; IT, intratumorally; PFU, plaque-forming unit; scIL12, single-chain IL12.

    To assess the systemic antitumor immune responses elicited by the Ellen-ΔORF8-tet-off-scIL12 virus in the bilateral B16-F10-nectin1 model, spleen cells and tumors were harvested 8 days after treatment. Interferon (IFN)-γ-Elispot analysis revealed that spleen cells showed a positive immune response against B16-F10-nectin1 tumor cells and tumor antigen peptides (TRP2180-188/gp10025-33) in the group treated with Ellen-ΔORF8. The introduction of scIL12 in the Ellen-ΔORF8-tet-off-scIL12 virus further intensified these antitumor immune responses (figure 7A,B). Additionally, the immune cells that infiltrated both the injected and uninjected tumors were characterized using flow cytometry, applying various immune cell markers. This analysis indicated that the use of the two Ellen viruses did not significantly alter the counts of natural killer (NK) cells within the tumors (figure 7C). However, there was a notable increase in both tumor-associated CD11b+ myeloid cells and CD11b+/F4/80+ macrophages in injected and uninjected tumors post Ellen-ΔORF8 treatment. The Ellen-ΔORF8-tet-off-scIL12 virus further significantly increased the levels of CD11b+/F4/80+ macrophages in injected tumors and CD11b+ myeloid cells in uninjected tumors (figure 7D,E). Furthermore, counts of CD3+ T cells and CD3+/granzyme B+ cytotoxic T cells were elevated in both injected and uninjected tumors following Ellen-ΔORF8 treatment, with the scIL12 expression further significantly boosting the abundance of both T cell types (figure 7F,G). Overall, these findings suggest that the Ellen-ΔORF8-tet-off-scIL12 virus successfully triggered antitumor immune responses, enhancing the presence of CD11b+/F4/80+ macrophages, T cells, and granzyme B+ cytotoxic T cells within the tumors in the B16-F10-nectin1 model.

    Figure 7
    Figure 7

    Oncolytic VZV induces systemic antitumor immune responses in the bilateral B16-F10-nectin1 melanoma syngeneic model. B16-F10-nectin1 cells were implanted subcutaneously on both flanks of C57BL/6J mice on day 0. Viruses were IT delivered (1×105 PFUs per injection for Ellen-ΔORF8 and Ellen-ΔORF8-tet-off-scIL12) into tumors on the right flank on days 11, 13, and 15. On day 8 post-treatment, spleens, injected tumors, and uninjected tumors were collected. (A, B) IFN-γ Elispot analysis of virus-induced antitumor immune responses. A total of 1×105 spleen cells per well were cocultured with 1×104 B16-F10-nectin1 cells or antigen peptide (Trp2 180-188 or gp100 25-33) for 3 days before signal development (n=4). Spot numbers for each group were counted and compared (unpaired t-test). SFU, spot-forming unit. (C) Abundance of CD3−/NK1.1+ natural killer (NK) cells in injected and uninjected tumors (n=4, unpaired t-test). (D, E) Abundance of CD11b+ myeloid cells and CD11b+/ F4/80+ macrophages in injected and uninjected tumors (n=4, unpaired t-test). (F, G) Abundance of CD3+ T cells and CD3+/granzyme B+ cytotoxic T cells in injected and uninjected tumors (n=4, unpaired t-test). ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. IFN, interferon; IL12, interleukin-12; IT, intratumorally; PFU, plaque-forming unit; scIL12, single-chain IL12; VZV, varicella-zoster virus.

    Discussion

    For decades, extensive research has focused on oncolytic HSVs. However, the effectiveness of existing virotherapies derived from HSVs, such as T-VEC and DELYTACT, remains constrained.39 Remarkably, despite indications of potential links between natural VZV infections and favorable outcomes in diverse cancer types,10 11 the oncolytic properties of this prevalent human α-herpesvirus have not been extensively explored, with only one prior publication on the topic.40 In this study, we constructed new oncolytic VZVs and tested them both in a human melanoma xenograft model and a syngeneic mouse melanoma model. Encouragingly, both the vOka and Ellen strains of VZV exhibited robust antitumor effects in the human MeWo melanoma model. We found that removing the ORF8 gene attenuated VZV’s virulence without compromising its antitumor potency. Tet-off controllable scIL12 was further added to the VZV backbone as a therapeutic transgene. Notably, in the immune-competent B16-F10 melanoma model, the VZV Ellen-ΔORF8-tet-off-scIL12 virus triggered systemic antitumor immune responses. These findings collectively underscore the potential of VZV as a pioneering platform for oncolytic virotherapy.

    We deliberately selected the vOka and Ellen strains for our study, taking into consideration the potential risks associated with clinical isolates, which could potentially lead to disseminated and visceral infections in patients with cancer, especially those with compromised immune systems.41 The vOka strain was developed through a series of sequential passages involving human embryonic lung cells, guinea pig embryo fibroblasts, WI-38 cells, and MRC-5 cells from the parental Oka strain. This process led to the accumulation of mutations within the vOka genome, resulting in the attenuation of the virus’s ability to cause disease.42 On the other hand, the Ellen strain represents a lab-adapted variant that has undergone over 90 passages in human cell lines. Both vOka and Ellen were proven to be less virulent in implanted human skin tissues than low passage clinical isolates,43 44 supporting the safety profile of vOka and Ellen strains as potential oncolytic vectors. Nevertheless, our findings demonstrate that the Ellen strain offers greater suitability to accommodate therapeutic transgenes compared with the vOka strain. As a result, we opted to advance our research using the Ellen strain for constructing oncolytic VZV variants that incorporate both viral gene deletions and therapeutic genes.

    The transmission of VZV relies on direct cell-to-cell contact between infected cells and uninfected cells. VZV particles, released through exocytosis, adhere to the plasma membrane of infected cells. Subsequently, these particles enter neighboring cells either through endocytosis or by directly fusing with the cell membrane.45 To augment the local spread of the virus, we engineered a hyperfusogenic variant by introducing a Y881F mutation into the gB protein, which is part of the core fusion machinery of gB/gH–gL.46 While the hyperfusogenic phenotype was confirmed in laboratory settings, our results in the MeWo melanoma tumor model revealed a significant reduction both in the mutant virus’s replication and its oncolytic activity. This outcome could likely be attributed to the disruption caused by the enhanced cell-to-cell fusion, affecting both the virus and host gene transcriptome.27 In contrast to other oncolytic viral platforms, it appears that the hyperfusogenic mutation might not confer the same benefits in enhancing the oncolytic potential of VZV.

    Unlike HSV, all immune-regulatory factors encoded by VZV are important for the growth of the virus, and deletion of these genes would result in growth arrest or defects.29 Therefore, we turned our attention to VZV non-essential genes. VZV has around 14 non-essential genes: 3 of them (ORF1, ORF2, and ORF15) encode membrane proteins, 4 genes (ORF11, ORF12, ORF57, and ORF64) express tegument proteins, and another 4 genes (ORF8, ORF13, ORF36, and ORF59) are involved in virus DNA replication.29 Functions of ORF3 and ORF58 are unknown, while ORF65 is predicted to promote virus transneuronal transportation.30 Importantly, the gene ORF36, responsible for thymidine kinase, was not targeted for mutation due to its vital role in activating antiviral drugs.47 After a careful selection process that excluded viral structural proteins and proteins with unclear functions, ORF8 and ORF65 were chosen because potentially they are involved in virus neurovirulence.30 We found that the deletion of ORF8 significantly reduced virulence in the neuronal dSH-SY5Y cells, ARPE-19 epithelial cells, and MeWo xenograft tumors. Deletion of ORF65 didn’t reduce virus transmission in ARPE-19 epithelial cells. Moreover, the removal of ORF8 or ORF65 did not compromise the virus’s antitumor efficacy as demonstrated in the MeWo tumor model. As a result, we proceeded with the removal of ORF8 to enhance the virus’s safety profile without compromising its effectiveness.

    Arming oncolytic viruses with therapeutic transgenes holds significant importance in boosting antitumor immune responses. IL12 has been considered a strong candidate for cancer immunotherapy, as it orchestrates the Th1-type immune response and potentiates both cytotoxic T cells and NK cells.33 The ability of VZV to recruit lymphocytes to the infection sites suggests that the concurrent use of IL12 in conjunction with oncolytic VZV could potentially lead to synergistic antitumor effects. Our efforts led to the successful incorporation of scIL12 into the Ellen virus. However, the attempt to integrate scIL12 into the vOka virus was unsuccessful in reconstitution. This failure could potentially be attributed to the presence of accumulated mutations within the vOka genome, compounded by the expression of scIL12, which might have triggered a growth arrest in the vOka strain.

    In vivo experiments with the Ellen-scIL12 and Ellen-ΔORF8-scIL12 in the immunocompromised human MeWo melanoma model revealed IL12-induced toxicity and mortality at nanogram levels per milliliter of blood. Conversely, the immunocompetent B16-F10-nectin1 model showed no such toxicity, with IL12 levels in the hundreds of picograms per milliliter range. In addition, our in vitro Elispot data indicated that IL12 effectively activated immune cells at 50 pg/mL. These findings underscore the need for careful IL12 monitoring in clinical environments. To enable drug-mediated control over IL12 expression, the Ellen strain was engineered to include a tet-off system controlling scIL12 expression, enabling IL12 regulation via doxycycline administration. This doxycycline-regulated IL12 expression has been confirmed through both in vitro and in vivo studies.

    Assessing virus-induced antitumor immune responses necessitates immune-competent models. When investigating oncolytic VZV, a limitation arises from the virus’s human-specific nature, resulting in non-productive infection within mouse cell lines.36 37 Despite the efficacy of the scIL12-armed Ellen virus being observed in the B16-F10-nectin1 model, comprehensively evaluating its engagement with antitumor immunity within mouse models remains difficult.

    Another constraint associated with VZV pertains to its dosage. VZV is highly cell-associated, and the generation of high titers of cell-free VZV particles in the laboratory is difficult.48 To address this limitation and augment the viral payload for delivery, one potential approach might be to deliver cell carriers infected with VZV. Notably, human mesenchymal stem cells have effectively served as carriers for transporting VZV to glioma cell spheroids ex vivo,40 presenting a viable method for increasing the viral payload available for in vivo delivery.

    In summary, a novel oncolytic VZV platform was built based on the lab strain Ellen. This platform involved the removal of ORF8 to reduce virulence, along with the integration of a tet-off controllable scIL12 as a therapeutic transgene. The oncolytic potency of this VZV platform was validated through preclinical studies conducted in melanoma models.

    Materials and methods

    All primers were listed in online supplemental table 1.

    Recombinant VZVs

    To generate a VZV capturing BAC vector, two homology arms were amplified from the vOka episomal DNA using primers No 1–4 and then were cloned into the pSG2 vector using restriction sites BamHI and HindIII, obtained vector was pSG6. The chloramphenicol resistance gene (CmR) cassette in the pSG6 vector was then replaced with the neomycin resistance cassette pPGK-pEM7-NeoR using primers No 5–8, obtained vector was pSG7. pSG7 was linearized with BamHI and transfected into APRE-19 cells (2 µg of linearized pSG7 was added per 5×105 cells), at 24 hours post-transfection live VZV (VZV vOka virus from Merk (VARIVAX) or VZV Ellen virus from ATCC (VR-1367)) was added to the transfected cells with a multiplicity of infection=0.05. At 48 hours postinfection, G418 (Geneticin) was added to cells with a final concentration of 800 µg/mL to enrich the GFP+ infected cells. The circular VZV BAC DNA was extracted from the GFP+ cells and transferred to DH10B competent cells (C640003, Invitrogen). BAC colonies containing full-length VZV genome were selected. VZV-BAC DNA was then transformed into the SW102 cells (National Cancer Institute Biological Resources Branch). Sequence integrity of the VZV-BACs was verified by next-generation sequencing (Genewiz Illumina MiSeq, 2×250 bp configuration). The cytomegalovirus immediate-early gene promoter-NIS (pCMV-NIS) cassette and pCMV-bHCG cassette were amplified from plasmids pcSV40-pCMV-NIS and pcSV40-pCMV-bHCG using primers No 9 and 10, and then inserted between ORF60 and ORF61 of VZV genome within SW102 cells. The scIL12 expression cassette was amplified and inserted between ORF60 and ORF61 of VZV genome using primers No 11 and 13 or used to replace the AmpR sequence of the pcSV40-pCMV-bHCG cassette inserted using primers No 11 and 12. The mouse scIL12 cassette (mIL12BA) was constructed to encode IL12 p40 and p35 subunits as a fusion protein with an elastin linker between the subunits. The mouse IL12 p40, IL12 p35, and scIL12 coding sequences were then cloned into the pSelect-zeo vector (psetz-mcs, Invivogen) under the control of the EF1/HTLV composite promoter. The tet-off drug-controllable pTight-scIL12 cassette was amplified from vector pTet-off-mIL12BA-ZeoR and then was used to replace the ORF8 coding sequence in VZV genome using primers No 14 and 24. The pRSV-tTA cassette was inserted downstream of the pTight-scIL12 cassette using primers No 25 and 26. Doxycycline (D5207, Sigma) was added to inhibit the expression of the tet-off controllable scIL12 encoded by VZV. The vOka carrying gBY881F hyperfusogenic mutation was constructed using primers No 15–17. The ORF8 and ORF65 were deleted from VZV genome using primers No 18–20 and Primers No 21–23, respectively. Recombinant VZVs were reconstituted in ARPE-19 cells by FuGENE 6 Transfection Reagent (E2691, Promega) or in ARPE-19-cre cells (for removing the BAC sequence).

    Cell-free VZV was produced as described previously.48 In brief, VZV-infected ARPE-19 cells in T175 flasks were resuspended in cold phosphate-buffered saline (PBS)-sucrose-glutamate-serum buffer (PSGC) (6 mL PSGC buffer per T175 flask). The cells were then frozen in liquid nitrogen and thawed in a 37°C water bath for two times before being sonicated with a VCX-130 Vibra-Cell sonicator at 1 min intervals (6×2 min). The absence of intact cells was verified under a microscope. The large cell debris was then removed by centrifugation at 3000×g for 15 min. Virus in the supernatant was pelleted down using the Lenti-X Concentrator reagent (631231, Takara). Virus pellets were resuspended in PBS and were stored at −80°C. For animal studies that require cell preparations as the control group, uninfected ARPE-19 cells underwent processing using the identical procedure as described above for the production of cell-free virus. VZV titers (PFUs) were determined in ARPE-19 monolayers by plaque formation assay. The HSV-1 KOS-ΔICP34.5ΔICP47-gBsyn-scIL12 virus was grown in Vero cells and purified with the method as described previously.38

    Cell lines

    The ARPE-19 cells (CRL-2302, ATCC), ARPE-19-cre cells, MeWo cells (HTB-65, ATCC), B16-F10 cells (CRL-6475, ATCC), B16-F10-nectin1 cells, and Vero cells (CCL-81, ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 100 units/mL of penicillin/streptomycin. B16-F10-nectin1 cells were constructed to stably express the human nectin1 and support HSV-1 replication.38 The SH-SY5Y cells (CRL-2266, ATCC) were cultured in DMEM/F12 Medium with 100 units/mL of penicillin/streptomycin and 10% FBS. Differentiated human neuronal dSH-SY5Y cells were obtained by culturing SH-SY5Y cells in DMEM/F12 medium containing 2% FBS and 10 µM retinoic acid (R2625, Sigma) for 3 days followed by a 5-day differentiation in DMEM/F12 medium supplemented with 100 ng/mL beta-nerve growth factor (788506, Biolegend) and 50 ng/mL brain-derived neurotrophic factor (788904, Biolegend).32 All tumor cell lines were authenticated by IDEXX BioAnalytics.

    Animal studies

    For the human MeWo melanoma immune-comprised tumor model, MeWo cells suspended in PBS (5×107 cells/mL) were subcutaneously implanted to the right flanks (100 µL per mouse) of 6 weeks old female nude mice (NCRNU-F, Taconic). For the syngeneic mouse B16-F10-nectin1 melanoma tumor model, B16-F10-nectin1 tumor cells suspended in PBS (1×107 cells/mL) were subcutaneously implanted to both flanks (100 µL per side of flank) of 5–6 weeks old female C57BL/6J mice (000664, The Jackson Laboratory). When the average tumor diameter reached 5 mm, viruses or control cell preparations were IT delivered. Totally three doses were given for each mouse at indicated time points. The body weight and tumor volumes were monitored three times per week postvirus injection. Blood and tumor samples were collected at indicated time points. Animals were euthanized when mice had weight loss equal to or exceeding 20% of baseline, tumor burden that equals or exceeds 10% of body weight, and development of hind limb paralysis or focal motor deficits. Spleens were collected when animals were euthanized. For each experimental group, there were no exclusions of animals, experimental units, or data points.

    ELISA analysis

    Human hCG-beta ELISA (ELH-hCGb-1, RayBiotech), Mouse IL-12 p70/p40 DuoSet ELISA (DY419-05, R&D systems), and Mouse IL-12 p35 ELISA (MBS2515782, MyBioSource) analyses were performed according to the manufacturer’s instructions.

    Immunohistochemistry analysis

    Tumors were fixed in 4% paraformaldehyde (PFA) for 3 days and were dehydrated with 30% sucrose in PBS at 4°C for 2 days. The tumor was then subjected to cryosectioning with a Leica CM1860 Cryostat (tumor slices were set at 40 µm). Tumor slices were penetrated with 1% Triton X-100 in Tris-buffered saline (TBS) buffer and stained with 4′,6-diamidino-2-phenylindole (62248, Invitrogen) and antibodies (Rabbit Anti-Varicella Zoster Virus ORF68 Polyclonal Antibody (VRX-0233J, Creative-Biolabs) and Alexa Fluor 594 tagged Goat Anti-Rabbit IgG (ab150080, Abcam)). Images were collected with a Zeiss LSM 780 confocal microscope.

    Elispot assays

    IFN-γ Elispot assays were conducted using the Mouse IFN-gamma ELISpot Kit (XEL485, R&D Systems) as according to the manufacturer’s instructions.49 Spleens were collected on day 8 post-treatment. Spleen cells suspended in Roswell Park Memorial Institute (RPMI)-1640 Medium (supplemented with 10% FBS, 10 ng/mL mIL2, and 1 µM 2-mercaptoethanol) were loaded to the 96-well plates (1×105 spleen cells per well). Antitumor cell immunity was analyzed by loading live tumor cells (1×104 tumor cells per well) into the spleen cell preseeded wells. Antitumor antigen immunity was analyzed by loading tumor antigen peptides (Trp2 180–188 or gp100 25–33, a final concentration of 5 µM for each peptide) to the spleen cell preseeded wells. After tumor cells, peptides, or IL12s were loaded, the plates were cultured at 5% CO2 and 37°C for 3 days before spots were developed and counted. Phytohemagglutinin P (inh-phap, Invivogen) (100 µg/mL) was added to the positive control wells.

    Quantitative PCR (qPCR)

    Total DNA and RNA were extracted from VZV-infected cells using the hirt method50 and the Rneasy plus mini kit (74134, QIAGEN). The LunaScript RT SuperMix Kit (E3010, NEB) was used for first-strand cDNA synthesis. qPCR quantitation of VZV genome copy numbers in total DNA and ORF62, ORF28, and ORF68 mRNA levels in total RNA was performed using the Luna Universal qPCR Master Mix (M3003, NEB) by a Roche LightCycler 96 System. VZV genome was quantified using primers No 27 and 28. ORF62, ORF28, and ORF68 mRNA levels were determined using primers No 29–34. The qPCR thermocycling protocol was set up as: (1) initial denaturation (95℃, 60 s, 1 cycle); (2) 45 cycles of two-step amplification (95℃ denaturation for 15 s, 60℃ extension for 30 s); (3) melt curve (60−95℃, 1 cycle). The purified Ellen-BAC plasmid was diluted as standards to generate the standard curves, and copy numbers were calculated with the LightCycler Software.

    Flow cytometry

    B16-F10-nectin1 tumors were minced into small pieces and digested for 30 min at 37°C with RPMI medium containing 0.1% collagenase type I (SCR103, Sigma), 0.2% dispase type II (D4693, Sigma), and 1% DNAse I (11284932001, Roche). Cell suspension was first passed through a 70 µm cell strainer and then incubated in 3 mL red blood cell lysis buffer (420301, Biolegend) per tumor sample. Cell suspension was pelleted by centrifugation for 5 min at 350×g and resuspended in flurescence-activated cell sorting (FACS) buffer (1 mM EDTA, and 2% FBS in Ca/Mg2+ free Hanks Buffer) before flow cytometry analysis.

    To determine the abundance of tumor-associated myeloid cells, macrophages, NK cells, and T cells in B16-F10-nectin1 tumors, dissociated tumor cells were stained with Zombie Aqua dye (423101, Biolegend) and fixed with 4% PFA. For analyzing myeloid cells and macrophages, cells were stained with Pacific Blue tagged antimouse/human CD11b antibody (101223, Biolegend) and Alexa Fluor 647 tagged antimouse F4/80 antibody (123122, Biolegend), or with isotype controls. For phenotyping NK cells and T cells, cells were stained with Pacific Blue antimouse NK-1.1 antibody (108722, Biolegend), Alexa Fluor 647 tagged antimouse CD3 antibody (100209, Biolegend) and fluorescein isothiocyanate (FITC) tagged antimouse granzyme B antibody (372206, Biolegend), or with isotype controls. Stained cells were analyzed with a BD LSRFortessa X-20 Cell Analyzer.

    Statistics

    Statistical analyses of data were performed using GraphPad Prism 8 software. Values were presented as mean±SEM. All data sets passed the normality test (Shapiro-Wilk test or Kolmogorov-Smirnov test). Data sets were subjected to one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test or Sidak’s multiple comparisons test, two-way ANOVA (Tukey’s multiple comparisons test or Dunnett’s multiple comparisons test), unpaired t-test (two-tailed), or multiple t-tests to calculate p values for group comparisons. p<0.05 was considered significant. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Data availability statement

    Data are available upon reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information.

    Ethics statements

    Patient consent for publication

    Ethics approval

    All mice experiments were approved by Mayo Clinic Institutional Animal Care and Use Committee (IACUC) and were performed in compliance with Mayo Clinic IACUC guidelines (IACUC protocol A00003155-18).

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    Footnotes

    • Contributors HJ and SJR designed the study, interpreted the data, and prepared the manuscript. HJ constructed the recombinant viruses and stable cell lines and performed the ELISA, immunohistochemistry, quantitative PCR, and flow cytometry analyses. HJ and LZ prepared and analyzed the recombinant viruses. HJ and RN conducted the animal studies. TFC prepared the spleen cells, and HJ conducted the Elispot analyses. KWP interpreted the data. HJ is responsible for the overall content as guarantor.

    • Funding This work was supported by New Frontiers Program of Vyriad, Inc. (Funding ID: FP00108613).

    • Competing interests None declared.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.