Vaccinia Virus Copenhagen Research Articles

Abstract 6657: Oncolytic Vaccinia Virus Carrying OPCML Tumor Suppressor is active in Epithelial Ovarian Cancer

Abstract Objective: Epithelial Ovarian cancer (EOC) has the highest mortality rate among gynecologic malignancies, necessitating novel therapeutic approaches. Viral oncolytic immunotherapy with recombinant oncolytic vaccinia virus (OVV) offers promise for advanced-stage cancer treatment. OPCML is a GPI-anchored 3 Ig-domain tumor suppressor inactivated by somatic methylation in 87% of EOC. OVV with OPCML (OVV-OPCML) gene insertion may have potential as a therapeutic agent. This study explores the oncolytic, signaling-inhibitory, anti-angiogenic and synergistic immunotherapeutic properties of OVV-OPCML in EOC treatment, evaluating its therapeutic potential. Methods: To create OVV-OPCML, the virulent vaccinia virus Copenhagen strain F3L site was modified with OPCML cDNA, confirmed by qPCR and western blot. Oncolytic potential was assessed through in vitro and in vivo EOC cell infection with OVV-OPCML. Real-time cell analysis (RTCA) assessed cell proliferation. Xenograft tumor models in nude mice were used to evaluate in vivo oncolytic activity, while OVV-OPCML combined with anti-PD-1 treatment was tested using a syngeneic orthotopic EOC mouse model (ID8) in C57BL/6 immunocompetent mice. Serum IFN-γ and TNF-α were quantified via ELISA. Receptor Tyrosine Kinases, angiogenesis and phosphorylation together with immune markers were examined by western blotting and by multiplex immunohistochemistry (mIHC) analysis of tumor sections. Results: OVV-OPCML selectively infected OC cells, leading to viral replication and tumor cell death, while sparing normal ovarian cells in-vitro. OPCML protein expression increased post-infection. OVV-OPCML reduced ERBB2, EGFR, and FGFR1 expression in SKVO3 OV cells. Intratumoral or intraperitoneal (IP) OVV-OPCML administration in human ovarian cancer nude mouse models reduced tumor growth and improved survival. IP in-vivo OVV-OPCML in combination with anti-PD-1 treatment in vivo, in the IP ID8 syngeneic model demonstrated synergy with survival improvement. H&E staining showed tumor cell damage without significant harm to normal tissues. Protein expression of ERBB2, EGFR, FGFR1, and their phosphorylated forms decreased. CD31, VEGFR2, p-VEGFR2 (Tyr1175), and p-VEGFR2 (Tyr1214) downregulation suggested tumor angiogenesis disruption. OVV-OPCML treatment elevated serum TNF-α and INF-γ levels and increased CD8+ T cells and PD-L1 expression in OVV-OPCML treated syngeneic tumors. Conclusions: OPCML gene enhances OVV selectivity against OC cells through direct oncolysis and OPCML protein production, inhibiting specific RTKs, suppressing angiogenesis, and activating antitumor immune responses synergistically with anti-PD-1. OVV-OPCML therapy shows promise as a targeted strategy for ovarian cancer treatment. Citation Format: Yong Tang, Yingzhao Liu, Mingjing Deng, Fei Huang, Jiahu Wang, Paul Blake, Hani Gabra, Qi Wang. Oncolytic Vaccinia Virus Carrying OPCML Tumor Suppressor is active in Epithelial Ovarian Cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6657.

High Oncolytic Activity of a Double-Deleted Vaccinia Virus Copenhagen Strain against Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a cancer of the pleura that lacks efficient treatment. Oncolytic immunotherapy using oncolytic vaccinia virus (VV) may represent an alternative therapeutic approach for the treatment of this malignancy. Here, we studied the oncolytic activity of VV thymidine kinase (TK)-ribonucleotide reductase (RR)-/green fluorescent protein (GFP) against MPM. This virus is a VV from the Copenhagen strain that is deleted of two genes encoding the TK (J2R) and the RR (I4L) and that express the GFP. First, we show in vitro that VVTK-RR-/GFP efficiently infects and kills the twenty-two human MPM cell lines used in this study. We also show that the virus replicates in all eight tested MPM cell lines, however, with approximately a 10-fold difference in the amplification level from one cell line to another. Then, we studied the therapeutic efficiency of VVTK-RR-/GFP in non-obese diabetic (NOD) severe combined immunodeficient (SCID) mice that bear peritoneal human MPM tumors. One intraperitoneal infection of VVTK-RR-/GFP reduces the tumor burden and significantly increases mice survival compared to untreated animals. Thus, VVTK-RR- may be a promising oncolytic virus (OV) for the oncolytic immunotherapy of MPM.

Abstract 4576: Oncolytic vaccinia virus TG6010 expressing cytidine deaminase induces DNA damage and shows potent anti-tumor effects

Abstract The recombinant Vaccinia virus Copenhagen strain deleted in Thymidine Kinase and Ribonucleotide Reductase (VVCOPTK−RR−) is a potent and versatile oncolytic platform that has demonstrated strong activity in various preclinical models. The deletion of such VV genes inhibits viral replication in normal cells, while retaining therapeutic replication in tumor cells. TG6002, a VVCOPTK−RR− expressing the suicide gene FCU1, is under investigation in Phase I trials in patients with advanced gastrointestinal tumors. We have developed a new product based on the VVCOPTK−RR− vector, named TG6010, expressing the human cytidine deaminase (CDA) that efficiently catalyzes the deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. The tumor specific expression of CDA by the VV leads to a depletion of cytidine and deoxycytidine. This cytidine/deoxycytidine depletion resulting from CDA overexpression, activated a DNA damage response highlighted by an induction of H2AX phosphorylation. Next, to validate the potential therapeutic use of TG6010, we analyzed the effects of the virus on human xenograft tumors implanted in mice. We observed, after systemic injection of TG6010, high expression of CDA in the tumors with a significant increase in DNA damage in vivo as revealed by the γH2AX foci assay. In addition, intraperitoneal administration of TG6010 in a xenograft model of human ovarian carcinomatosis resulted in significantly prolonged animal survival. In conclusion, the oncolytic vaccinia virus expressing the cytidine deaminase (TG6010) has shown potent anti-tumor effects. Mechanistically, due to the cytidine deaminase overexpression, we observed induction of a DNA damage response. These are data encouraging of clinical development for this highly potent oncolytic Vaccinia virus TG6010 against ovarian cancer. Citation Format: Johann Foloppe, Juliette Kempf, Christelle Pichon, Annie Findeli, Isabelle Farine, Julie Hortelano, Baptiste Moreau, Michèle Klein, Sandrine Cochin, Renée Brandely, Véronique Koerper, Nathalie Silvestre, Christelle Machon, Jérôme Guitton, Eric Quéméneur, Philippe Erbs. Oncolytic vaccinia virus TG6010 expressing cytidine deaminase induces DNA damage and shows potent anti-tumor effects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4576.

Canine Adipose-Derived Mesenchymal Stem Cells (cAdMSCs) as a "Trojan Horse" in Vaccinia Virus Mediated Oncolytic Therapy against Canine Soft Tissue Sarcomas.

Several oncolytic viruses (OVs) including various human and canine adenoviruses, canine distemper virus, herpes-simplex virus, reovirus, and members of the poxvirus family, such as vaccinia virus and myxoma virus, have been successfully tested for canine cancer therapy in preclinical and clinical settings. The success of the cancer virotherapy is dependent on the ability of oncolytic viruses to overcome the attacks of the host immune system, to preferentially infect and lyse cancer cells, and to initiate tumor-specific immunity. To date, several different strategies have been developed to overcome the antiviral host defense barriers. In our study, we used canine adipose-derived mesenchymal stem cells (cAdMSCs) as a “Trojan horse” for the delivery of oncolytic vaccinia virus Copenhagen strain to achieve maximum oncolysis against canine soft tissue sarcoma (CSTS) tumors. A single systemic administration of vaccinia virus-loaded cAdMSCs was found to be safe and led to the significant reduction and substantial inhibition of tumor growth in a CSTS xenograft mouse model. This is the first example that vaccinia virus-loaded cAdMSCs could serve as a therapeutic agent against CSTS tumors.

A highly specific monoclonal antibody against monkeypox virus detects the heparin binding domain of A27

The eradication of smallpox and the cessation of global vaccination led to the increased prevalence of human infections in Central Africa. Serologic and protein-based diagnostic assay for MPXV detection is difficult due to cross-reactive antibodies that do not differentiate between diverse orthopoxvirus (OPXV) species. A previously characterized monoclonal antibody (mAb 69-126-3-7) against MPXV [1] was retested for cross-reactivity with various OPXVs. The 14.5kDa band protein that reacted with mAb 69-126-3 was identified to be MPXV A29 protein (homolog of vaccinia virus Copenhagen A27). Amino acid sequence analysis of the MPXV A29 with other OPXV homologs identified four amino acid changes. Peptides corresponding to these regions were designed and evaluated for binding to mAb 69-126-3 by ELISA and BioLayer Interferometry (BLI). Further refinement and truncations mapped the specificity of this antibody to a single amino acid difference in a 30-mer peptide compared to other OPXV homologs. This particular residue is proposed to be essential for heparin binding by VACV A27 protein. Despite this substitution, MPXV A29 bound to heparin with similar affinity to that of VACV A27 protein, suggesting flexibility of this motif for heparin binding. Although binding of mAb 69-126-3-7 to MPXV A29 prevented interaction with heparin, it did not have any effect on the infectivity of MPXV. Characterization of 69-126-3-7 mAb antibody allows for the possibility of the generation of a serological based species-specific detection of OPXVs despite high proteomic homology.

Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKɛ-mediated IRF activation

Viruses are detected by different classes of pattern recognition receptors (PRRs), such as Toll-like receptors and RIG-like helicases. Engagement of PRRs leads to activation of interferon (IFN)-regulatory factor 3 (IRF3) and IRF7 through IKKepsilon and TBK1 and consequently IFN-beta induction. Vaccinia virus (VACV) encodes proteins that manipulate host signalling, sometimes by targeting uncharacterised proteins. Here, we describe a novel VACV protein, K7, which can inhibit PRR-induced IFN-beta induction by preventing TBK1/IKKepsilon-mediated IRF activation. We identified DEAD box protein 3 (DDX3) as a host target of K7. Expression of DDX3 enhanced Ifnb promoter induction by TBK1/IKKepsilon, whereas knockdown of DDX3 inhibited this, and virus- or dsRNA-induced IRF3 activation. Further, dominant-negative DDX3 inhibited virus-, dsRNA- and cytosolic DNA-stimulated Ccl5 promoter induction, which is also TBK1/IKKepsilon dependent. Both K7 binding and enhancement of Ifnb induction mapped to the N-terminus of DDX3. Furthermore, virus infection induced an association between DDX3 and IKKepsilon. Therefore, this study shows for the first time the involvement of a DEAD box helicase in TBK1/IKKepsilon-mediated IRF activation and Ifnb promoter induction.

A real time PCR assay for the detection and quantification of orf virus

A real time quantitative PCR assay based on TaqMan ® technology was developed for orf virus (ORFV) DNA quantification in clinical samples, infected cells and organotypic cultures. This method was based on the amplification of a 70 bp fragment from the ORFV B2L gene (orthologue of the Vaccinia virus Copenhagen F13L gene) that encodes the major envelope protein. Both intra- and inter-assay variability were well within ±0.25 log 10 S.D. showing the high efficiency and reproducibility of the assay. The TaqMan ® PCR was subsequently used to determine the titre of several batches of the ORFV strain NZ-2, with it being possible to quantify virus solutions in the range of 1 × 10 1 to 1 × 10 6 TCID 50/ml. A good correlation between the titre determined by the TaqMan ® PCR and by conventional endpoint dilution was found. The PCR assay is reproducible and can be used for a rapid quantification of ORFV in vitro and ex vivo, being readily achievable within 1 h.

Amplification refractory mutation system PCR assays for the detection of variola and Orthopoxvirus

PCR assays that can identify the presence of variola virus (VARV) sequences in an unknown DNA sample were developed using principles established for the amplification refractory mutation system (ARMS). The assay’s specificity utilised unique single nucleotide polymorphisms (SNP) identified among Orthopoxvirus (OPV) orthologs of the vaccinia virus Copenhagen strain A13L and A36R genes. When a variola virus specific primer was used with a consensus primer in an ARMS assay with different O rthopoxvirus genomes, a PCR product was only amplified from variola virus DNA. Incorporating a second consensus primer into the assay produced a multiplex PCR that provided Orthopoxvirus generic and variola-specific products with variola virus DNA. We tested two single nucleotide polymorphisms with a panel of 43 variola virus strains, collected over 40 years from countries across the world, and have shown that they provide reliable markers for variola virus identification. The variola virus specific primers did not produce amplicons with either assay format when tested with 50 other Orthopoxvirus DNA samples. Our analysis shows that these two polymorphisms were conserved in variola virus genomes and provide a reliable signature of Orthopoxvirus species identification.

The vaccinia virus C12L protein inhibits mouse IL-18 and promotes virus virulence in the murine intranasal model.

A bioassay that measured the interleukin (IL)-12-induced production of interferon (IFN)-gamma from mouse splenocytes was used to identify a soluble factor in the supernatants of vaccinia virus (VV)-infected cells that inhibited the production of IFN-gamma. This soluble factor was expressed by 14 out of 16 VV strains including the Western Reserve (WR) strain, but strains Copenhagen and Tashkent and a mutant of strain WR called 6/2 lacked this activity. The gene encoding this activity was identified as C12L by transferring DNA present in VV WR but missing in VV WR 6/2 into VV Copenhagen and testing for expression of the soluble factor. The C12L protein shows amino acid similarity to IL-18 binding proteins that are encoded by poxviruses, mice and humans, and C12L protein produced from VV or baculovirus inhibited the biological activity of mouse IL-18 in vitro. Thus the inhibition of IL-12-induced IFN-gamma production was due to indirect effects of C12L on IL-18, illustrating the synergistic action of these pro-inflammatory cytokines. To study the role of the C12L protein in the virus life-cycle, we constructed a deletion mutant lacking the C12L gene and a revertant virus in which the gene was reinserted into the deletion mutant. In vitro the replication and plaque size of these viruses were indistinguishable. However, infection of BALB/c mice by the intranasal route showed that the deletion mutant was attenuated and induced lower weight loss and signs of illness compared to controls.

Vaccinia virus WR gene A5L is required for morphogenesis of mature virions.

The vaccinia virus WR A5L open reading frame (corresponding to open reading frame A4L in vaccinia virus Copenhagen) encodes an immunodominant late protein found in the core of the vaccinia virion. To investigate the role of this protein in vaccinia virus replication, we have constructed a recombinant virus, vA5Li, in which the endogenous gene has been deleted and an inducible copy of the A5 gene dependent on isopropyl-beta-D-thiogalactopyranoside (IPTG) for expression has been inserted into the genome. In the absence of inducer, the yield of infectious virus was dramatically reduced. However, DNA synthesis and processing, viral protein expression (except for A5), and early stages in virion formation were indistinguishable from the analogous steps in a normal infection. Electron microscopy revealed that the major vaccinia virus structural form present in cells infected with vA5Li in the absence of inducer was immature virions. Viral particles were purified from vA5Li-infected cells in the presence and absence of inducer. Both particles contained viral DNA and the full complement of viral proteins, except for A5, which was missing from particles prepared in the absence of inducer. The particles prepared in the presence of IPTG were more infectious than those prepared in its absence. The A5 protein appears to be required for the immature virion to form the brick-shaped intracellular mature virion.

Vaccinia virus strains Lister, USSR and Evans express soluble and cell-surface tumour necrosis factor receptors.

Poxviruses encode a broad range of proteins that interfere with host immune functions such as soluble versions of cytokine receptors. Soluble virus tumour necrosis factor receptors (vTNFRs) were described originally in myxoma and Shope fibroma viruses. Cowpox virus (CPV) encodes three vTNFRs (CrmB, CrmC and CrmD). The genes equivalent to CrmB and CrmC in vaccinia virus (VV) Copenhagen are mutated and are named B28R/C22L and A53R, respectively. CrmD was identified recently in CPV and ectromelia virus but the gene is absent in VV Copenhagen. We have tested for expression of soluble binding activity for human TNF in cultures infected with 18 orthopoxviruses and have found that TNFRs are mostly absent but are produced by VV strains Lister, USSR and Evans, by the CPV elephantpox and by camelpox virus. Interestingly, we also found TNFR activity on the surface of cells infected with VV Lister, USSR and Evans. Sequence analysis of the relevant regions in VV Lister identified an intact A53R gene and an inactive B28R gene. Expression of VV Lister A53R in baculovirus and VV Western Reserve demonstrated that gene A53R encodes an active soluble vTNFR of 22 kDa. Expression and characterization of recombinant vTNFRs from VV Lister (A53R) and CPV (CrmB and CrmC) showed a similar binding specificity, with each receptor binding TNF from man, mouse and rat, but not human lymphotoxin-alpha. Lastly, the VV Lister and CPV vTNFRs bind human TNF with high affinity and prevent the binding of TNF to cellular receptors.

Recombinant vaccine-induced protection against the highly pathogenic simian immunodeficiency virus SIV(mac251): dependence on route of challenge exposure.

Vaccine protection from infection and/or disease induced by highly pathogenic simian immunodeficiency virus (SIV) strain SIV(mac251) in the rhesus macaque model is a challenging task. Thus far, the only approach that has been reported to protect a fraction of macaques from infection following intravenous challenge with SIV(mac251) was the use of a live attenuated SIV vaccine. In the present study, the gag, pol, and env genes of SIV(K6W) were expressed in the NYVAC vector, a genetically engineered derivative of the vaccinia virus Copenhagen strain that displays a highly attenuated phenotype in humans. In addition, the genes for the alpha and beta chains of interleukin-12 (IL-12), as well as the IL-2 gene, were expressed in separate NYVAC vectors and inoculated intramuscularly, in conjunction with or separate from the NYVAC-SIV vaccine, in 40 macaques. The overall cytotoxic T-lymphocyte (CTL) response was greater, at the expense of proliferative and humoral responses, in animals immunized with NYVAC-SIV and NYVAC-IL-12 than in animals immunized with the NYVAC-SIV vaccine alone. At the end of the immunization regimen, half of the animals were challenged with SIV(mac251) by the intravenous route and the other half were exposed to SIV(mac251) intrarectally. Significantly, five of the eleven vaccinees exposed mucosally to SIV(mac251) showed a transient peak of viremia 1 week after viral challenge and subsequently appeared to clear viral infection. In contrast, all 12 animals inoculated intravenously became infected, but 5 to 6 months after viral challenge, 4 animals were able to control viral expression and appeared to progress to disease more slowly than control animals. Protection did not appear to be associated with any of the measured immunological parameters. Further modulation of immune responses by coadministration of NYVAC-cytokine recombinants did not appear to influence the outcome of viral challenge. The fact that the NYVAC-SIV recombinant vaccine appears to be effective per se in the animal model that best mirrors human AIDS supports the idea that the development of a highly attenuated poxvirus-based vaccine candidate can be a valuable approach to significantly decrease the spread of human immunodeficiency virus (HIV) infection by the mucosal route.