Replication efficiency of oncolytic vaccinia virus in cell cultures prognosticates the virulence and antitumor efficacy in mice

<p>Abstract</p> <p>Background</p> <p>We have shown that insertion of the three vaccinia virus (VACV) promoter-driven foreign gene expression cassettes encoding <it>Renilla </it>luciferase-<it>Aequorea </it>GFP fusion protein, β-galactosidase, and β-glucuronidase into the <it>F14.5L</it>, <it>J2R</it>, and <it>A56R </it>loci of the VACV LIVP genome, respectively, results in a highly attenuated mutant strain GLV-1h68. This strain shows tumor-specific replication and is capable of eradicating tumors with little or no virulence in mice. This study aimed to distinguish the contribution of added VACV promoter-driven transcriptional units as inserts from the effects of insertional inactivation of three viral genes, and to determine the correlation between replication efficiency of oncolytic vaccinia virus in cell cultures and the virulence and antitumor efficacy in mice</p> <p>Methods</p> <p>A series of recombinant VACV strains was generated by replacing one, two, or all three of the expression cassettes in GLV-1h68 with short non-coding DNA sequences. The replication efficiency and tumor cell killing capacity of these newly generated VACV strains were compared with those of the parent virus GLV-1h68 in cell cultures. The virus replication efficiency in tumors and antitumor efficacy as well as the virulence were evaluated in nu/nu (nude) mice bearing human breast tumor xenografts.</p> <p>Results</p> <p>we found that virus replication efficiency increased with removal of each of the expression cassettes. The increase in virus replication efficiency was proportionate to the strength of removed VACV promoters linked to foreign genes. The replication efficiency of the new VACV strains paralleled their cytotoxicity in cell cultures. The increased replication efficiency in tumor xenografts resulted in enhanced antitumor efficacy in nude mice. Similarly, the enhanced virus replication efficiency was indicative of increased virulence in nude mice.</p> <p>Conclusions</p> <p>These data demonstrated that insertion of VACV promoter-driven transcriptional units into the viral genome for the purpose of insertional mutagenesis did modulate the efficiency of virus replication together with antitumor efficacy as well as virulence. Replication efficiency of oncolytic VACV in cell cultures can predict the virulence and therapeutic efficacy in nude mice. These findings may be essential for rational design of safe and potent VACV strains for vaccination and virotherapy of cancer in humans and animals.</p

Poxvirus-Encoded Gamma Interferon Binding Protein Dampens the Host Immune Response to Infection

Ectromelia virus (ECTV), a natural mouse pathogen and the causative agent of mousepox, is closely related to variola virus (VARV), which causes smallpox in humans. Mousepox is an excellent surrogate small-animal model for smallpox. Both ECTV and VARV encode a multitude of host response modifiers that target components of the immune system and that are thought to contribute to the high mortality rates associated with infection. Like VARV, ECTV encodes a protein homologous to the ectodomain of the host gamma interferon (IFN-γ) receptor 1. We generated an IFN-γ binding protein (IFN-γbp) deletion mutant of ECTV to study the role of viral IFN-γbp (vIFN-γbp) in host-virus interaction and also to elucidate the contribution of this molecule to the outcome of infection. Our data show that the absence of vIFN-γbp does not affect virus replication per se but does have a profound effect on virus replication and pathogenesis in mice. BALB/c mice, which are normally susceptible to infection with ECTV, were able to control replication of the mutant virus and survive infection. Absence of vIFN-γbp from ECTV allowed the generation of an elective host immune response that was otherwise diminished by this viral protein. Mice infected with a vIFN-γbp deletion mutant virus, designated ECTV-IFN-γbpΔ, produced increased levels of IFN-γ and generated robust cell-mediated and antibody responses. Using several strains of mice that exhibit differential degrees of resistance to mousepox, we show that recovery or death from ECTV infection is determined by a balance between the host's ability to produce IFN-γ and the virus' ability to dampen its effects

Oncolytic vaccinia therapy of squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Novel therapies are necessary to improve outcomes for patients with squamous cell carcinomas (SCC) of the head and neck. Historically, vaccinia virus was administered widely to humans as a vaccine and led to the eradication of smallpox. We examined the therapeutic effects of an attenuated, replication-competent vaccinia virus (GLV-1h68) as an oncolytic agent against a panel of six human head and neck SCC cell lines.</p> <p>Results</p> <p>All six cell lines supported viral transgene expression (β-galactosidase, green fluorescent protein, and luciferase) as early as 6 hours after viral exposure. Efficient transgene expression and viral replication (>150-fold titer increase over 72 hrs) were observed in four of the cell lines. At a multiplicity of infection (MOI) of 1, GLV-1h68 was highly cytotoxic to the four cell lines, resulting in ≥ 90% cytotoxicity over 6 days, and the remaining two cell lines exhibited >45% cytotoxicity. Even at a very low MOI of 0.01, three cell lines still demonstrated >60% cell death over 6 days. A single injection of GLV-1h68 (5 × 10⁶pfu) intratumorally into MSKQLL2 xenografts in mice exhibited localized intratumoral luciferase activity peaking at days 2–4, with gradual resolution over 10 days and no evidence of spread to normal organs. Treated animals exhibited near-complete tumor regression over a 24-day period without any observed toxicity, while control animals demonstrated rapid tumor progression.</p> <p>Conclusion</p> <p>These results demonstrate significant oncolytic efficacy by an attenuated vaccinia virus for infecting and lysing head and neck SCC both <it>in vitro </it>and <it>in vivo</it>, and support its continued investigation in future clinical trials.</p

Genomic sequence and analysis of a vaccinia virus isolate from a patient with a smallpox vaccine-related complication

BACKGROUND: Vaccinia virus (VACV)-DUKE was isolated from a lesion on a 54 year old female who presented to a doctor at the Duke University Medical Center. She was diagnosed with progressive vaccinia and treated with vaccinia immune globulin. The availability of the VACV-DUKE genome sequence permits a first time genomic comparison of a VACV isolate associated with a smallpox vaccine complication with the sequence of culture-derived clonal isolates of the Dryvax vaccine. RESULTS: This study showed that VACV-DUKE is most similar to VACV-ACAM2000 and CLONE3, two VACV clones isolated from the Dryvax(® )vaccine stock confirming VACV-DUKE as an isolate from Dryvax(®). However, VACV-DUKE is unique because it is, to date, the only Dryvax(® )clone isolated from a patient experiencing a vaccine-associated complication. The 199,960 bp VACV-DUKE genome encodes 225 open reading frames, including 178 intact genes and 47 gene fragments. Between VACV-DUKE and the other Dryvax(® )isolates, the major genomic differences are in fragmentation of the ankyrin-like, and kelch-like genes, presence of a full-length Interferon-α/β receptor gene, and the absence of a duplication of 12 ORFs in the inverted terminal repeat. Excluding this region, the DNA sequence of VACV-DUKE differs from the other two Dryvax(® )isolates by less than 0.4%. DNA sequencing also indicated that there was little heterogeneity in the sample, supporting the hypothesis that virus from an individual lesion is clonal in origin despite the fact that the vaccine is a mixed population. CONCLUSION: Virus in lesions that result from progressive vaccinia following vaccination with Dryvax are likely clonal in origin. The genomic sequence of VACV-DUKE is overall very similar to that of Dryvax(® )cell culture-derived clonal isolates. Furthermore, with the sequences of multiple clones from Dryvax(® )we can begin to appreciate the diversity of the viral population in the smallpox vaccine

Regression of Human Prostate Tumors and Metastases in Nude Mice following Treatment with the Recombinant Oncolytic Vaccinia Virus GLV-1h68

Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In the current study, we analyzed the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 against two human prostate cancer cell lines DU-145 and PC-3 in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 was able to infect, replicate in, and lyse these prostate cancer cells in culture. In DU-145 and PC-3 tumor xenograft models, a single intravenous injection with GLV-1h68 resulted in a significant reduction of primary tumor size. In addition, the GLV-1h68-infection led to strong inflammatory and oncolytic effects resulting in drastic reduction of regional lymph nodes with PC-3 metastases. Our data documented that the GLV-1h68 virus has a great potential for treatment of human prostate carcinoma

VAI TRÒ CỦA HỆ VI SINH VẬT ĐƯỜNG RUỘT TRONG ĐIỀU TRỊ KHỐI U CỦA VACCINIA VIRUS GLV-1h68

GLV-1h68 là một vaccinia virus tái tổ hợp nhược độc mang ba gen ngoại lai mã hóa cho Renilla luciferase–GFP protein dung hợp (ruc-gfp), b-galactosidase (lacZ), và b-glucuronidase (gusA) được lần lượt được chèn vào các vị trí F14.5L, J2R (mã hóa cho thymidine kinase) và A56R (mã hóa cho hemagglutinin) của hệ gen vaccinia virus LIVP. GLV-1h68 đã chứng minh là một tác nhân sinh học có khả năng đặc hiệu định hướng khối u, nhân lên và phá hủy nhiều dòng tế bào ung thư của người. Trong nghiên cứu này chúng tôi sử dụng kết hợp 4 loại kháng sinh kháng sinh để tạm thời loại bỏ hầu hết vi sinh vật đường ruột của những lô chuột được gây u thực nghiệm với dòng tế bào ung thư phổi của người (A549), tiếp đó chuột mang khối u được chữa trị bằng GLV-1h68. Kết quả cho thấy ở khối u của những lô chuột được dùng kháng sinh, kích thước khối u nhỏ hơn, đồng thời số lượng virus nhân lên nhiều hơn so với lô đối chứng không sử dụng kháng sinh. Phát hiện này mở ra một hướng nghiên cứu mới trong việc sử dụng kháng sinh kết hợp với virus để nâng cao hiệu quả điều trị ung thư

Functional hyper-IL-6 from vaccinia virus-colonized tumors triggers platelet formation and helps to alleviate toxicity of mitomycin C enhanced virus therapy

<p>Abstract</p> <p>Background</p> <p>Combination of oncolytic vaccinia virus therapy with conventional chemotherapy has shown promise for tumor therapy. However, side effects of chemotherapy including thrombocytopenia, still remain problematic.</p> <p>Methods</p> <p>Here, we describe a novel approach to optimize combination therapy of oncolytic virus and chemotherapy utilizing virus-encoding hyper-IL-6, GLV-1h90, to reduce chemotherapy-associated side effects.</p> <p>Results</p> <p>We showed that the hyper-IL-6 cytokine was successfully produced by GLV-1h90 and was functional both in cell culture as well as in tumor-bearing animals, in which the cytokine-producing vaccinia virus strain was well tolerated. When combined with the chemotherapeutic mitomycin C, the anti-tumor effect of the oncolytic virotherapy was significantly enhanced. Moreover, hyper-IL-6 expression greatly reduced the time interval during which the mice suffered from chemotherapy-induced thrombocytopenia.</p> <p>Conclusion</p> <p>Therefore, future clinical application would benefit from careful investigation of additional cytokine treatment to reduce chemotherapy-induced side effects.</p

Significant Growth Inhibition of Canine Mammary Carcinoma Xenografts following Treatment with Oncolytic Vaccinia Virus GLV-1h68

Canine mammary carcinoma is a highly metastatic tumor that is poorly responsive to available treatment. Therefore, there is an urgent need to identify novel agents for therapy of this disease. Recently, we reported that the oncolytic vaccinia virus GLV-1h68 could be a useful tool for therapy of canine mammary adenoma in vivo. In this study we analyzed the therapeutic effect of GLV-1h68 against canine mammary carcinoma. Cell culture data demonstrated that GLV-1h68 efficiently infected and destroyed cells of the mammary carcinoma cell line MTH52c. Furthermore, after systemic administration, this attenuated vaccinia virus strain primarily replicated in canine tumor xenografts in nude mice. Finally, infection with GLV-1h68 led to strong inflammatory and oncolytic effects resulting in significant growth inhibition of the tumors. In summary, the data showed that the GLV-1h68 virus strain has promising potential for effective treatment of canine mammary carcinoma

Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus

Introduction: Oncolytic viruses show promise for treating cancer. However, to assess therapeutic efficacy and potential toxicity, a noninvasive imaging modality is needed. This study aimed to determine if insertion of the human sodium iodide symporter (hNIS) cDNA as a marker for non-invasive imaging of virotherapy alters the replication and oncolytic capability of a novel vaccinia virus, GLV-1h153. Methods: GLV-1h153 was modified from parental vaccinia virus GLV-1h68 to carry hNIS via homologous recombination. GLV-1h153 was tested against human pancreatic cancer cell line PANC-1 for replication via viral plaque assays and flow cytometry. Expression and transportation of hNIS in infected cells was evaluated using Westernblot and immunofluorescence. Intracellular uptake of radioiodide was assessed using radiouptake assays. Viral cytotoxicity and tumor regression of treated PANC-1tumor xenografts in nude mice was also determined. Finally, tumor radiouptake in xenografts was assessed via positron emission tomography (PET) utilizing carrier-free (124)I radiotracer. Results: GLV-1h153 infected, replicated within, and killed PANC-1 cells as efficiently as GLV-1h68. GLV-1h153 provided dose-dependent levels of hNIS expression in infected cells. Immunofluorescence detected transport of the protein to the cell membrane prior to cell lysis, enhancing hNIS-specific radiouptake (P < 0.001). In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts (P < 0.001). Finally, intratumoral injection of GLV-1h153 facilitated imaging of virus replication in tumors via (124)I-PET. Conclusion: Insertion of the hNIS gene does not hinder replication or oncolytic capability of GLV-1h153, rendering this novel virus a promising new candidate for the noninvasive imaging and tracking of oncolytic viral therapy