Semliki Forest Virus Expressing Interleukin-12 Induces Antiviral and Antitumoral Responses in Woodchucks with Chronic Viral Hepatitis and Hepatocellular Carcinoma

A vector based on Semliki Forest virus (SFV) expressing high levels of interleukin-12 (SFV-enhIL-12) has previously demonstrated potent antitumoral efficacy in small rodents with hepatocellular carcinoma (HCC) induced by transplantation of tumor cells. In the present study, the infectivity and antitumoral/antiviral effects of SFV vectors were evaluated in the clinically more relevant woodchuck model, in which primary HCC is induced by chronic infection with woodchuck hepatitis virus (WHV). Intratumoral injection of SFV vectors expressing luciferase or IL-12 resulted in high reporter gene activity within tumors and cytokine secretion into serum, respectively, demonstrating that SFV vectors infect woodchuck tumor cells. For evaluating antitumoral efficacy, woodchuck tumors were injected with increasing doses of SFV-enhIL-12, and tumor size was measured by ultrasonography following treatment. In five (83%) of six woodchucks, a dose-dependent, partial tumor remission was observed, with reductions in tumor volume of up to 80%, but tumor growth was restored thereafter. Intratumoral treatment further produced transient changes in WHV viremia and antigenemia, with ≥1.5-log₁₀ reductions in serum WHV DNA in half of the woodchucks. Antitumoral and antiviral effects were associated with T-cell responses to tumor and WHV antigens and with expression of CD4 and CD8 markers, gamma interferon, and tumor necrosis factor alpha in peripheral blood mononuclear cells, suggesting that immune responses against WHV and HCC had been induced. These experimental observations suggest that intratumoral administration of SFV-enhIL-12 may represent a strategy for treatment of chronic HBV infection and associated HCC in humans but indicate that this approach could benefit from further improvements.Hepatocellular carcinoma (HCC) is a major public health problem worldwide, representing the fifth most common type of cancer. HCC is also the third leading cause of cancer-related death, mainly because only surgical and local ablative therapeutic options have shown efficacy in patients with this type of cancer (21). Approximately 80% of all HCC cases are attributed to chronic infection with hepatitis C virus and/or hepatitis B virus (HBV). Chronic carriers of HBV have a greater than 100-fold-increased relative risk of developing HCC compared to HBV-uninfected humans, with an annual incidence rate of 2 to 6% in cirrhotic patients. The high incidence of HCC, together with its poor prognosis and limited therapeutic options, warrants the development of new treatment strategies for this disease.There is increasing evidence that stimulation of the immune system for subsequent recognition and killing of tumor cells may be a valuable treatment option for liver cancer. In general, HCC appears to be an attractive target for immunotherapy because cases of spontaneous tumor regression have been reported, HCC is often infiltrated with lymphocytes, and HCC-associated proteins such as alpha-fetoprotein may be used as targets for immune-mediated killing of tumors (5, 49).A promising strategy to stimulate the deficient antitumoral immune response is based on the transfer and subsequent expression of immunostimulatory genes in tumor cells using viral or nonviral delivery vectors. One of the most effective immunostimulatory cytokines is interleukin-12 (IL-12), a protein usually expressed by macrophages and dendritic cells. IL-12 has been demonstrated to induce strong antitumoral effects that are mediated by the stimulation of T-helper cell type 1 (Th1) responses, including the activation of cytolytic T lymphocytes (CTL) and natural killer cells, and by the inhibition of angiognesis (48, 50). All of these effects are dependent on the production of gamma interferon (IFN-γ). Viral vectors that are based on adenovirus have been used to deliver IL-12 into several animal models with transplantable HCC, resulting in a localized expression of this cytokine and usually leading to antitumoral effects (3, 14, 37). However, and despite successful treatment of HCC in preclinical studies, a phase I clinical trial with a first-generation adenoviral vector for delivery and expression of IL-12 in patients with primary and metastatic liver cancer produced only a modest antitumoral effect (41). This poor response was probably due to the low and transient IL-12 expression in tumors. These results in humans indicated a need for vectors with higher potency and for preclinical testing in relevant models of HCC (i.e., large animals with spontaneous tumors).Vectors based on Semliki Forest virus (SFV), a member of the alphavirus group, are highly efficient in inducing antitumoral responses in a variety of animal models (2, 9, 10, 39, 44, 53). The SFV vector used in the present study is based on a viral RNA genome in which the region coding for the structural proteins has been replaced by a heterologous gene (24). Recombinant SFV RNA can be transcribed in vitro and transfected into cells, resulting in viral replication and subsequent production of a subgenomic RNA from which the heterologous protein is expressed at very high levels. Recombinant SFV RNA can be packaged into viral particles (vp) by cotransfecting it into cells together with two helper RNAs coding for the capsid and the envelope proteins (43). Compared to adenoviral vectors expressing IL-12, tumor treatment with SFV vectors expressing the same cytokine resulted in greater antitumoral effects in a murine colon adenocarcinoma model and also in a rat orthotopic HCC model (16, 39). The greater antitumoral effect mediated by SFV vectors has been attributed to the higher expression of IL-12 and to the induction of apoptosis caused by SFV replication within tumor cells. Apoptosis leads to the release of tumor antigens that can be taken up by antigen-presenting cells, thereby potentiating the antitumoral response induced by IL-12 (54). Furthermore, SFV vectors have low immunogenicity when delivered intratumorally, allowing repetitive administrations into the same tumor, which is not possible with adenoviral vectors (38).In the present study, the antitumoral efficacy of an SFV vector expressing IL-12 (SFV-enhIL-12) was investigated in woodchucks with HCC. The Eastern woodchuck (Marmota monax) is frequently infected with the woodchuck hepatitis virus (WHV), which is closely related to the human HBV in its structure, genomic organization, mechanism of replication, and course of infection (29). The woodchuck has been used as a mammalian model for research on HBV, including the pathogenesis of acute and chronic HBV infection, and for preclinical evaluation of the safety and efficacy of candidate antiviral drugs and therapeutic immunomodulators for the treatment of chronic HBV infection (29) and prevention of HCC (47).All woodchucks chronically infected with WHV as neonates develop HCC, and the median time for tumor appearance is 24 months of age (34, 47). After identification of HCC, the median survival time of woodchucks is 6 months, a situation similar to that for patients with HCC. In addition, WHV-induced hepatocarcinogenesis shows strong similarity to HBV-induced carcinogenesis in humans (34, 47). These features of HCC that are associated with persistent hepatitis virus infection make the woodchuck model unique compared to other animal models, in which HCC is induced by a chemical carcinogen or by transplantation of established tumor cell lines into immune-deficient or immune-compatible hosts. Woodchucks with large liver tumors that acquire malignant characteristics in a stepwise process similar to HCC in humans are an attractive and suitable model for the preclinical evaluation of new treatment strategies for HBV-induced HCC in humans (47).The antitumoral efficacy of a SFV vector expressing high levels of IL-12 (SFV-enhIL-12) was investigated in six woodchucks with established chronic WHV infection and primary HCC. The results demonstrate that SFV-delivered IL-12 expression produced a dose-dependent, partial tumor remission that was associated with a general activation of cellular immune responses against HCC. The antitumoral activity, in addition to an antiviral activity against WHV, and the favorable safety profile in woodchucks suggest that a therapeutic approach based on SFV-enhIL-12 may represent a treatment strategy for HCC in patients with chronic HBV infection, but the overall results also indicate that this approach needs further improvement for inducing a complete tumor remission.