The goal of this study was to determine the oncolytic efficiency of H-1PV against intracerebral gliomas in animal models. The data show the capacity of H-1PV to eliminate even advanced and symptomatic glioma xenografts in the absence of pathogenic effects on normal tissues, including adjacent brain areas. This is in line with the innocuousness of H-1PV for adult rats in natural infections.22
In most studies of virus-based local therapy of ic gliomas, the treatment was initiated within only a few days after tumor cell implantation.23–27
Recently, a prolonged survival of symptomatic animals after viral therapy with an oncolytic herpesvirus was reported.28
Although in this setting the virus (HSV-1) was able to cure animals when administered 14 days after tumor implantation, it prolonged survival, but failed to eradicate the tumor in cases of advanced and symptomatic disease after 19 days. Also in the models used for iv treatment of gliomas with VSV, the lethal effect of the virus prevented assessment of long-term survival. As shown here for oncolytic parvo-(H-1PV)virotherapy, a complete cure of animals symptomatic from gliomas at late tumor stages could be achieved, resulting in long-term survival.
The efficiency of H-1PV in inducing glioma regression is likely to be boosted by an increase in the initial virus dose as a result of the replication of input virus in primarily infected tumor cells, followed by spreading of progeny virus to and secondary infection of remaining tumor cells. In keeping with this mechanism, H-1PV DNA, transcripts, and proteins (in particular the nonstructural viral products driving replication) accumulated in the tumor (remnants), starting from a few days after virus injection. Furthermore, indication of transient viremia originating from the tumor was given by the time-dependent transfer of viral DNA to nontumoral tissues, first in the periphery of the lesion and subsequently in distant organs. Infection of nontumoral tissues appeared to be cryptic as no H-1PV gene expression could be detected even when these tissues tested positive for the presence of viral DNA, which vanished within 2 weeks p.i. This specificity of H-1PV replication and cytopathogenicity for neoplastic tissues, in particular gliomas, shows that the parvovirus keeps under in vivo
conditions the oncotropism that it displays in cell cultures.13,16
The spreading of the virus through the brain and to other organs could be of therapeutic significance. One main obstacle in the treatment of malignant gliomas lies in the early migration of tumor cells away from the primary tumor site, resulting in reduced accessibility for local therapies. Secondary rounds of infection by oncolytic viruses without further spreading of progeny particles beyond the margins of the tumor might therefore not be sufficient for successful virotherapy.
An additional advantage of H-1PV lies in its ability to circumvent the mechanisms allowing glioma cells to resist other cytotoxic agents. This feature was traced back to the capacity of the parvovirus to activate a nonconventional, cathepsin-mediated death pathway as recently demonstrated in cultures of H-1PV-infected glioma cells.13
The selective up-regulation of cathepsin B in tumor tissues of animals treated with H-1PV is in line with these in vitro
findings and is most likely induced by the virus. However, the detailed analysis of death pathways after H-1PV infection in vivo
was beyond the scope of this proof of concept study.
The antiglioma effect of H-1PV could be achieved both by direct ic injection of the virus inoculum into the tumor and by repeated iv injection. This study shows for the first time the capacity of H-1PV to cross the blood–brain barrier and target and destroy ic gliomas. To achieve this effect, the virus dose was 50-fold higher than by stereotactic treatment, and even this high concentration of viral particles did not cause any side effects in treated animals. H-1PV infection resulted in oncolytic effects within the tumor in all animals with progressing gliomas. As obviously not all animals could be cured with either local or systemic therapy, the current methodology may be further improved, for example, by administering the virus through intratumoral convection-enhanced delivery, by further increasing the virus dose, or by combining intratumoral and systemic virotherapy. This possibility for the combination of local and systemic H-1PV application could be a major advantage for future therapy.
The RG-2 model system used in this study allowed viral therapy to be assessed in immunocompetent animals, mimicking in this regard the patient's situation. This setup raises the question of the possible involvement of the immune system in the tumor suppression observed. In our study, implantation of only 3000 RG-2 glioma cells led to the development of a lethal brain tumor in all untreated animals and even in some of the rats that received H-1PV therapy. This is in contrast to Mariani et al.29
who showed spontaneous regression of RG-2 tumors in this system, correlating with the occurrence of antitumor immune responses. Under our experimental conditions, glioma suppression proved to fully depend on H-1PV infection and to be associated with the expression of viral cytotoxic proteins in regressing tumor areas. Infection of large tumors with subsequent virus production and spreading within the brain and to other organs did not lead to inflammatory changes or related pathology. No obvious infiltration of treated tumors with immune cells was noticed in histological sections. In particular, the number of CD3-positive cells in the tumor area (cryosections) of glioma-bearing rats did not differ significantly between H-1PV and mock-treated animals (11 days post-tumor cell implantation, 4 days p.i.; data not shown). The possibility still needs to be considered that oncolytic H-1PV serves, at least in part, as an adjuvant to promote anticancer vaccination through the release of tumor-associated antigens and additional immunostimulating activities.30
In keeping with this possibility, a tumor challenge of all 4 cured animals 1 year after successful treatment failed to result in the development of RG-2 gliomas even when cells were injected in 30-fold greater numbers into the contralateral hemisphere (data not shown). Investigations addressing this observation are currently in progress to assess the capacity of the immune system to take over from the initial oncolytic activity of H-1PV and complete tumor eradication. It should, however, be stated that H-1PV treatment leads to oncolysis and prolonged survival without side effects in immunodeficient animals bearing human glioma xenografts, pointing to the fact that an adaptive cell-mediated immune response is not an absolute prerequisite for H-1PV-induced glioma suppression.
In conclusion, this study produces proof of the concept that H-1PV can be used as an efficient and safe oncolytic virus for glioma treatment in humans using different routes of infection. Although this study was not designed to determine the minimum efficient dose of the virus, it can serve as a basis to calculate the dose range to be administered to humans, by considering a rat to human body weight ratio of 1:300. The titers calculated in this way can be achieved using the H-1PV production system presently available. It should also be noted that, in a previous trial, the inoculation of cancer patients with H-1PV was found to be devoid of significant harmful side effects.14
In this pilot study, purified virus was injected in accessible skin metastases from different kinds of primary solid tumors. Each patient received 2 or 3 injections of H-1PV, at intervals of 10 days. Single doses administered ranged from 108
pfu. Toxicity was absent or mild, and a maximal tolerated dose was not reached. The presence of H-1PV could be demonstrated by a transient viremia and seroconversion as well as by in situ virus replication in neoplastic lesions. This innocuousness of H-1PV to humans, together with the fact that preexisting antiviral immunity (a possible reason for a low efficiency of virus-based treatments) is rare or even nonexistent in the case of H-1PV in the human population,7
render this parvovirus a very promising candidate for virotherapy.
Altogether, the data presented and discussed in this study lay the foundation for the initiation of a phase I/IIa clinical trial using H-1PV as a novel therapeutic agent for the treatment of patients with high-grade gliomas.