The immunogenicity of the tumor-associated antigen α-fetoprotein is enhanced by a fusion with a transmembrane domain.

AIM: To investigate the ability of recombinant modified vaccinia virus Ankara (rMVA) vector to induce an immune response against a well-tolerated self-antigen. METHODS: rMVA vectors expressing different form of α-fetoprotein (AFP) were produced and characterized. Naïve mice were vaccinated with MVA vectors expressing the AFP antigen in either a secreted, or a membrane-bound, or an intracellular form. The immune response was monitored by an IFNΓ ELISpot assay and antibody detection. RESULTS: Vaccination with the membrane-associated form of AFP induced a stronger CD8(+) T-cell response compared to the ones obtained with the MVA encoding the secreted or the intracellular forms of AFP. Moreover, the vaccination with the membrane-bound AFP elicited the production of AFP-specific antibodies. CONCLUSIONS: The AFP transmembrane form is more immunogenic. Expressing a membrane-bound form in the context of an MVA vaccination could enhance the immunogenicity of a self-antigen.

AFP-specific immunotherapy impairs growth of autochthonous hepatocellular carcinoma in mice.

BACKGROUND AND AIMS: In this study, we have assessed the potential of antigen-specific immunotherapy against hepatocellular carcinoma (HCC) in conditions of low tumour burden, in an autochthonous HCC model. METHODS: Diethylnitrosamine (DEN) injected into infant mice results in the development of multi-nodular HCC in which alpha-fetoprotein (AFP) is re-expressed. DEN-injected animals received an antigen-specific immunization with a synthetic vector consisting of a low dose of AFP-encoding plasmid formulated with the amphiphilic block copolymer 704 (DNAmAFP/704). Animals were treated at 4 and 5 months, before macroscopic nodules were detected, and were sacrificed at 8 months. The tumour burden, as well as liver histology, was assessed. AFP and MHC class I molecule expression in the nodules were monitored by qRT-PCR. RESULTS: The AFP-specific immunotherapy led to a significant (65%) reduction in tumour size. The reduced expression of AFP and MHC class I molecules was measured in the remaining nodules taken from the DNAmAFP/704-treated group. CONCLUSIONS: This is the first study demonstrating the relevance of antigen-specific immunotherapy in an autochthonous HCC model. In this context, we validated the use of an anti-tumour immunotherapy based on vaccination with nanoparticles consisting of low dose antigen-encoding DNA formulated with a block copolymer. Our results demonstrate the potential of this strategy as adjuvant immunotherapy to reduce the recurrence risk after local treatment of HCC patients.

Differential biodistribution of oncolytic poxvirus administered systemically in an autochthonous model of hepatocellular carcinoma.

BACKGROUND: Preclinical studies have demonstrated that, unlike oncolytic adenoviruses, oncolytic vaccinia viruses can reach implanted tumors upon systemic injection. However, the biodistribution of this oncolytic agent in in situ autochthonous tumor models remains poorly characterized. In the present study, we assessed this biodistribution in a model of mouse hepatocellular carcinoma (HCC) obtained after injection of the carcinogen diethylnitrosamine (DEN). METHODS: Twelve months after DEN administration, histology, quantitative reverse transcription-polymerase chain reaction, in situ hybridization and viral titration were used to characterize tumors, as well as to assess the viral load of the livers upon either intravenous or intraperitoineal injection. RESULTS: The results obtained showed that the architecture of the liver was lost, with a noticeable absence of sinusoids, as well as the presence of steatosis and α-fetoprotein-positive HCC tumor nodules. Bioluminescence imaging and measures of the infective virus load demonstrated that intravenous injection of 10(8) plaque-forming units of the recombinant vaccinia virus led to a predominant transduction of the liver, whereas intraperitoneal injection resulted in a lower level of liver transduction accompanied by an increased infection of the lungs, spleen, kidneys and bowels. Immunohistochemical analysis of liver sections of animals injected intravenously with the virus revealed a preferential localization of vaccinia-specific immunoreactivity in the tumors. CONCLUSIONS: The findings of the present study emphasize the importance of the route of administration of the vector and highlight the relevance of systemic injection of oncolytic vaccinia virus in the context of hepatocellular carcinoma.

Assessment of the Na/I symporter as a reporter gene to visualize oncolytic adenovirus propagation in peritoneal tumours.

PURPOSE: In vivo imaging of the spread of oncolytic viruses using the Na/I symporter (NIS) has been proposed. Here, we assessed whether the presence of NIS in the viral genome affects the therapeutic efficacy of the oncolytic adenovirus dl922-947 following intraperitoneal administration, in a mouse model of peritoneal ovarian carcinoma. METHODS: We generated AdAM7, a dl922-947 oncolytic adenovirus encoding the NIS coding sequence. Iodide uptake, NIS expression, infectivity and cell-killing activity of AdAM7, as well as that of relevant controls, were determined in vitro. In vivo, the propagation of this virus in the peritoneal cavity of tumour-bearing mice was determined using SPECT/CT imaging and its therapeutic efficacy was evaluated. RESULTS: In vitro infection of ovarian carcinoma IGROV-1 cells with ADAM7 led to functional expression of NIS. However, the insertion of NIS into the viral genome resulted in a loss of efficacy of the virus in terms of replication and cytotoxicity. In vivo, on SPECT/CT imaging AdAM7 was only detectable in the peritoneal cavity of animals bearing peritoneal ovarian tumours for up to 5 days after intraperitoneal administration. Therapeutic experiments in vivo demonstrated that AdAM7 is as potent as its NIS-negative counterpart. CONCLUSION: This study demonstrated that despite the detrimental effect observed in vitro, insertion of the reporter gene NIS in an oncolytic adenovirus did not affect its therapeutic efficacy in vivo. We conclude that NIS is a highly relevant reporter gene to monitor the fate of oncolytic adenovectors in live subjects.

Adrenal gland infection by serotype 5 adenovirus requires coagulation factors.

Recombinant, replication-deficient serotype 5 adenovirus infects the liver upon in vivo, systemic injection in rodents. This infection requires the binding of factor X to the capsid of this adenovirus. Another organ, the adrenal gland is also infected upon systemic administration of Ad, however, whether this infection is dependent on the cocksackie adenovirus receptor (CAR) or depends on the binding of factor X to the viral capsid remained to be determined. In the present work, we have used a pharmacological agent (warfarin) as well as recombinant adenoviruses lacking the binding site of Factor X to elucidate this mechanism in mice. We demonstrate that, as observed in the liver, adenovirus infection of the adrenal glands in vivo requires Factor X. Considering that the level of transduction of the adrenal glands is well-below that of the liver and that capsid-modified adenoviruses are unlikely to selectively infect the adrenal glands, we have used single-photon emission computed tomography (SPECT) imaging of gene expression to determine whether local virus administration (direct injection in the kidney) could increase gene transfer to the adrenal glands. We demonstrate that direct injection of the virus in the kidney increases gene transfer in the adrenal gland but liver transduction remains important. These observations strongly suggest that serotype 5 adenovirus uses a similar mechanism to infect liver and adrenal gland and that selective transgene expression in the latter is more likely to be achieved through transcriptional targeting.

In vivo positron emission tomography imaging using the sodium iodide symporter as a reporter gene.

Information regarding the biodistribution and kinetics of spread of oncolytic viruses is crucial for safety considerations in the design of future, more efficient reagents. Although optical imaging can be used to gain this information in rodent models, imaging with radioactive isotopes presents the advantage of being directly applicable to humans, and preclinical imaging data can provide a basis for the design of clinical protocols. In this chapter, we describe the use of the Na/I symporter as a reporter gene, associated with (124)I-NaI as a radiotracer, to monitor the anatomical localization, as well as the propagation, of oncolytic viruses using dedicated, preclinical positron emission tomography scanners.

Immunotherapy of hepatocellular carcinoma: is there a place for regulatory T-lymphocyte depletion?

Immunotherapy represents a potential therapeutic option for patients with hepatocellular carcinoma (HCC), especially as secondary treatment to prevent recurrence. It has been shown that a patient's survival is directly correlated to the type and number of tumor-infiltrating immune cells, indicating that immune responses have a direct effect on the clinical course of the disease. We have assessed the potential of immunotherapy against HCC in preclinical models of low tumor burden. An antigen-specific strategy targeting α-fetoprotein, and consisting of immunization with a DNA-based synthetic vector (DNAmAFP/704), was tested on an autochthonous model of chemical hepatocarcinogenesis and led to an important (65%) reduction of the tumor burden. A nonspecific approach of CD25(+) T-cell depletion by injection of PC61 antibody was also tested on an orthotopic HCC model and led to a significant protection against tumor development. Antigen-specific immunotherapy and Treg depletion are promising strategies in physiologically relevant HCC preclinical models. Future clinical trials will demonstrate if a combination of Treg depletion with an antigen-specific immunotherapy will also translate into clinical responses in HCC patients.