The oncolytic virus H101 combined with GNAQ siRNA-mediated knockdown reduces uveal melanoma cell viability.

BACKGROUND: Uveal melanoma (UM) is a severe human malignancy with a high mortality rate, as well as high metastasis and recurrence potential. The active mutation of G protein subunit alpha q (GNAQ) or G protein subunit alpha 11 (GNA11) is a major trigger for UM. Oncolytic adenovirus H101 (H101) is the first oncolytic virus approved for clinical applications in cancer therapy by the China Food and Drug Administration. We investigated whether combining H101 with the downregulation of GNAQ expression would act synergistically in UM therapy. METHODS: Three UM cell lines OMM2.3 and 92.1, harboring GNAQ mutation, and OCM1, harboring B-Raf proto-oncogene mutation, were chosen for our research. The cellular toxicity of adenoviral infection and the cell growth rate were measured with a Cell Counting Kit-8. Western blot analysis was used to detect GNAQ, p-MEK1/2, YAP, and p-YAP expression. The apoptosis and cell-cycle distribution of cells were evaluated with annexin-V and propidium iodide staining. RESULTS: Our results revealed that OMM2.3 and 92.1 cells were more sensitive to H101 infection than OCM1 cells. GNAQ expression was markedly reduced by small interfering RNA, siGNAQ. Combined treatment of siGNAQ and H101 inhibited the proliferation and activated the apoptosis of OMM2.3 and 92.1 cells by blocking the phosphorylation of MEK1/2 and increasing the phosphorylation of YAP. CONCLUSIONS: In summary, a therapy combining H101 and siGNAQ is feasible, with potential utility as a novel targeted molecular therapy for UM, especially those carrying a GNAQ mutation.

CANT1 lncRNA Triggers Efficient Therapeutic Efficacy by Correcting Aberrant lncing Cascade in Malignant Uveal Melanoma.

Uveal melanoma (UM) is an intraocular malignant tumor with a high mortality rate. Recent studies have shown the functions of long non-coding RNAs (lncRNAs) in tumorigenesis; thus, targeting tumor-specific lncRNA abnormalities has become an attractive approach for developing therapeutics to treat uveal melanoma. In this study, we identified a novel nuclear CANT1 lncRNA (CASC15-New-Transcript 1) that acts as a necessary UM suppressor. CANT1 significantly reduced tumor metastatic capacity and tumor formation, either in cell culture or in animals harboring tumor xenograft. Intriguingly, XIST lncRNA serves as a potential target of CANT1, and JPX or FTX lncRNA subsequently serves as a contextual hinge to activate a novel CANT1-JPX/FTX-XIST long non-coding (lncing) pathway in UM. Moreover, CANT1 triggers the expression of JPX and FTX by directly binding to their promoters and promoting H3K4 methylation. These observations delineate a novel lncing cascade in which lncRNAs directly build a lncing cascade without coding genes that aims to modulate UM tumorigenesis, thereby specifying a novel "lncing-cascade renewal" anti-tumor therapeutic strategy by correcting aberrant lncing cascade in uveal melanoma.

Restoration of IGF2 imprinting by polycomb repressive complex 2 docking factor SUZ12 in colon cancer cells.

The insulin-like growth factor II (IGF2) gene is aberrantly expressed in tumors as a result of loss of imprinting (LOI). Reactivation of the normally-suppressed maternal allele may lead to IGF2 upregulation and increased tumor growth, particularly in colon cancer. However, the mechanisms underlying IGF2 LOI in tumors are poorly defined. In this report, we identified polycomb repressive complex 2 (PRC2) docking factor SUZ12 as a critical factor in regulating IGF2 imprinting in tumors. Human colon cancer cell lines (HRT18 and HT29) show loss of IGF2 imprinting. Ectopic expression of SUZ12 restored normal monoallelic expression of IGF2 in these two colon cancer cell lines. Using chromatin immunoprecipitation (ChIP) and chromatin conformation capture (3C), we found that the virally-expressed SUZ12 bound to IGF2 promoters, coordinating with endogenous CTCF to orchestrate a long range intrachromosomal loop between the imprinting control region (ICR) and the IGF2 promoters. The histone methyltransferase EZH2 was recruited to the IGF2 promoters, where it induced H3K27 hypermethylation, suppressing one allele, leading to the restoration of IGF2 imprinting. These data demonstrate that SUZ12 is a key molecule in the regulation of monoallelic expression of IGF2, suggesting a novel epigenetic therapeutic strategy for modulating IGF2 production in human tumors.

IL-1ß and TNF-α induce neurotoxicity through glutamate production: a potential role for neuronal glutaminase.

Glutaminase 1 is the main enzyme responsible for glutamate production in mammalian cells. The roles of macrophage and microglia glutaminases in brain injury, infection, and inflammation are well documented. However, little is known about the regulation of neuronal glutaminase, despite neurons being a predominant cell type of glutaminase expression. Using primary rat and human neuronal cultures, we confirmed that interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), two pro-inflammatory cytokines that are typically elevated in neurodegenerative disease states, induced neuronal death and apoptosis in vitro. Furthermore, both intracellular and extracellular glutamate levels were significantly elevated following IL-1ß and/or TNF-α treatment. Pre-treatment with N-Methyl-D-aspartate (NMDA) receptor antagonist MK-801 blocked cytokine-induced glutamate production and alleviated the neurotoxicity, indicating that IL-1ß and/or TNF-α induce neurotoxicity through glutamate. To determine the potential source of excess glutamate production in the culture during inflammation, we investigated the neuronal glutaminase and found that treatment with IL-1ß or TNF-α significantly upregulated the kidney-type glutaminase (KGA), a glutaminase 1 isoform, in primary human neurons. The up-regulation of neuronal glutaminase was also demonstrated in situ in a murine model of HIV-1 encephalitis. In addition, IL-1ß or TNF-α treatment increased the levels of KGA in cytosol and TNF-α specifically increased KGA levels in the extracellular fluid, away from its main residence in mitochondria. Together, these findings support neuronal glutaminase as a potential component of neurotoxicity during inflammation and that modulation of glutaminase may provide therapeutic avenues for neurodegenerative diseases.

The regulation of Toll-like receptor 2 by miR-143 suppresses the invasion and migration of a subset of human colorectal carcinoma cells.

BACKGROUND: The Toll-like receptor 2 (TLR2)-driven tissue response may promote neoangiogenesis and tumour growth by mechanisms that are poorly understood. METHODS: We investigated the expression levels of TLR2 and associated-miRNAs in colorectal carcinoma (CRC) tissues and cell lines using real-time PCR, northern blotting and western blotting. Survival curver was generated by Log-Rank test and the role of TLR2 signalling in tumour invasion and migration was determined by transwell analysis kits. RESULTS: We observed that the tissues from CRC patients express relatively high levels of TLR2. Targeting TLR2 markedly reduces the invasion and migration of CRC cells. We also found that miR-143, a putative tumour suppressor that is down-regulated in CRC tissues, reduces the invasion and migration of CRC cells primarily via TLR2. Utilising a xenograft mouse model, we demonstrated that re-expression of miR-143 inhibits CRC cell colonisation in vivo. CONCLUSION: miR-143 blocks the TLR2 signalling pathway in human CRC cells. This knowledge may pave the way for new clinical applications utilising miR-143 mimics in the treatment of patients with CRC.

Knockdown of TIGAR by RNA interference induces apoptosis and autophagy in HepG2 hepatocellular carcinoma cells.

Apoptosis and autophagy are crucial mechanisms regulating cell death, and the relationship between apoptosis and autophagy in the liver has yet to be thoroughly explored. TIGAR (TP53-induced glycolysis and apoptosis regulator), which is a p53-inducible gene, functions in the suppression of ROS (reactive oxygen species) and protects U2OS cells from undergoing cell death. In this study, silencing TIGAR by RNAi (RNA interference) in HepG2 cells down-regulated both TIGAR mRNA (∼75%) and protein levels (∼80%) and led to the inhibition of cell growth (P<0.01) by apoptosis (P<0.001) and autophagy. We demonstrated that TIGAR can increase ROS levels in HepG2 cells. The down-regulation of TIGAR led to the induction of LC-3 II (specific autophagic marker), the formation of the autophagosome, and increased Beclin-1 expression. 3-MA (3-Methyladenine), an inhibitor of autophagic sequestration blocker, inhibited TIGAR siRNA-enhanced autophagy, as indicated by the decrease in LC-3 II levels. Consequently, these data provide the first evidence that targeted silencing of TIGAR induces apoptotic and autophagic cell death in HepG2 cells, and our data raise hope for the future successful application of TIGAR siRNA in patients with hepatocellular carcinoma (HCC).

Hypoxia inducible factor-1α-mediated activation of survivin in cervical cancer cells.

AIM: Hypoxia, a characteristic of almost all types of solid tumors, has been associated with poor outcome in a number of human malignancies. The aim of this study was to investigate the molecular mechanisms involved in hypoxia-induced activation of the human survivin gene promoter in cervical HeLa cells. MATERIAL AND METHODS: Immunohistochemical staining was used to detect the expression of HIF-1α and survivin in cervical cancer samples and normal cervical samples. Under normoxic and hypoxic conditions, the expression of hypoxia inducible factor (HIF)-1α and survivin in cervical cancer HeLa cells was detected by quantitative reverse transcription polymerase chain reaction and Western blotting. Luciferase reporter assays was used to investigate the molecular mechanisms in hypoxia-induced survivin activation. We also studied the effect of HIF-1α overexpression on the expression of survivin in cervical cancer HeLa cells. RESULTS: Significant HIF-1α and survivin overexpression is associated with cervical cancer, and HIF-1α protein expression is strongly correlated with survivin protein expression. In cervical cancer cell line (HeLa), hypoxia upregulated both HIF-1α and survivin expression. Moreover, luciferase reporter assays using survivin core promoter demonstrated that survivin transcription was activated under hypoxia conditions and was associated with HIF-1α overexpression. The transcriptional activation of reporter genes in response to hypoxia is independent of potential HIF-1α-responsive element, located between -86 and -82 regions. HIF-1α overexpression significantly activated survivin expression. CONCLUSION: Our results demonstrate that survivin expression is upregulated following the induction of HIF-1α by hypoxia resulting from tumor formation, possibly leading to tumor progression. These findings have potential implication in developing novel cancer therapy targeting HIF-1.

Potentiation of tumor radiotherapy by a radiation-inducible oncolytic and oncoapoptotic adenovirus in cervical cancer xenografts.

The p53 tumor suppressor pathway is impaired in more than 90% of cervical cancers and cancer-derived cell lines as a result of infection by human papillomavirus (HPV). The HPV E6 oncoprotein forms complexes with p53 and promotes its degradation via ubiquitin-dependent mechanism. In our study, we attempted to improve the clinical outcomes of this combined therapy by modifying the p53-targeted adenovirus to become radiation-responsive. The antitumor adenovirus was constructed by inserting a radiation-responsive expression cassette composed of the promoter of early growth response-1 (Egr-1) and the proapoptotic protein TRAIL. We showed that the addition of adenovirus containing Egr-1/TRAIL significantly increased cell death and apoptosis caused by radiotherapy. In mice bearing xenograft tumors, intratumoral administration of the Egr-1/TRAIL adenovirus followed by radiation significantly reduced tumor growth and enhanced tumor survival. Our Egr-1/TRAIL adenoviral gene product may offer a novel "one-two punch" tumor therapy for cervical cancers not only by potentiating radiation treatment but also by preserving p53 defect-specific tumor killing of the oncolytic adenovirus.

Combined treatment with an oncolytic adenovirus and antitumor activity of vincristine against retinoblastoma cells.

Treatment trends of retinoblastoma (RB) have gradually evolved from eye enucleation and external radiation to local treatment. Combined treatment with an oncolytic virus and chemotherapy is currently a new method in RB treatment. To investigate the therapeutic effect of oncolytic adenovirus SG600 in combination with vincristine (VCR) on retinoblastoma in vitro, the cell viability, cell cycle effects and apoptotic activity of HXO-RB(44) cells treated with SG600, VCR or SG600 plus VCR were measured using a cell counting kit-8-based procedure and flow cytometry. Western blot analysis for Akt, p-Akt, p-p53 and p-Rb protein was performed to investigate the underlying mechanisms of combined therapy. The combination therapy exerted a synergistic antitumor effect via a type of G(2)/M and S phase arrest rather than the induction of apoptosis. The combination of VCR and SG600 further reduced Akt phosphorylation compared with cells treated with VCR alone, suggesting that SG600 could overcome chemoresistance, perhaps by down-regulating Akt in RB cells. An increase in the expression of p-p53 and decrease in p-Rb expression in HXO-RB(44) after co-treatment might be associated with cell cycle block. Western blot examination revealed that VCR might enhance SG600 replication. These results suggest that viro-chemo combination therapy is a feasible and potentially promising approach for the treatment of retinoblastoma.

SP1 suppresses phorbol 12-myristate 13-acetate induced up-regulation of human regucalcin expression in liver cancer cells.

There is a growing evidence that regucalcin (RGN) plays a multifunctional role in liver cancer cells. Previous reports showed that the presence of phorbol 12-myristate 13-acetate (PMA) caused a significant increase in RGN mRNA expression and promoter activity in rat hepatoma cells. In this study, we confirmed that human RGN is also up-regulated by PMA treatment independent of translation, and we identified the mechanism by which PMA up-regulates the expression of human RGN via driving SP1 away from a SP1 motif located within -188/-180 of the promoter in HepG2 cells. Overexpression of SP1 dramatically reduces PMA-induced up-regulation of both internal expression of mRNA and promoter activity, whereas knockdown of SP1 has the opposite effect. Therefore, the present study delineates the fundamental elements in the promoter which will be helpful in the future studies on the regulation of RGN expression in liver cancer.

MiR-145, a new regulator of the DNA fragmentation factor-45 (DFF45)-mediated apoptotic network.

BACKGROUND: MicroRNA-145 (miR-145) is considered to play key roles in many cellular processes, such as proliferation, differentiation and apoptosis, by inhibiting target gene expression. DNA Fragmentation Factor-45 (DFF45) has been found to be the substrate of Caspase-3, and the cleavage of DFF45 by caspase-3 during apoptosis releases DFF40 that degrades chromosomal DNA into nucleosomal fragments. There are currently no in-depth studies on the relationship between miR-145 and the DFF45 gene. RESULTS: In this study, we identified DFF45 as a novel target of miR-145. We demonstrated that miR-145 targets a putative binding site in the coding sequence (CDS) of DFF45, and its abundance is inversely associated with DFF45 expression in colon cancer cells. Using a luciferase reporter system, we found that miR-145 suppresses the expression of the luciferase reporter gene fused to the putative binding site of DFF45. The level of DFF45 protein, but not DFF45 mRNA, was decreased by miR-145, suggesting a mechanism of translational regulation. Furthermore, we demonstrate that this specific silencing of DFF45 by miR-145 accounts, at least in part, for the staurosporine-induced tumor cell apoptosis in vitro. CONCLUSIONS: Our study reveals a previously unrecognized function of miR-145 in DFF45 processing, which may underlie crucial aspects of cancer biology.

Targeted knockdown of Bcl2 in tumor cells using a synthetic TRAIL 3'-UTR microRNA.

Targeting tumor-related overexpression of anti-apoptotic Bcl2 protein by RNAi has been suggested as a potential treatment for cancer. However, the stability of RNAi and its delivery are still major obstacles to the clinical testing of Bcl2 RNAi. Here, we explore a novel strategy of expressing a synthetic Bcl2 microRNA (smRNA) in the 3' untranslated region (UTR) of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), an apoptosis-inducing protein without apparent toxic effects in normal cells. TRAIL was specifically expressed from the human telomerase reverse transcriptase promoter (pTRT) that is active in many human tumors. Using this approach, we demonstrated that pTRT drove the tumor-specific expression of Bcl2 smRNA, which was processed by the host RNAi machinery and silenced endogenous Bcl2 expression in tumor cells. Bcl2 smRNA induced tumor cell apoptosis by activating caspase-3 and led to significant sensitization of tumor cells to TRAIL-induced apoptosis, while normal cells were spared. We also showed that the combined therapy of TRAIL-induced apoptosis and Bcl2 downregulation was superior to the mono-therapy of TRAIL or Bcl2 smRNA alone. This study proves a general paradigm for cancer therapy by using 3' UTR microRNA technology.

Enhanced therapeutic efficacy by simultaneously targeting two genetic defects in tumors.

Targeting tumor-specific gene abnormalities has become an attractive approach in developing therapeutics to treat cancer. Overexpression of Bcl2 and mutations of p53 represent two of the most common molecular defects in tumors. In the nucleus, p53 induces cell cycle arrest, while it interacts with Bcl2 outside of the nucleus to regulate signal pathways involved in apoptosis. To potentiate antitumor activity, we tested a "double target" approach to antitumor therapy by combining H101, a recombinant oncolytic adenovirus that targets the inactive p53 in tumors, with a small interfering RNA (siBCL2) that targets Bcl2. In cell culture, the combined treatment significantly enhanced apoptosis and cytotoxicity as compared with treatment with either H101 or siBCL2 alone. In animals carrying tumor xenographs, combined H101 and siBCL2 treatment significantly inhibited tumor growth and prolonged survival. At the end of the study, all animals in the combined therapy group survived and two of the five animals showed complete eradication of their tumors. Interestingly, siBCL2 treatment increased H101 viral replication in both treated cells and tumor tissues. Simultaneously targeting two tumor-specific gene abnormalities using an oncolytic adenovirus and siRNA potentiates total antitumor activity.

Co-expression of P1A35-43/beta2m fusion protein and co-stimulatory molecule CD80 elicits effective anti-tumor immunity in the P815 mouse mastocytoma tumor model.

A strong CTL response is dependent upon a high level of expression of specific class I major histocompatibility complex (MHC)/peptide complexes at the cell surface. An epitope-linked beta2-microglobulin (beta2m) molecule could provide a simple and more efficient means to enhance the formation of defined MHC/peptide complexes. However, the ability of an epitope-linked beta2m molecule to elicit primary CTL responses in vivo is still unknown. In this study, we modified the P1A tumor cell vaccine by addition of the tumor-associated epitope (TAE)-linked beta2m molecule and co-stimulatory molecule CD80 to improve the efficiency in the application of the vaccine. A eukaryotic co-expression vector consisting of the P1A35-43-linked beta2m molecule and the murine CD80 gene was constructed. P815 cell lines stably expressing P1A35-43-linked beta2m molecule and/or CD80 were established after transfection, by selection under G418. Administration of these inactivated tumor cell vaccines allowed the TAE-specific CD8+ T cell responses to be examined in vivo. Our results indicate that immunization with P815 cells expressing both the P1A35-43-linked beta2m molecule and the murine CD80 gene elicited a significantly stronger antitumor immune response than the single-modified tumor cell vaccines (expressing either P1A35-43-linked beta2m or CD80 alone). These findings support the feasibility and effectiveness of developing a dual-modified tumor cell vaccine consisting of the epitope-linked beta2m molecule and a co-stimulatory molecule.

A study on anti-tumor immunity induced by gene-modified melanoma B16 cells.

T cell-mediated cell immunity is the main anti-tumor immunity in which the effector T cells need specific antigen and costimulatory signals. One of the vaccines applied in tumor immunotherapy is the gene-modified tumor cell vaccine. One potential method to increase cell epitope density is to link the antigen with the major histcompatibility complex subunit beta2m. Our previous research indicated that the strategy of epitope fusion gene OVA-linker-beta2m can promote the formation of specific compounds on the tumor cell surface in vitro. In this study, we constructed two coexpression vectors pGL3-CD80-OVA-linker-beta2m and pGL3-IL21-OVA-linker-beta2m, in order to explore the cooperative action of CD80 or interleukin-21 (IL21) with the epitope fusion gene in anti-tumor immunity. Results showed that gene-modified B16 cells (B16/OVA, B16/CD80-OVA and B16/IL21-OVA) grew slower than B16 cells in vitro and in vivo, especially the B16/IL21-OVA subline, which illustrated that such gene modification decreased oncogenicity of malignant tumor cells. On the other hand, gene-modified tumor cell subline immunization can induce effective long-term anti-tumor immunity defending tumor cell attacks. IL21 played a more cooperative role with the OVA-linker-beta2m than CD80 in this study. This strategy might lay foundations for the research of a new type of tumor vaccine.

The effect of co-expression costimulatory molecule CD80 on uptake of antigen peptide-MHC class I-GFP complex by specific T cells.

CD80, a costimulatory molecule, plays an important role in eliciting antitumor immunity. Without costimulation, recognition of antigens by T cells may not cause a response, even if tumor cells express MHC class I molecules and specific antigens. On the basis of the recombinant GFP-tagged Kb molecule, we constructed a co-expression vector of CD80 and GFP-tagged Kb molecules. The recombinant fusion was transfected into mouse melanoma B16 cells by electroporation; positive cells were obtained by G418 screening. Highly expressing monoclonal cells, irradiated by 137Cs, were used to immunize mice to obtain specific T cells, which were then cultivated with tumor cells in vitro and examined with a laser confocal microscope. The evident and intense uptake of the antigen peptide-MHC class I-GFP complex by specific T cells was visualized from the culture of B16/CD80-Kb-GFP and T cells. However, little uptake was observed from the culture of B16/Kb-GFP and T cells. These results show that co-expression of CD80 molecules with Kb, an MHC class I molecule, on the surface of B16 tumor cells can enhance the response of specific T cells and thus increase the uptake of the antigen peptide-MHC class I-GFP complex. The absorbed green fluorescence was concentrated mainly on the T cell surface, and this result might pave the way to eluting specific antigen peptides directly from T cells to find and isolate novel tumor-specific antigen peptides.

Differential expression of TYRP1 in adult human retinal pigment epithelium and uveal melanoma cells.

Uveal melanoma (UM) is the most frequently occurring primary intraocular malignancy in adults. Tyrosinase (TYR) is a copper-containing enzyme and a type I membrane protein that is involved in the generation of melanin, the main pigment in vertebrates. TYR-related protein 1 (TYRP1) is regarded to have a crucial role in the immunotherapy of melanoma. As biomarkers, the TYR-related proteins, TYRP1 and TYRP2, exhibit specific expression in melanocytes, while also contributing to melanin synthesis within melanosomes. In the present study, the differential expression of TYRP1 was investigated at the mRNA, protein and morphological levels in four human UM cell lines (SP6.5, OM431, OCM1 and OCM290) and the human retinal pigment epithelium (RPE) cell line, using polymerase chain reaction, western blotting, immunocytochemistry and immunofluorescence staining. It was found that SP6.5 cells expressed the highest level of TYRP1, in comparison to SP6.5 OCM1 and OM431 cells, which produced less TYRP1, and OCM290 cells, which produced almost no TYRP1. No TYRP1 protein expression was identified in the RPE cell line. These findings indicate the potential use of TYRP1 in the development of therapy for UM.

Long non-coding RNA ROR decoys gene-specific histone methylation to promote tumorigenesis.

BACKGROUND: Long non-coding RNAs (lncRNAs) are not translated into proteins and were initially considered to be part of the 'dark matter' of the genome. Recently, it has been shown that lncRNAs play a role in the recruitment of chromatin modifying complexes and can influence gene expression. However, it is unknown if lncRNAs function in a similar way in cancer. RESULTS: Here, we show that the lncRNA ROR occupies and activates the TESC promoter by repelling the histone G9A methyltransferase and promoting the release of histone H3K9 methylation. Suppression of ROR in tumors results in silencing of TESC expression, and G9A-mediated histone H3K9 methylation in the TESC promoter is restored, which significantly reduces tumor growth and metastasis. Without ROR silencing, TESC knockdown presents consistent and significant reductions in tumor progression. CONCLUSIONS: Our results reveal a novel mechanism by which ROR may serve as a decoy oncoRNA that blocks binding surfaces, preventing the recruitment of histone modifying enzymes, thereby specifying a new pattern of histone modifications that promote tumorigenesis.

[Antigen presentation efficiency of recombinant Vaccinia virus expressing minigene product OVA(257-264) in eukaryotic cells].

rVV (recombinant vaccinia virus) expressing minigene products and rVV expressing full length OVA protein were used to infect several eukaryotic cells. Monoclonal antibody 25.D-1.16 (anti-OVA(257-264)/K(b)) was used to detect the conformation of H-2K(b)-OVA complex on the surface of these cells. The tetramer technique was used to determine their ability to induce antigen specific T cells. The results showed that the tetramer had been generated successfully. Moreover, L929-K(b) cells infected by rVV-OVA can be used as APC to induce CTL response. Specific CTL response was demonstrated by H-2K(b-OVA tetramer staining and cytotoxicity assay. Extracelluar IFN-gamma was also quantitatively analyzed. The results from H-2Kb)-OVA tetramer staining were consistent with cytotoxicity assay. It can be concluded that rVV expressing minigene product is a more efficient source of peptides for class I molecules, and can be more immunogenic than rVV expressing full length protein.