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Oncolytic adenoviruses (Ads) are cancer selective tumoricidal agents; however their mechanism of Ad-mediated cancer cell lysis, or oncolysis, remains undefined. This report focuses upon the autophagy mediator c-JUN n-terminal kinase (JNK) and its effects upon Ad oncolysis and replication. Previously, E1b-deleted Ads have been used to treat several hundred cancer patients with limited clinical efficacy. We hypothesize that by studying the potential interactions between E1b and JNK, mechanisms to improve oncolytic Ad design and cancer therapeutic efficacy may be elucidated. To test this hypothesis, E1b was selectively deleted from the Ad genome. These studies indicated that Ads encoding E1b induced JNK phosphorylation predominately occurred via E1b-19K. The expression of another crucial Ad gene E1a was then overexpressed by the CMV promoter via the replication competent Ad vector Adhz69; these data indicated that E1A also induced JNK phosphorylation. To assess the effects of host cell JNK expression upon Ad oncolysis and replication, siRNA targeting JNK1 and JNK2 (JNK1/2) were utilized. The oncolysis and replication of the E1b-19K wild-type Ads Ad5 and Adhz63 were significantly attenuated following JNK1/2 siRNA transfection. However the oncolytic effects and replication of the E1b-19K deleted Ad Adhz60 were not altered by JNK1/2 siRNA transfection, further implicating the crucial role of E1b-19K for Ad oncolysis and replication via JNK phosphorylation. This study has demonstrated for the first time that JNK is an intriguing molecular marker associated with enhanced Ad virotherapy efficacy, influencing future Ad vector design.

Adenoviruses (Ads) have been extensively manipulated for the development of cancer selective replication, leading to cancer cell death or oncolysis. Clinical studies using E1-modified oncolytic Ads have shown that this therapeutic platform was safe, but with limited efficacy, indicating the necessity of targeting other viral genes for manipulation. To improve the therapeutic efficacy of oncolytic Ads, we treated the entire Ad genome repeatedly with UV-light and have isolated AdUV which efficiently lyses cancer cells as reported previously (Wechman, S. L. et al. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection. Viruses 2016, 8, 6). In this report, we show that no mutations were observed in the early genes (E1 or E4) of AdUV while several mutations were observed within the Ad late genes which have structural or viral DNA packaging functions. This study also reported the increased release of AdUV from cancer cells. In this study, we found that AdUV inhibits tumor growth following intratumoral injection. These results indicate the potentially significant role of the viral late genes, in particular the DNA packaging genes, to enhance Ad oncolysis.

E2F-1-deleted mutant, ‘truncated E2F’ (E2Ftr, E2F-1[1–375]), lacking the carboxy-terminal transactivation domain, was shown to be more potent at inducing cancer cell apoptosis than wild-type E2F-1 (wtE2F-1; full-length E2F-1). Mechanisms by which wtE2F-1 and E2Ftr induce apoptosis, however, are not fully elucidated. Our study demonstrates molecular effects of pro-apoptotic BH3-only Bcl-2 family member Harakiri (Hrk) in wtE2F-1- and E2Ftr-induced melanoma cell apoptosis. We found that Hrk mRNA and Harakiri (HRK) protein expression was highly up-regulated in melanoma cells in response to wtE2F-1 and E2Ftr overexpression. HRK up-regulation did not require the E2F-1 transactivation domain. In addition, Hrk gene up-regulation and HRK protein expression did not require p53 in cancer cells. Hrk knockdown by Hrk siRNA was associated with significantly reduced wtE2F-1- and E2Ftr-induced apoptosis. We also found that an upstream factor, ‘downstream regulatory element antagonist modulator’ (DREAM), may be involved in HRK-mediated apoptosis in response to wtE2F-1 and E2Ftr overexpression. DREAM expression levels increased following wtE2F-1 and E2Ftr overexpression. Western blotting detected increased DREAM primarily in dimeric form. The homodimerization of DREAM resulting from wtE2F-1 and E2Ftr overexpression may contribute to the decreased binding activity of DREAM to the 3′-untranslated region of the Hrk gene as shown by electromobility shift assay. Results showed wtE2F-1- and E2Ftr-induced apoptosis is partially mediated by HRK. HRK function is regulated in response to DREAM. Our findings contribute to understanding the mechanisms that regulate wtE2F-1- and E2Ftr-induced apoptosis and provide insights into the further evaluation of how E2Ftr-induced apoptosis may be used for therapeutic gain.

Adenoviruses (Ads) with E1B55K mutations can selectively replicate in and destroy cancer cells. However, the mechanism of Ad-selective replication in tumor cells is not well characterized. We have shown previously that expression of several cell cycle-regulating genes is markedly affected by the Ad E1b gene in WI-38 human lung fibroblast cells (X. Rao, et al., Virology 350:418-428, 2006). In the current study, we show that the Ad E1B55K region is required to enhance cyclin E expression and that the failure to induce cyclin E overexpression due to E1B55K mutations prevents viral DNA from undergoing efficient replication in WI-38 cells, especially when the cells are arrested in the G 0 phase of the cell cycle by serum starvation. In contrast, cyclin E induction is less dependent on the function encoded in the E1B55K region in A549 and other cancer cells that are permissive for replication of E1B55K-mutated viruses, whether the cells are in the S phase or G 0 phase. The small interfering RNA that specifically inhibits cyclin E expression partially decreased viral replication. Our study provides evidence suggesting that E1B55K may be involved in cell cycle regulation that is important for efficient viral DNA replication and that cyclin E overexpression in cancer cells may be associated with the oncolytic replication of E1B55K-mutated viruses.

Adenoviruses with deletion of E1b gene can selectively replicate in cancer cells. The underlying mechanisms in tumor-selective replication of E1b-deleted adenoviruses are insufficiently understood. Identifying genes with altered expression patterns caused by the E1B proteins in virus-infected cells will further increase our understanding of E1B functions and provide insight into the tumor-selective replication of E1b-mutated adenoviruses on the molecular level. An approach based on large-scale gene array was applied to analyze molecular changes affected by viral E1B. We identified a total of 345 genes with expression changes of two-fold or greater affected by wild-type adenovirus compared with its E1b-deleted counterpart. The gene array data were confirmed by quantitative real-time PCR and Western blot. E1B proteins affect the expression of a diverse range of genes involved in cell cycle regulation, apoptosis, stress responses and angiogenesis. This is the first study of the global profile of gene expression altered by the viral E1B proteins in human lung cells, and the majority of the genes were previously not known to be affected by the viral proteins. The data presented in this study will lead to more detailed analysis of E1B functions and may also lead to development of new agents and approaches for oncolytic therapy.

E1B55K-deleted dl1520 could selectively replicate in cancer cells and has been used in clinical trials as an antitumor agent. The mechanism of virus selective replication in cancer cells, including a possible role of p53, is unclear. Studies with established cancer cell lines have demonstrated that some cancer cells are resistant to dl1520 replication, regardless of the p53 status. Hep3B cells supported the E1b-deleted adenoviruses to replicate, whereas Saos2 cells were resistant to viral replication. We applied p53-null Hep3B and Saos2 cells as models to clarify the replication ability of E1B55K-deleted adenoviruses with different expression levels of E1a. We show that lower E1A expression in Saos2 may be the reason for the poor replication in some cancer cells due to the fact that E1a promoter was less activated in Saos2 than in Hep3B. We also demonstrate that the E1B55K protein can increase E1A expression in Saos2 cells for efficient virus replication. In addition, the upstream regions of the E1a promoter have transcriptional activity in Hep3B cells but not in Saos2 cells. The viral E1B55K protein may activate cancer cellular factor(s) that targets the upstream regions of the E1a gene to increase its expression. This is the first study demonstrating that E1B55K protein affects the E1A production levels that is related to cancer selective replication. Our studies have suggested that increase of E1A expression from E1b-deleted adenoviruses may enhance killing cancer cells that otherwise are resistant to viral replication.

One of the promising strategies for targeting replication of oncolytic adenovirus in tumor cells is to regulate the expression of essential viral genes such as E1a by using tumor- or tissue-specific promoters that are preferentially active in cancer cells. However, this approach may lead to some degree of viral replication in normal cells other than in cancer cells if the viral gene also expresses in normal cells. In this study, we investigated the effect of E1a expression levels on the virus replication ability in human cells. Three vectors, all with mutated E1B55K, were created, one without any promoter controlling the E1a gene and two vectors with the E1a gene being controlled by either its endogenous promoter or a strong CMV promoter. We observed that the CMV promoter-mediated high levels of E1A expression could increase virus replication, resulting in the titers of the E1B55K-mutated virus being even higher than the wild-type virus in some cancer cells. However, the strong CMV promoter could not always enhance virus replication, such as in cancer cells OE33 and OsACL. The results suggest that whether increased E1A levels would enhance E1B55K-mutated virus replication may be also depended on cellular factors or pathways in cancer cells. We also observed that the virus without any promoter for the E1a gene could still express leaky levels of E1A which can lead to viral replication in normal and cancer cells. Future efforts in the development of transcription-controlled oncolytic adenoviruses should focus on how to completely block E1a expression in normal cells.

Oncolytic virotherapy can selectively destroy cancer cells and is a potential approach in cancer treatment. A strategy to increase tumor-specific selectivity is to control the expression of a key regulatory viral gene with a tumor-specific promoter. We have previously found that cyclin E expression is augmented in cancer cells after adenovirus (Ad) infection. Thus, the cyclin E promoter that is further activated by Ad in cancer cells may have unique properties for enhancing oncolytic viral replication. We have shown that high levels of viral E1a gene expression are achieved in cancer cells infected with Ad-cycE, in which the endogenous Ad E1a promoter was replaced with the cyclin E promoter. Ad-cycE shows markedly selective oncolytic efficacy in vitro and destroys various types of cancer cells, including those resistant to ONYX-015/dl1520. Furthermore, Ad-cycE shows a strong capacity to repress A549 xenograft tumor growth in nude mice and significantly prolongs survival. This study suggests the potential of Ad-cycE in cancer therapy and indicates the advantages of using promoters that can be upregulated by virus infection in cancer cells in development of oncolytic viruses. Key messages: Cyclin E promoter activity is high in cancer cells and enhanced by adenovirus infection. Cyclin E promoter is used to control the E1a gene of a tumor-specific oncolytic adenovirus. Ad-cycE efficiently targets cancer cells and induces oncolysis. Ad-cycE significantly repressed xenograft tumor and prolonged survival.

Epidemiological studies suggest that high intake of cruciferous vegetables is associated with a lower risk of cancer. Experiments have shown that indole-3-carbinol (I3C), a naturally occurring compound derived from cruciferous vegetables, exhibits potent anticarcinogenic properties in a wide range of cancers. In this study, we showed that higher doses of I3C (≥400 μM) induced apoptotic cancer cell death and lower doses of I3C (≤200 μM) repressed cancer cell growth concurrently with suppressed expression of cyclin E and its partner CDK2. Notably, we found that pretreatment with low doses of I3C enhanced Ad-mediated oncolysis and cytotoxicity of human carcinoma cells by synergistic upregulation of apoptosis. Thus, the vegetable compound I3C as a dietary supplement may benefit cancer prevention and improve Ad oncolytic therapies.

The gene for HER2/neu is overexpressed in 30–40% of breast and ovarian cancers, and this overexpression correlates with increased metastasis and poor prognosis. The HER2/neu gene product, a transmembrane protein kinase member of the EGF receptor family, has significant potential as a tumor antigen for vaccination. We inserted the sequence for neu into a novel plasmid called ELVIS containing a Sindbis virus replicon that reproduces multiple copies of mRNA. Mice vaccinated one time intramuscularly demonstrated a strong antibody response against A2L2, a murine breast cancer cell line transfected to express neu. Vaccinated mice challenged in the mammary fatpad with A2L2 had reduced tumor incidence and reduced tumor mass compared to mice challenged with tumor cells lacking the neu insert. Intradermal vaccination was also protective and required 80% less plasmid for a similar level of protection. Vaccination reduced the incidence of lung metastasis from mammary fatpad tumors and reduced the number of lung metastases resulting from intravenous injection of A2L2 cells. Cytotoxic T lymphocytes cultures of immune spleen cells with P815-neu cells produced high levels of interferon-γ indicating an antigen-specific Th1-type immune response resulting from the vaccination. In a spontaneous breast tumor model using neu transgenic mice, vaccination with ELVIS-neu protected against development of spontaneous breast tumors. Our preclinical data indicate that therapeutic vaccination of patients with ELVIS-neu may reduce metastasis from HER2/neu-expressing breast and ovarian tumors. Cancer Gene Therapy (2001) 8, 259–268

Vaccination against Helicobacter pylori using DNA sequences encoding Urease A and B subunits was compared to immunization with urease antigen and MTP-PE in a liposome formulation. To determine the effectiveness of a vaccine against H. pylori in a mouse model it is essential to quantify the number of H. pylori remaining in the stomachs following challenge with an inoculum of live bacteria. Culture assays and enzymatic assays produce inconsistent results often unsuitable to conclude if vaccine candidates are protective. To overcome this problem, we developed two assays: 1) a competitive quantitative PCR using a colorimetric readout and 2) a non-competitive direct quantitative PCR using a highly sensitive bioluminescent readout. The competitive PCR requires coamplification of a segment of the urease C sequence and an internal control standard in a competitive manner using a single set of primers. PCR products were quantified colorimetrically by an enzyme-linked immunosorbent assay and compared with known quantities of the internal control standard added to the PCR reaction. The highly sensitive, bioluminescent assay measures the amplified DNA directly using a flash-type luminescent tag and a specific probe. The Sydney strain of H. pylori was used for the mouse infection model. Quantification of H. pylori by either the bioluminescent assay or the competitive PCR was reliable, specific and sensitive compared to quantitative growth assays which often gave false results. The bioluminescent assay was much more sensitive and less labor/time intensive than the competitive PCR. The bioluminescent assay was able to quantitate as few as 100 bacteria, while the competitive assay could not detect less than 10(3) bacteria per mouse stomach. Quantification of H. pylori by bioluminescent assay was superior to the competitive assay and may be used for research applications, such as the development of vaccines, pathogenesis of gastric disease and monitoring of antibiotic treatment.

Lung cancer is the leading cause of cancer-related death worldwide and kills more patients than the next four cancers combined (Colon, breast, prostate, and pancreas). Currently available lung cancer treatments have not been effective. Therefore the development of novel lung cancer therapeutics is of critical importance. One emerging treatment option is the use of E1b-deleted adenoviruses (Ads) which preferentially lyse cancer cells. Although E1b-deleted Ads have been applied in clinical trials, their efficacy remains low. We have developed a novel oncolytic vector, AdUV, via the bioselection of the cancer selective E1b-deleted Ad Adhz60. We have demonstrated that AdUV is much more potent against multiple cancer cell lines than the parental vector Adhz60 in vitro and suppressed tumor growth in vivo. We compared AdUV treated cells to cells treated with the wildtype Ad5 and the E1b-deleted Adhz60. AdUV lysed A549 lung cancer cells more efficiently than Adhz60, but neither AdUV nor Adhz60 killed MRC5 and HBEC normal lung cells. Possible mechanisms of action were then investigated to determine how AdUV lysed A549 cells more effectively. AdUV virions were shown to release from infected A549 cells and induce autophagy more effectively than both Adhz60 and Ad5. Autophagy induction is known to support Ad replication and spread through cancer cell monolayers. The plaque assay was used to verify if AdUV could spread more effectively than Ad5 and Adhz60 because viruses which efficiently replicate and release from cells are known to form larger plaques. The average size of plaques formed by AdUV is 340% and 214% larger than those plaques formed by Adhz60 and Ad5, respectively. In vivo, AdUV was non-lethal to athymic nude mice and strongly suppressed A549-xenograft tumor growth, prolonging murine survival. These results indicate that AdUV is a more effective, safe oncolytic Ad vector. Our findings also demonstrate the utility of bioselection of the generation of more effective cancer therapeutic vectors. Citation Format: Stephen L. Wechman, Xiao-Mei Rao, Kelly Marc McMasters, Heshan Sam Zhou. Improved oncolytic virotherapy by increasing adenovirus spread. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3753.

Adenovirus-mediated gene transfer into a tumor mass can be improved by combining it with conditionally-replicating adenovirus (CRAd) when both vectors co-infect the same cancer cell. We investigated the efficiency of enhancing transgene expression and effectiveness of cancer killing of two advenoviruses (Ads), one expressing E2F-1 (AdE2F-1) and another expressing a truncated form of E2F-1 that lacks the transactivation domain (AdE2Ftr), when combined with oncolytic Adhz60. We found that AdE2F-1 with Adhz60 actually decreased E2F-1 expression and viral replication through a mechanism apparently involving repression of the cyclin-E promoter and decreased expression of early and late structural proteins necessary for viral replication. In contrast, AdE2Ftr with Adhz60 resulted in increased E2Ftr expression, AdE2Ftr replication, and cancer cell death both in vitro and in vivo. These results indicate that AdE2Ftr coupled with a CRAd enhances AdE2Ftr-mediated cancer cell death.

Apoptotic pathways are initiated as a cellular defense mechanism to eliminate adenovirus-infected cells. We have investigated how E1A-induced apoptosis interferes with viral replication in cancer cells. We found that E1B19K alone can efficiently suppress E1A-induced apoptosis in cancer cells. Viruses deleted for both E1B19K and E1B55K resulted in cellular DNA degradation. However, less than 20% of human lung cancer cells infected with a virus deleted for both E1B19K and E1B55 K had evidence of chromatin condensation and multiple-micronuclei formation (apoptotic hallmarks); these cells could not produce infectious viral particles. The majority of cancer cells infected with viruses deleted for the entire E1b gene did not undergo extended apoptosis and produced abundant viral progeny. Thus, only a fraction of cancer cells underwent apoptosis and did not allow E1b-deleted viruses to replicate, while the majority of cancer cells were resistant to E1A-induced apoptosis and could support virus-selective replication. The results of this study imply that, in addition to inhibiting E1A-induced apoptosis, E1B proteins may contribute other important roles in the viral life cycle. Our results also suggest that combining virus-induced apoptosis and selective viral replication into one vector will be a novel approach to destroy cancer cells.

The adenovirus E1A proteins are involved in the transcriptional activation of viral and cellular genes needed for controlling cell cycle and virus replication. Undifferentiated embryonic carcinoma cells have the ability to produce an E1A-like activity that can induce the expression of E1A-targeted adenoviral and cellular genes in the absence of the E1A products. Differentiated embryonic carcinoma cells lose the ability to produce the E1A-like activity. In this study, we investigated the E1A-like activity in cancer cells with an adenovirus having a mutated E1a gene. The mutation is generated by the insertion of a large DNA fragment in the E1a gene and interrupts the COOH-terminal region of both the E1A 12S and 13S proteins. The E1a-mutated virus can efficiently replicate in HepG2 and Hep3B liver cancer cells and produce high titers of virus. Replication of the E1a-mutated virus inhibits tumor formation and destroys tumors in vivo. The results obtained in this study imply that cancer cells may produce an E1A-like activity to support the selective replication of mutated virus in cancer cells. In addition, we found that although the E1a-mutated virus could not replicate in Huh1.cl2 liver cells, the viral DNA could amplify in the cells. This result suggests that replication of adenoviral DNA is necessary, but not sufficient, for generating infectious viral progeny and destroying tumor cells.

Immunoliposomes containing monoclonal antibodies (MAbs) to the costimulatory molecules CD28 and CTLA4 and their counterreceptors B7-1 (CD80) and B7-2 (CD86) were evaluated for the ability to increase the immune response to recombinant envelope protein rgp120 of the MN strain of human immunodeficiency virus type 1 (HIV-1) during vaccination. MAbs were attached to rgp120-containing liposomes via a biotin-avidin-biotin bridge. Mice vaccinated with immunoliposomes were found to have a strong delayed-type hypersensitivity (DTH) response to the weakly immunogenic gp120 that was dependent on the presence of the MAbs. However, this vaccination protocol did not induce humoral immunity. The DTH response was not accompanied by increased production of interferon gamma (IFN-gamma) or interleukin 4 (IL-4), implying that the primary cellular interaction was between the immunoliposomes and cells of the reticuloendothelial system and not helper T (Th) cells. This strategy of incorporating antibodies to costimulatory molecules on the surface of antigen-containing particulates, such as liposomes or microspheres, can be used to increase DTH immune responses to protein or peptide vaccines.

Background. A murine model for Helicobacter pylori infection could facilitate vaccine development. This study was designed to determine the effect of various conditions of dose, frequency of administration, and fasting on H. pylori infection of mice. Materials and Methods. Balb/c and C3H/HeN mice were inoculated orogastrically with clinical isolates of H. pylori grown in liquid culture. At 2-week intervals, the stomachs were removed for secondary culture on horse blood agar and for histological analysis. H. pylori from secondary cultures or homogenized stomach tissue from infected mice was inoculated a second time in naive animals. Results. H. pylori was cultured with high frequency only from the stomachs of C3H/HeN mice. Fasting the mice and increasing the number of organisms inoculated did not increase the rate of infection. Histological analysis detected no inflammation, but mucus depletion and erosion were present in the stomachs of C3H/HeN mice. H. pylori organisms were not observed. Secondary cultures of H. pylori or homogenized infected stomach tissue did not cause infection when inoculated in naive mice. Conclusions. Clinical isolates of H. pylori transiently infect C3H/HeN mice. This murine model is suitable for testing oral vaccines. Effective vaccination against H. pylori could prevent transient infection and reduce subsequent gastritis.

Background The density of Helicobacter pylori is thought to correlate with the degree of inflammation and thus indirectly with the outcome of the infection. Rapid quantitative assays of H. pylori in gastric or duodenal mucosa are lacking. The aim was to develop quantitative assays using the polymerase chain reaction to assess the quantity of H. pylori in the gastric mucosa. Methods Competitive PCR was based on coamplification of a segment of the ureC sequence and an internal control using a single set of primers. PCR products were quantified colorimetrically by an enzyme-linked immunosorbent assay and compared with known quantities of the internal control standard added to the PCR reaction. The highly sensitive, noncompetitive PCR assay does not use coamplification and measures the amplified DNA sequence using a flash-type luminescent tag and a specific probe. The mouse infected model using H. pylori strain SS-1 was used to develop the assays. Results Quantification of H. pylori using either the competitive or noncompetitive PCR was reliable, highly sensitive and specific. Conclusions The ability to rapidly quantitate H. pylori from gastric mucosa should be useful to investigate the role of H. pylori density and infection outcome, as well as to monitor the effectiveness of antibiotic treatment or vaccines against H. pylori.