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DNA viruses are responsible for many diseases in humans. Current treatments are often limited by toxicity, as in the case of cidofovir (CDV, Vistide), a compound used against cytomegalovirus (CMV) and adenovirus (AdV) infections. CDV is a polar molecule with poor bioavailability, and its overall clinical utility is limited by the high occurrence of acute nephrotoxicity. To circumvent these disadvantages, we designed nine CDV prodrug analogues. The prodrugs modulate the polarity of CDV with a long sulfonyl alkyl chain attached to one of the phosphono oxygens. We added capping groups to the end of the alkyl chain to minimize β-oxidation and focus the metabolism on the phosphoester hydrolysis, thereby tuning the rate of this reaction by altering the alkyl chain length. With these modifications, the prodrugs have excellent aqueous solubility, optimized metabolic stability, increased cellular permeability, and rapid intracellular conversion to the pharmacologically active diphosphate form (CDV-PP). The prodrugs exhibited significantly enhanced antiviral potency against a wide range of DNA viruses in infected human foreskin fibroblasts. Single-dose intravenous and oral pharmacokinetic experiments showed that the compounds maintained plasma and target tissue levels of CDV well above the EC

Treatments with cytotoxic agents or viruses may cause Immunogenic Cell Death (ICD) that immunize tumor-bearing hosts but do not cause complete regression of tumor. We postulate that combining two ICD inducers may cause durable regression in immunocompetent mice. ICD was optimized in vitro by maximizing calreticulin externalization in human colorectal carcinoma (CRC) cells by exposure to mixtures of Oxaliplatin (OX) and human adenovirus (AdV). Six mm diameter CT26 or 4T1 carcinomas in flanks of BALB/c mice were injected once intratumorally (IT) with OX, AdV or their mixture. Tumor growth, Tumor-Infiltrating Lymphocytes (TIL), nodal cytotoxicity, and rejection of a viable cell challenge were measured. Tumors injected IT once with an optimum mixture of 80 µM OX – AdV 25 Multiplicity of Infection (MOI) in PBS buffer were 17–29% the volume of control tumors. When buffer was changed from PBS to 5% dextrose in water (D5W), volumes of tumors injected IT with 80 µM OX-AdV 25 MOI were 10% while IT OX or AdV alone were 32% and 40% the volume of IT buffer-treated tumors. OX-AdV IT increased CD3+ TIL by 4-fold, decreased CD8+ PD-1+ TIL from 79% to 19% and induced cytotoxicity to CT26 cells in draining node lymphocytes while lymphocytes from CT26-bearing untreated mice were not cytotoxic. OX-AdV IT in D5W caused complete regression in 40% of mice. Long-term survivors rejected a contralateral challenge of CT26. The buffer for Oxaliplatin is critical. The two ICD inducer mixture is promising as an agnostic sensitizer for carcinomas like colorectal carcinoma.

Human adenovirus (HAdV) infection is common in the general population and can cause a range of clinical manifestations, among which pneumonia and keratoconjunctivitis are the most common. Although HAdV infections are mostly self-limiting, infections in immunocompromised individuals can be severe. No antiviral drug has been approved for treating adenoviruses. Filociclovir (FCV) is a nucleoside analogue which has successfully completed phase I human clinical safety studies and is now being developed for treatment of human cytomegalovirus (HCMV)-related disease in immunocompromised patients. In this report, we show that FCV is a potent broad-spectrum inhibitor of HAdV types 4 to 8, with 50% effective concentrations (EC(50)s) ranging between 1.24 and 3.6 μM and a 50% cytotoxic concentration (CC(50)) of 100 to 150 μM in human foreskin fibroblasts (HFFs). We also show that the prophylactic oral administration of FCV (10 mg/kg of body weight) 1 day prior to virus challenge and then daily for 14 days to immunosuppressed Syrian hamsters infected intravenously with HAdV6 was sufficient to prevent morbidity and mortality. FCV also mitigated tissue damage and inhibited virus replication in the liver. The 10-mg/kg dose had similar effects even when the treatment was started on day 4 after virus challenge. Furthermore, FCV administered at the same dose after intranasal challenge with HAdV6 partially mitigated body weight loss but significantly reduced pathology and virus replication in the lung. These findings suggest that FCV could potentially be developed as a pan-adenoviral inhibitor.

Model animals are indispensable for the study of human diseases, and in general, of complex biological processes. The Syrian hamster is an important model animal for infectious diseases, behavioral, and metabolic science for which more experimental tools are becoming available. Here, we describe the generation and characterization of an interleukin-2 receptor subunit gamma (IL2RG) knockout (KO) Syrian hamster strain. In humans, mutations in IL2RG can result in a total failure in T and natural killer (NK) lymphocyte development and nonfunctional B lymphocytes (X-linked severe combined immunodeficiency; XSCID). Therefore, we sought to develop a non-murine model to study XSCID and the infectious diseases associated with IL2RG deficiency. We demonstrated that the IL2RG KO hamsters have a lymphoid compartment that is greatly reduced in size and diversity and is impaired in function. As a result of the defective adaptive immune response, IL2RG KO hamsters developed a more severe human adenovirus infection and cleared virus less efficiently than immune competent wild-type (wt) hamsters. Due to this enhanced virus replication, IL2RG KO hamsters developed more severe adenovirus-induced liver pathology than wt hamsters. This novel hamster strain will provide researchers with a new tool to investigate human XSCID and its related infections.

Infections of immunocompromised patients with human adenoviruses (hAd) can develop into life-threatening conditions, whereas drugs with anti-adenoviral efficiency are not clinically approved and have limited efficacy. Small double-stranded RNAs that induce RNAi represent a new class of promising anti-adenoviral therapeutics. However, as yet, their efficiency to treat hAd5 infections has only been investigated in vitro. In this study, we analyzed artificial microRNAs (amiRs) delivered by self-complementary adeno-associated virus (scAAV) vectors for treatment of hAd5 infections in immunosuppressed Syrian hamsters. In vitro evaluation of amiRs targeting the E1A, pTP, IVa2, and hexon genes of hAd5 revealed that two scAAV vectors containing three copies of amiR-pTP and three copies of amiR-E1A, or six copies of amiR-pTP, efficiently inhibited hAd5 replication and improved the viability of hAd5-infected cells. Prophylactic application of amiR-pTP/amiR-E1A- and amiR-pTP-expressing scAAV9 vectors, respectively, to immunosuppressed Syrian hamsters resulted in the reduction of hAd5 levels in the liver of up to two orders of magnitude and in reduction of liver damage. Concomitant application of the vectors also resulted in a decrease of hepatic hAd5 infection. No side effects were observed. These data demonstrate anti-adenoviral RNAi as a promising new approach to combat hAd5 infection. Keywords: adenovirus infection, RNA interference, antiviral therapy, AAV vector

Taterapox virus (TATV) is phylogenetically the closest related virus to variola&mdash, the etiological agent of smallpox. Despite the similarity, few studies have evaluated the virus. In vivo, TATV can infect several animals but produces an inapparent infection in wild-type mice, however, TATV does cause morbidity and mortality in some immunocompromised strains. We employed in vitro techniques to compare TATV to ectromelia (ECTV) and vaccinia (VACV) viruses. Both ECTV and TATV replicate efficiently in primate cell lines but TATV replicates poorly in murine cells lines. Furthermore, TATV induces cytopathic effects, but to a lesser extent than ECTV, and changes cytoskeletal networks differently than both ECTV and VACV. Bioinformatic studies revealed differences in several immunomodulator open reading frames that could contribute to the reduced virulence of TATV, which were supported by in vitro cytokine assays.

The accumulating evidence demonstrates that Syrian hamsters have advantages as models for various diseases. To develop a Syrian hamster (Mesocricetus auratus) model of human immunodeficiency caused by RAG1 gene mutations, we employed the CRISPR/Cas9 system and introduced an 86-nucleotide frameshift deletion in the hamster RAG1 gene encoding part of the N-terminal non-core domain of RAG1. Histological and immunohistochemical analyses demonstrated that these hamsters (referred herein as RAG1-86nt hamsters) had atrophic spleen and thymus, and developed significantly less white pulp and were almost completely devoid of splenic lymphoid follicles. The RAG1-nt86 hamsters had barely detectable CD3+ and CD4+ T cells. The expression of B and T lymphocyte-specific genes (CD3γ and CD4 for T cell-specific) and (CD22 and FCMR for B cell-specific) was dramatically reduced, whereas the expression of macrophage-specific (CD68) and natural killer (NK) cell-specific (CD94 and KLRG1) marker genes was increased in the spleen of RAG1-nt86 hamsters compared to wildtype hamsters. Interestingly, despite the impaired development of B and T lymphocytes, the RAG1-86nt hamsters still developed neutralizing antibodies against human adenovirus type C6 (HAdV-C6) upon intranasal infection and were capable of clearing the infectious viruses, albeit with slower kinetics. Therefore, the RAG1-86nt hamster reported herein (similar to the hypomorphic RAG1 mutations in humans that cause Omenn syndrome), may provide a useful model for studying the pathogenesis of the specific RAG1-mutation-induced human immunodeficiency, the host immune response to adenovirus infection and other pathogens as well as for evaluation of cell and gene therapies for treatment of this subset of RAG1 mutation patients.

The Syrian golden hamster is an attractive animal for research on infectious diseases and other diseases. We report here the sequencing, assembly, and annotation of the Syrian hamster transcriptome. We include transcripts from ten pooled tissues from a naïve hamster and one stimulated with lipopolysaccharide. Our data set identified 42,707 non-redundant transcripts, representing 34,191 unique genes. Based on the transcriptome data, we generated a custom microarray and used this new platform to investigate the transcriptional response in the Syrian hamster liver following intravenous adenovirus type 5 (Ad5) infection. We found that Ad5 infection caused a massive change in regulation of liver transcripts, with robust up-regulation of genes involved in the antiviral response, indicating that the innate immune response functions in the host defense against Ad5 infection of the liver. The data and novel platforms developed in this study will facilitate further development of this important animal model.

The main characteristic of cancers, including breast cancer, is the ability of cancer cells to proliferate uncontrollably. However, the underlying mechanisms of cancer cell proliferation, especially those regulated by the RNA binding protein tristetraprolin (TTP), are not completely understood. In this study, we found that TTP inhibits cell proliferation in vitro and suppresses tumor growth in vivo through inducing cell cycle arrest at the S phase. Our studies demonstrate that TTP inhibits c-Jun expression through the C-terminal Zn finger and therefore increases Wee1 expression, a regulatory molecule which controls cell cycle transition from the S to the G2 phase. In contrast to the well-known function of TTP in regulating mRNA stability, TTP inhibits c-Jun expression at the level of transcription by selectively blocking NF-κB p65 nuclear translocation. Reconstitution of NF-κB p65 completely abolishes the inhibition of c-Jun transcription by TTP. Moreover, reconstitution of c-Jun in TTP-expressing breast tumor cells diminishes Wee1 overexpression and promotes cell proliferation. Our results indicate that TTP suppresses c-Jun expression that results in Wee1 induction which causes cell cycle arrest at the S phase and inhibition of cell proliferation. Our study provides a new pathway for TTP function as a tumor suppressor which could be targeted in tumor treatment.

Human adenoviruses can cause serious disseminated infections including death in immunosuppressed patients, especially pediatric allogeneic hematopoietic stem cell transplant (allo-HSCT) patients. There are no drugs approved to treat such infections. Cidofovir is used intravenously in many transplant clinics, probably with some effect, but controlled trials have not been completed. Cidofovir is an acyclic nucleoside phosphonate analog of cytidine monophosphate. Following conversion to its diphosphate form within cells, cidofovir is a preferred substrate for the adenovirus DNA polymerase, leading to viral DNA chain termination. Problems with cidofovir include poor cellular uptake and nephrotoxicity. Brincidofovir, a lipid-linked derivative of cidofovir which is active against five families of double-stranded DNA viruses, represents a major advance in anti-adenovirus therapy. It is administered orally, taken up readily by cells followed by release of cidofovir within cells, and is not nephrotoxic. Brincidofovir, under development by Chimerix, Inc., is being evaluated against adenovirus infections in transplant patients including allo-HSCT patients in a phase III clinical trial (AdVise Study). Preliminary results indicate that brincidofovir is safe and very effective at decreasing adenovirus viremia and adenovirus-induced pathogenicity and mortality. Anti-adenovirus adoptive T cell therapy is another very promising approach to treating allo-HSCT patients as demonstrated in clinical studies.

Adenovirus infections of immunocompromised pediatric hematopoietic stem cell transplant patients can develop into serious and often deadly multi-organ disease. There are no drugs approved for adenovirus infections. Cidofovir (an analog of 2-deoxycytidine monophosphate) is used at times but it can be nephrotoxic and its efficacy has not been proven in clinical trials. Brincidofovir, a promising lipid-linked derivative of cidofovir, is in clinical trials. Ganciclovir, an analog of 2-deoxyguanosine, has been employed occasionally but with unknown efficacy in the clinic. In this study, we evaluated valganciclovir against disseminated adenovirus type 5 (Ad5) infection in our permissive immunosuppressed Syrian hamster model. We administered valganciclovir prophylactically, beginning 12 h pre-infection or therapeutically starting at Day 1, 2, 3, or 4 post-infection. Valganciclovir significantly increased survival, reduced viral replication in the liver, and mitigated the pathology associated with Ad5 infection. In cultured cells, valganciclovir inhibited Ad5 DNA replication and blocked the transition from early to late stage of infection. Valganciclovir directly inhibited Ad5 DNA polymerase in vitro, which may explain, at least in part, its mechanism of action. Ganciclovir and valganciclovir are approved to treat infections by certain herpesviruses. Our results support the use of valganciclovir to treat disseminated adenovirus infections in immunosuppressed patients.

Human adenoviruses (AdV) cause generally mild infections of the respiratory and GI tracts as well as some other tissues. However, AdV can cause serious infection in severely immunosuppressed individuals, especially pediatric patients undergoing allogeneic hematopoietic stem cell transplantation, where mortality rates are up to 80% with disseminated disease. Despite the seriousness of AdV disease, there are no drugs approved specifically to treat AdV infections. We report here that USC-087, an N-alkyl tyrosinamide phosphonate ester prodrug of HPMPA, the adenine analog of cidofovir, is highly effective against multiple AdV types in cell culture. USC-087 is also effective against AdV-C6 in our immunosuppressed permissive Syrian hamster model. In this model, hamsters are immunosuppressed by treatment with high dose cyclophosphamide. Injection of AdV-C6 (or AdV-C5) intravenously leads to a disseminated infection that resembles the disease seen in humans, including death. We have tested the efficacy of orally-administered USC-087 against the median lethal dose of intravenously administered AdV-C6. USC-087 completely prevented or significantly decreased mortality when administered up to 4 days post challenge. USC-087 also prevented or significantly decreased liver damage caused by AdV-C6 infection, and suppressed virus replication even when administered 4 days post challenge. These results imply that USC-087 is a promising candidate for drug development against HAdV infections.

Oncolytic viruses based on adenovirus type 5 (Ad5) have been developed as a new class of therapeutic agents for cancers that are resistant to conventional therapies. Clinical experience shows that these agents are safe, but virotherapy alone has not achieved long-term cure in cancer patients. The vast majority of oncolytic adenoviruses used in clinical trials to date have deletion of the E3B genes. It has been demonstrated that the antitumor potency of the E3B-deleted mutant (dl309) is inferior to adenovirus with E3B genes intact. Tumors treated with dl309 show markedly greater macrophage infiltration than E3B-intact adenovirus. However, the functional mechanisms for this were not previously known. Here, we demonstrate that deletion of E3B genes increases production of chemokines by monocytes after adenovirus infection and increases monocyte migration. The E3B 14,700-Da protein (E3B-14.7K) inhibits STAT1 function by preventing its phosphorylation and nuclear translocation. The STAT1 inhibitor, fludarabine, rescues the effect of E3B-14.7K deletion by downregulating target chemokine expression in human and murine monocytes and results in an enhanced antitumor efficacy with dl309 in vivo. These findings have important implications for clinical use of E3B-deleted oncolytic adenovirus and other E3B-deleted adenovirus vector-based therapy.

Recently, increasing attention has been focused on the influence of sex on the course of infectious diseases. Thus far, the best-documented examples point toward an immune-mediated mechanism: the generally stronger immune response in females can result in a faster clearance of the pathogen or, conversely, a more severe immune-mediated pathology. Here, we report that human species C adenoviruses replicate more and cause more pathology in male Syrian hamsters than in females. We also show that this sex disparity is not caused by a stronger immune response to the infection by the female hamsters. Rather, the liver of male hamsters is more susceptible to adenovirus infection: after intravenous injection, more hepatocytes become infected in male animals than in females. We hypothesize that Kupffer cells (hepatic tissue macrophages) of female animals are more active in sequestering circulating virions, and thus protect hepatocytes more efficiently than those of males.

Adenovirus infections of immunocompromised patients can cause a severe multi-organ disease that often results in the patients’ death. Presently, there are no drugs specifically approved to treat adenovirus infections, and clinicians resort to the off-label use of antivirals that are approved to treat other DNA virus infections, most frequently cidofovir (CDV). CDV, however, has considerable nephrotoxicity, thus it is recommended only for the most severe cases of adenovirus infections. To facilitate the development of effective, non-toxic antivirals against adenovirus, we have developed a permissive animal model based on the Syrian hamster that can be used to test the efficacy of antiviral compounds. Here, we show that in the hamster model, HAdV-C6 is a more useful challenge virus than the previously described HAdV-C5, because it is filtered out by tissue macrophages to a lesser extent. HAdV-C6 has a 10-fold lower LD50 in hamsters than HAdV-C5 and the pathology is caused by virus replication to a larger extent. We show that valganciclovir (VGCV), a drug that was shown to be active against intravenous HAdV-C5 infection previously, is efficacious against HAdV-C6 when administered either prophylactically or therapeutically. Further, we show for the first time that VGCV, and to a lesser extent CDV, can be used to treat respiratory adenovirus infections in the hamster model. These results extend the utility of the hamster model, and demonstrate the efficacy of two drugs available for clinicians to treat adenovirus infections.

Syrian hamsters are permissive for the replication of species C human adenoviruses (HAdV-C). The virus replicates to high titers in the liver of these animals after intravenous infection, while respiratory infection results in virus replication in the lung. Here we show that two types belonging to species C, HAdV-C5 and HAdV-C6, replicate to significantly different extents and cause pathology with significantly different severities, with HAdV-C6 replicating better and inducing more severe and more widespread lesions. The virus burdens in the livers of HAdV-C6-infected hamsters are higher than the virus burdens in HAdV-C5-infected ones because more of the permissive hepatocytes get infected. Furthermore, when hamsters are infected intravenously with HAdV-C6, live, infectious virus can be isolated from the lung and the kidney, which is not seen with HAdV-C5. Similarly to mouse models, in hamsters, HAdV-C6 is sequestered by macrophages to a lesser degree than HAdV-C5. Depletion of Kupffer cells from the liver greatly increases the replication of HAdV-C5 in the liver, while it has only a modest effect on the replication of HAdV-C6. Elimination of Kupffer cells also dramatically increases the pathology induced by HAdV-C5. These findings indicate that in hamsters, pathology resulting from intravenous infection with adenoviruses is caused mostly by replication in hepatocytes and not by the abortive infection of Kupffer cells and the following cytokine storm. IMPORTANCE Immunocompromised human patients can develop severe, often lethal adenovirus infections. Respiratory adenovirus infection among military recruits is a serious problem, in some cases requiring hospitalization of the patient. Furthermore, adenovirus-based vectors are frequently used as experimental viral therapeutic agents. Thus, it is imperative that we investigate the pathogenesis of adenoviruses in a permissive animal model. Syrian hamsters are susceptible to infection with certain human adenoviruses, and the pathology accompanying these infections is similar to what is observed with adenovirus-infected human patients. We demonstrate that replication in permissive cells in a susceptible host animal is a major part of the mechanism by which systemic adenovirus infection induces pathology, as opposed to the chiefly immune-mediated pathology observed in nonsusceptible hosts. These findings support the use of compounds inhibiting adenovirus replication as a means to block adenovirus-induced pathology.

Adenovirus vectors are the most commonly employed vector for cancer gene therapy. They are also used for gene therapy and as vaccines to express foreign antigens. Adenovirus vectors can be replication-defective; certain essential viral genes are deleted and replaced by a cassette that expresses a foreign therapeutic gene. Such vectors are used for gene therapy, as vaccines, and for cancer therapy. Replication-competent (oncolytic) vectors are employed for cancer gene therapy. Oncolytic vectors are engineered to replicate preferentially in cancer cells and to destroy cancer cells through the natural process of lytic virus replication. Many clinical trials indicate that replication-defective and replication-competent adenovirus vectors are safe and have therapeutic activity.

We have previously reported that intratumoral injection of VRX-007--an Ad5 (a species C adenovirus)-based vector overexpressing adenovirus death protein--can suppress the growth of subcutaneous HaK (hamster renal cancer) tumors. VRX-007 replication and tumor growth inhibition are enhanced when the hamsters are immunosuppressed by a high dose of cyclophosphamide (CP), an immunosuppressive and chemotherapeutic agent. Here, we report that continuous immunosuppression with CP was not required for increased oncolytic activity of VRX-007 because short-term dosing or continuous dosing with the drug yielded similar antitumor results. Prolonged viral replication was found only in animals on continuous CP treatment. We used 007-Luc, a replication-competent, luciferase-expressing vector similar to VRX-007, to investigate the replication of the vector over time. Tumor growth inhibition was similar in hamsters given CP treatment either 1 week before or 1 week after 007-Luc injection, which suggests that CP exerts its antitumor efficacy independently of vector therapy. 007-Luc did not spread far from the inoculation site, even in immunosuppressed, CP-treated animals. Our results indicate that the enhanced effectiveness that is produced by the combination of VRX-007 and CP therapies is due to their two independent mechanisms and that they do not have to be given simultaneously for the improved outcome.

Human adenoviruses have been studied extensively in cell culture and have been a model for studies in molecular, cellular, and medical biology. However, much less is known about adenovirus replication and pathogenesis in vivo in a permissive host because of the lack of an adequate animal model. Presently, the most frequently used permissive immunocompetent animal model for human adenovirus infection is the Syrian hamster. Species C human adenoviruses replicate in these animals and cause pathology that is similar to that seen with humans. Here, we report findings with a new Syrian hamster strain in which the STAT2 gene was functionally knocked out by site-specific gene targeting. Adenovirus-infected STAT2 knockout hamsters demonstrated an accentuated pathology compared to the wild-type control animals, and the virus load in the organs of STAT2 knockout animals was 100- to 1000-fold higher than that in wild-type hamsters. Notably, the adaptive immune response to adenovirus is not adversely affected in STAT2 knockout hamsters, and surviving hamsters cleared the infection by 7 to 10 days post challenge. We show that the Type I interferon pathway is disrupted in these hamsters, revealing the critical role of interferon-stimulated genes in controlling adenovirus infection. This is the first study to report findings with a genetically modified Syrian hamster infected with a virus. Further, this is the first study to show that the Type I interferon pathway plays a role in inhibiting human adenovirus replication in a permissive animal model. Besides providing an insight into adenovirus infection in humans, our results are also interesting from the perspective of the animal model: STAT2 knockout Syrian hamster may also be an important animal model for studying other viral infections, including Ebola-, hanta-, and dengue viruses, where Type I interferon-mediated innate immunity prevents wild type hamsters from being effectively infected to be used as animal models.

Oncolytic adenovirus (Ad) vectors present a new modality to treat cancer. These vectors attack tumors via replicating in and killing cancer cells. Upon completion of the vector replication cycle, the infected tumor cell lyses and releases progeny virions that are capable of infecting neighboring tumor cells. Repeated cycles of vector replication and cell lysis can destroy the tumor. Numerous Ad vectors have been generated and tested, some of them reaching human clinical trials. In 2005, the first oncolytic Ad was approved for the treatment of head-and-neck cancer by the Chinese FDA. Oncolytic Ads have been proven to be safe, with no serious adverse effects reported even when high doses of the vector were injected intravenously. The vectors demonstrated modest anti-tumor effect when applied as a single agent; their efficacy improved when they were combined with another modality. The efficacy of oncolytic Ads can be improved using various approaches, including vector design, delivery techniques, and ancillary treatment, which will be discussed in this review.

Primary and some transformed hepatocytes undergo apoptosis in response to transforming growth factor beta1 (TGFbeta). We report that infection with species C human adenovirus conferred resistance to TGFbeta-induced apoptosis in human hepatocellular carcinoma cells (Huh-7). Protection against TGFbeta-mediated cell death in adenovirus-infected cells correlated with the maintenance of normal nuclear morphology, lack of pro-caspases 8 and 3 processing, maintenance of the mitochondrial membrane potential, and lack of cellular DNA degradation. The TGFbeta pro-apoptotic signaling pathway was blocked upstream of mitochondria in adenovirus-infected cells. Both the N-terminal sequences of the E1A proteins and the E1B-19K protein were necessary to protect infected cells against TGFbeta-induced apoptosis.

Syrian hamster is a practical animal model for studying the systemic effects of oncolytic vectors derived from adenovirus serotype 5 (Ad5). Ad5 replicates well in Syrian hamster tissues, and Syrian hamster cell lines are available that are known to support Ad5 replication. In this study, we established four new Syrian hamster cell lines from transplantable pancreatic, renal, hepatic and lung tumors. The pancreatic cell line (SHPC6) and the renal cell line were highly permissive for Ad5 replication. The SHPC6 cell line formed disseminated intraperitoneal tumors when cells were injected into the peritoneal cavity. INGN 007, an oncolytic Ad5-based vector, completely reversed the growth of disseminated intraperitoneal SHPC6 tumor nodules following intraperitoneal injection of the vector, leading to 100% survival of the treated animals. SHPC6 cells also formed subcutaneous tumors, whose growth was suppressed by INGN 007 following intratumoral injection. INGN 007 replicated in both the intraperitoneal and subcutaneous SHPC6 tumors. Following intraperitoneal injection, INGN 007 did not replicate in the livers of hamsters with intraperitoneal SHPC6 tumors, and was not hepatotoxic. These studies suggest that the SHPC6 cell line may be useful as a model for disseminated pancreatic cancer, and that INGN 007 may be a safe and effective vector to treat these tumors.

Immune responses against adenovirus (Ad) vectors pose a possible concern for the outcome of treatment efficacy. To address the role of preexisting immunity in oncolytic Ad vector antitumor efficacy following intratumoral injection of vector as well as tumor-to-tissue spread of the vector, we employed the Syrian hamster model. These animals are immunocompetent, and their tumors and tissues are permissive for replication of Ad type 5 (Ad5). We used the adenovirus death protein-overexpressing Ad5-based vector INGN 007. Subcutaneous tumors were established in groups of hamsters that were or were not immunized with Ad5. Half of the hamsters in these groups were immunosuppressed with cyclophosphamide. For all groups, tumors injected with INGN 007 grew significantly more slowly than those injected with buffer. Under immunocompetent conditions, there was no significant effect of preexisting immunity on vector antitumor efficacy. Soon after the tumors in naïve animals were injected with vector, the hamsters developed neutralizing antibody (NAb) and the difference in NAb titers between the naïve and immunized groups diminished. Under immunosuppressed conditions, preexisting NAb did significantly reduce vector efficacy. Thus, NAb do reduce vector efficacy to some extent, but immunosuppression is required to observe the effect. Regarding vector toxicity, there was spillover of vector from the tumor to the liver and lungs in naïve immunocompetent hamsters, and this was nearly eliminated in the immunized hamsters. Thus, preexisting immunity to Ad5 does not affect INGN 007 antitumor efficacy following intratumoral injection, but immunity prevents vector spillover from the tumor to the liver and lungs.

Preclinical biodistribution studies with INGN 007, an oncolytic adenovirus (Ad) vector, supporting an early stage clinical trial were conducted in Syrian hamsters, which are permissive for Ad replication, and mice, which are a standard model for assessing toxicity and biodistribution of replication-defective (RD) Ad vectors. Vector dissemination and pharmacokinetics following intravenous administration were examined by real-time PCR in nine tissues and blood at five time points spanning 1 year. Select organs were also examined for the presence of infectious vector/virus. INGN 007 (VRX-007), wild-type Ad5 and AdCMVpA (an RD vector) were compared in the hamster model, whereas only INGN 007 was examined in mice. DNA of all vectors was widely disseminated early after injection, but decayed rapidly in most organs. In the hamster model, DNA of INGN 007 and Ad5 was more abundant than that of the RD vector AdCMVpA at early times after injection, but similar levels were seen later. An increased level of INGN 007 and Ad5 DNA but not AdCMVpA DNA in certain organs early after injection, and the presence of infectious INGN 007 and Ad5 in lung and liver samples at early times after injection, strongly suggests that replication of INGN 007 and Ad5 occurred in several Syrian hamster organs. There was no evidence of INGN 007 replication in mice. In addition to providing important information about INGN 007, the results underscore the utility of the Syrian hamster as a permissive immunocompetent model for Ad5 pathogenesis and oncolytic Ad vectors.

We recently described an immunocompetent Syrian hamster model for oncolytic adenoviruses (Ads) that permits virus replication in tumor cells as well as some normal tissues. This model allows exploration of interactions between the virus, tumor, normal organs, and host immune system that could not be examined in the immunodeficient or nonpermissive animal models previously used in the oncolytic Ad field. Here we asked whether the immune response to oncolytic Ad enhances or limits antitumor efficacy. We first determined that cyclophosphamide (CP) is a potent immunosuppressive agent in the Syrian hamster and that CP alone had no effect on tumor growth. Importantly, we found that the antitumor efficacy of oncolytic Ads was significantly enhanced in immunosuppressed animals. In animals that received virus therapy plus immunosuppression, significant differences were observed in tumor histology, and in many cases little viable tumor remained. Notably, we also determined that immunosuppression allowed intratumoral virus levels to remain elevated for prolonged periods. Although favorable tumor responses can be achieved in immunocompetent animals, the rate of virus clearance from the tumor may lead to varied antitumor efficacy. Immunosuppression, therefore, allows sustained Ad replication and oncolysis, which leads to substantially improved suppression of tumor growth.

Adenoviruses (Ads) cause a wide array of end-organ and disseminated diseases in severely immunosuppressed patients. For example, ≈20% of pediatric allogeneic hematopoietic stem cell transplant recipients develop disseminated Ad infection, and the disease proves fatal in as many as 50–80% of these patients. Ad infections are a serious problem for solid-organ transplant recipients and AIDS patients as well. Unfortunately, there are no antiviral drugs approved specifically to treat these infections. A suitable animal model that is permissive for Ad replication would help in the discovery process. Here we identify an animal model to study Ad pathogenesis and the efficacy of antiviral compounds. We show that human serotype 5 Ad (Ad5) causes severe systemic disease in immunosuppressed Syrian hamsters that is similar to that seen in immunocompromised patients. We also demonstrate that hexadecyloxypropyl-cidofovir (CMX001) rescues the hamsters from a lethal challenge with Ad5. The antiviral drug provided protection both prophylactically and when given up to 2 days after i.v. exposure to Ad5. CMX001 acts by reducing Ad replication in key target organs. Thus, the immunosuppressed Syrian hamster is a powerful model to evaluate anti-Ad drugs, and its use can facilitate the entry of drugs such as CMX001 into clinical trials.

A short open reading frame named the “U exon,” located on the adenovirus (Ad) l -strand (for leftward transcription) between the early E3 region and the fiber gene, is conserved in mastadenoviruses. We have observed that Ad5 mutants with large deletions in E3 that infringe on the U exon display a mild growth defect, as well as an aberrant Ad E2 DNA-binding protein (DBP) intranuclear localization pattern and an apparent failure to organize replication centers during late infection. Mutants in which the U exon DNA is reconstructed have a reversed phenotype. Chow et al. (L. T. Chow et al., J. Mol. Biol. 134:265-303, 1979) described mRNAs initiating in the region of the U exon and spliced to downstream sequences in the late DBP mRNA leader and the DBP-coding region. We have cloned this mRNA (as cDNA) from Ad5 late mRNA; the predicted protein is 217 amino acids, initiating in the U exon and continuing in frame in the DBP leader and in the DBP-coding region but in a different reading frame from DBP. Polyclonal and monoclonal antibodies generated against the predicted U exon protein (UXP) showed that UXP is ∼24K in size by immunoblot and is a late protein. At 18 to 24 h postinfection, UXP is strongly associated with nucleoli and is found throughout the nucleus; later, UXP is associated with the periphery of replication centers, suggesting a function relevant to Ad DNA replication or RNA transcription. UXP is expressed by all four species C Ads. When expressed in transient transfections, UXP complements the aberrant DBP localization pattern of UXP-negative Ad5 mutants. Our data indicate that UXP is a previously unrecognized protein derived from a novel late l -strand transcription unit.

A molecular clone of Japanese encephalitis (JE) virus Nakayama strain was used to create intertypic viruses containing either the 5′-C-prM-E or the prM-E region of the attenuated JE SA14-14-2 virus in the JE Nakayama background. These two intertypic JE viruses, JE-X/5′CprME(S) and JE-X/prME(S), respectively, generally resembled the parental JE virus in cell culture properties. Similar to virus derived from the JE Nakayama molecular clone (JE-XJN), JE-X/prME(S) was highly neuroinvasive and neurovirulent for young adult mice, whereas JE-X/5′CprME(S) was attenuated for neuroinvasiveness and only partially attenuated for neurovirulence. Immunization of young mice with JE-X/5′CprME(S) virus elicited neutralizing antibodies against JE Nakayama virus and conferred protection against encephalitis following challenge with JE Nakayama virus. The sequence of the JE-X/5′CprME(S) virus differed from that of JE-X/prME(S) virus at two nucleotides in the 5′ UTR, 3 amino acid positions in the capsid protein, 4 positions in the prM protein and 1 in the envelope protein. For JE-X/prME(S) virus, the 4 differences in prM and the single substitution in the envelope represented reversions to the sequence of JE Nakayama virus. Overall, this study reveals that molecular determinants associated with the prM-E region of the attenuated JE SA14-14-2 virus are insufficient by themselves to confer an attenuation phenotype upon JE Nakayama virus. This suggests a role for determinants in the 5′ UTR and/or the capsid protein of the JE SA 14-14-2 virus genome in influencing the virulence properties of the JE Nakayama virus in the mouse model.

In this study, we show that adenoviral infection induced accumulation of the sphingolipid ceramide in a dose- and time-dependent manner. This accumulation preceded cell lysis, occurred in the absence of biochemical evidence of apoptosis, and was derived from de novo synthesis of ceramide. An adenovirus mutant that lacks the adenovirus death protein (ADP) produced ceramide accumulation in the absence of cell lysis. This suggested that ceramide accumulation was either driven by adenovirus or was a cellular stress response but was unlikely a result of cell death. The use of inhibitors of ceramide synthesis resulted in a significant delay in cell lysis, suggesting that ceramide was necessary for the lytic phase of the infection. Serine/arginine-rich (SR) proteins were dephosphorylated during the late phase of the viral cycle, and inhibitors of ceramide synthesis reversed this. These findings suggest that adenovirus utilizes the ceramide pathway to regulate SR proteins during infection.

Oncolytic adenoviruses represent an innovative approach to cancer therapy. These vectors are typically evaluated in immunodeficient mice with human xenograft tumors. However, in addition to being immunodeficient, this model is limited because normal and cancerous mouse tissues are poorly permissive for human adenovirus replication. This prompted us to search for a model that more accurately reflects the use of oncolytic adenoviruses in cancer patients. To this end, we developed a novel Syrian hamster model that is both immunocompetent and replication-permissive. We found that human adenovirus replicates well in Syrian hamster cell lines and confirmed replication in the lungs. Oncolytic adenovirus injection showed efficacy in three different hamster tumor models. Furthermore, i.t. oncolytic adenovirus injection resulted in suppression of primary and metastatic lesions, i.t. replication and necrosis, vector entrance into the bloodstream, replication in the liver and lungs, and anti-adenovirus antibody induction. Our findings show that the Syrian hamster is a promising immunocompetent model that is permissive to human adenovirus replication in tumors as well as normal tissues. Therefore, the Syrian hamster model may become a valuable tool for the field of oncolytic adenovirus vectors in which vector safety and efficacy can be evaluated. (Cancer Res 2006; 66(3): 1270-6)

The majority of proteins encoded in the early 3 (E3) region of human subgroup C adenoviruses function to modulate the host immune response. For example, gp19K, one of these E3 proteins, prevents the major histocompatibility complex type I (MHC-I) from presenting viral antigens on the surface of the infected cell. Other E3 proteins, such as the RID and 14.7K proteins, counteract the effector phase of the cellular immune response. In order to study further the effects of these proteins, we constructed an E1-/E3- adenovirus vector, Ad/E3, that contains all the E3 genes with the exception of the cytolytic adp gene, inserted into the deleted E1 region. The transcription of the E3 genes in this vector is driven by a CMV promoter in place of the native E3 promoter. Ad/E3 expressed close to wild-type adenovirus levels of all E3 proteins, and these proteins appear to function normally in cell culture. For example, in Ad/E3-infected cells, surface expression of MHC-I was down-regulated, as was cell surface display of death receptors Fas and TRAIL Receptor 1. A human cell line of lung origin (A549), which was rapidly rejected after transplantation into C57BL/6 mice, was protected for an extended time from the host immune response after infection with an Ad/E3, and went through a number of divisions in immunocompetent mice. These latter results indicate that the E3 proteins protect cells from destruction by the immune system.

Replication-competent (RC) adenoviruses (Ads) are increasingly being developed as oncolytic vectors and as vehicles for delivering vaccine antigens. Although the safety of such vectors in humans is of paramount importance, these vectors pose additional special concerns. Specifically, the prospect of causing Ad-mediated disease in the patient, the amount and sites of Ad replication, the possibility of virus shedding leading to unintended transmission to patient contacts, and the potential for persistence in the inoculated individual must be evaluated. Previous experience with administration of wild-type and RC recombinant Ads to humans may shed light on some of these issues. Experimental infections of humans with natural Ad isolates and RC recombinant vectors show that in adults Ads cause mild or no disease, particularly with Ad serotypes 2 and 5, the serotypes most often used to make recombinant constructs. Other studies show that Ad can replicate in experimentally infected persons, that in some situations Ads can be shed and transmitted to close contacts, and that there is evidence for persistent/latent Ad infection in naturally infected individuals. Overall, these studies indicate that Ads can be safely administered to humans for the treatment of cancer and as antigen delivery vehicles suggesting that the continued development of RC oncolytic and vaccine vectors should be pursued.

Transforming growth factor β1 (TGF-β1) signaling is compromised in many tumors, thereby allowing the tumor to escape the growth-inhibitory and proapoptotic activities of the cytokine. Human adenoviruses interfere with a number of cellular pathways involved in cell cycle regulation and apoptosis, initially placing the cell in a “tumor-like” state by forcing quiescent cells into the cell cycle and also inhibiting apoptosis. We report that adenovirus-infected cells resemble tumor cells in that TGF-β1 signaling is inhibited. The levels of TGF-β1 receptor II (TβRII) in adenovirus-infected cells were decreased, and this decrease was mapped, by using virus mutants, to the E1A gene and to amino acids 2 to 36 and the C-terminal binding protein binding site in the E1A protein. The decrease in the TβRII protein was accompanied by a decrease in TβRII mRNA. The decrease in TβRII protein levels in adenovirus-infected cells was greater than the decrease in TβRII mRNA, suggesting that downregulation of the TβRII protein may occur through more than one mechanism. Surprisingly in this context, the half-lives of the TβRII protein in infected and uninfected cells were similar. TGF-β1 signaling was compromised in cells infected with wild-type adenovirus, as measured with 3TP-lux, a TGF-β-sensitive reporter plasmid expressing luciferase. Adenovirus mutants deficient in TβRII downregulation did not inhibit TGF-β1 signaling. TGF-β1 pretreatment reduced the relative abundance of adenovirus structural proteins in infected cells, an effect that was potentiated when cells were infected with mutants incapable of modulating the TGF-β signaling pathway. These results raise the possibility that inhibition of TGF-β signaling by E1A is a means by which adenovirus counters the antiviral defenses of the host.

ADP (also known as E3-11.6K protein) is synthesized abundantly in late adenovirus infection and is required for efficient lysis of infected cells and release of viral progeny at the end of the viral replication cycle. ADP is a type III bitopic N endo C exo nuclear membrane and Golgi glycoprotein that is produced at high levels in late adenovirus infection (>24 h postinfection). We show pulse-chase and other studies indicating that ADP undergoes a complex process of N- and O-linked glycosylation and proteolytic cleavage. In order to further characterize ADP, a series of 23 deletion and point mutations has been constructed in the adenovirus serotype 2 adp gene and then built into a wild-type adenovirus background. These mutants were analyzed for processing and intracellular localization of ADP. Mutation of the single predicted N glycosylation site eliminated N glycosylation. Deletion of a region in ADP rich in serine and threonine residues reduced O glycosylation. In general, mutations within the lumenal domain of ADP resulted in lower protein stability; immunofluorescence assays indicated that these ADPs were primarily present in the Golgi apparatus. Viruses with mutations within the cytoplasmic-nucleoplasmic domain of ADP showed normal glycosylation patterns and protein abundance for ADP, but the protein was often found throughout cellular membranes rather than being localized specifically to the nuclear membrane and Golgi apparatus. The ADP virus mutants were analyzed by cell viability assays to determine the kinetics of cell lysis following infection of human A549 cells. In general, viruses with mutations within the lumenal domain of ADP display greatly reduced efficiencies of cell lysis. Viruses with large deletions in the cytoplasmic-nucleoplasmic domain of ADP retain much of their ability to lyse infected cells.

We have described three potential adenovirus type 5 (Ad5)-based replication-competent cancer gene therapy vectors named KD1, KD3, and VRX-007. All three vectors overexpress an Ad5 protein named Adenovirus Death Protein (ADP, also named E3-11.6 K protein). ADP is required for efficient lysis of Ad5-infected cells and spread of virus from cell to cell, and thus its overexpression increases the oncolytic activity of the vectors. KD1 and KD3 contain mutations in the Ad5 E1A gene that knock out binding of the E1A proteins to cellular p300/CBP and pRB; these mutations allow KD1 and KD3 to grow well in cancer cells but not in normal cells. VRX-007 has wild-type E1A. Here we report that radiation increases the oncolytic activity of KD1, KD3, and VRX-007. This increased activity was observed in cultured cells, and it was not because of radiation-induced replication of the vectors. The combination of radiation plus KD3 suppressed the growth of A549 lung adenocarcinoma xenografts in nude mice more efficiently than radiation alone or KD3 alone. The combination of ADP-overexpressing vectors and radiation may have potential in treating cancer.

The adenovirus-encoded receptor internalization and degradation (RID) protein (previously named E3-10.4K/14.5K), which is composed of RIDα and RIDβ subunits, down-regulates a number of cell surface receptors in the tumor necrosis factor (TNF) receptor superfamily, namely Fas, TRAIL receptor 1, and TRAIL receptor 2. Down-regulation of these “death” receptors protects adenovirus-infected cells from apoptosis induced by the death receptor ligands Fas ligand and TRAIL. RID also down-regulates certain tyrosine kinase cell surface receptors, especially the epidermal growth factor receptor (EGFR). RID-mediated Fas and EGFR down-regulation occurs via endocytosis of the receptors into endosomes followed by transport to and degradation within lysosomes. However, the molecular interactions underlying this function of RID are unknown. To investigate the molecular determinants of RIDβ that are involved in receptor down-regulation, mutations within the cytoplasmic tail of RIDβ were constructed and the mutant proteins were analyzed for their capacity to internalize and degrade Fas and EGFR and to protect cells from death receptor ligand-induced apoptosis. The results demonstrated the critical nature of a tyrosine residue near the RIDβ C terminus; mutation of this residue to alanine abolished RID function. Mutating the tyrosine to phenylalanine did not abolish the function of RID, arguing that phosphorylation of the tyrosine is not required for function. These data suggest that this tyrosine residue forms part of a tyrosine-based sorting signal (Yxxφ). Additional mutations that target another potential sorting motif and several possible protein-protein interaction motifs had no discernible effect on RID function. It was also demonstrated that mutation of serine 116 to alanine eliminated phosphorylation of RIDβ but did not affect any of the functions of RID that were examined. These results suggest a model in which the tyrosine-based sorting signal in RID plays a role in RID's ability to down-regulate receptors.

Previous research has indicated that the adenovirus protein complex named RID, derived from the E3 transcription unit, functions to remove the receptors named Fas/Apo1/CD95 (Fas) and epidermal growth factor receptor (EGFR) from the surface of cells. (The RID complex is composed of the RIDalpha and RIDbeta polypeptides, previously named 10.4K and 14.5K, respectively.) In response to RID, Fas and EGFR appear to be internalized into endosomes and degraded in lysosomes. Fas is a death receptor in the tumor necrosis factor (TNF) receptor superfamily. RID inhibits apoptosis via the Fas pathway, presumably because RID gets rid of Fas. Earlier work further showed that another adenovirus E3-coded protein, E3-14.7K, inhibits apoptosis induced by TNF. Most of the above studies have been conducted using viable virus mutants that lack one or more of the genes for RID, E3-14.7K, or E1B-19K (this protein, coded by the E1B transcription unit, also inhibits apoptosis via the TNF and Fas pathways). Some studies have also been conducted with the genes for RID or E3-14.7K transiently or stably transfected into cells. We now report a new approach to studying the E3 genes. We have constructed four E1-minus replication-defective vectors that have all the E3 genes deleted from their natural position and then reinserted, in different permutations, into the deleted E1 region under control of the cytomegalovirus immediate early promoter. Vector Ad/RID only has the genes for RIDalpha and RIDbeta. Vector Ad/14.7K only has the gene for E3-14.7K. Vector Ad/RID/14.7K only has the genes for RIDalpha, RIDbeta, and E3-14.7K. Vector Ad/E3 has all E3 genes, but there are two missense mutations in the gene for Adenovirus Death Protein. These vectors expressed RID and/or E3-14.7K, as expected. The RID-expressing vectors forced the internalization and degradation of Fas and EGFR, and they inhibited apoptosis induced through the Fas pathway. These vectors should be useful reagents to study the E3 proteins.

The successful clinical application of adenovirus (Ad) in cancer control has been of limited success because of the current inability to infect the majority of cancer cells with a large amount of vector. In this study, we show that when human lung tumors growing in immunodeficient nude mice were coinfected with a replication-defective (RD) Ad vector expressing green fluorescent protein and a replication-competent (RC) Ad vector named KD3, KD3 enhanced the expression of green fluorescent protein throughout the tumor. Also, KD3 and another RC vector named KD1 complemented the expression of luciferase from a RD vector in a human liver tumor xenotransplant in nude mice. Altogether, these results suggest that the combination of a RD vector with a RC vector might be a more effective treatment for cancer than either vector alone due to more widespread dissemination of the virus.

Adenovirus infections of immunocompromised patients can develop into deadly multiorgan or systemic disease. The virus is especially threatening for pediatric allogeneic hematopoietic stem cell transplant recipients; according to some studies, 10% or more of these patients succumb to disease resulting from adenovirus infection. At present, there is no drug approved for the treatment or prevention of adenovirus infections. Compounds that are approved to treat other virus infections are used off-label to combat adenovirus, but only anecdotal evidence of the efficacy of these drugs exists. Ganciclovir, a drug approved for the treatment of herpesvirus infection, was previously reported to be effective against human adenoviruses in vitro . To model adenovirus infections in immunocompromised humans, we examined ganciclovir's efficacy in immunosuppressed Syrian hamsters intravenously infected with type 5 human adenovirus (Ad5). This animal model is permissive for Ad5 replication, and the animals develop symptoms similar to those seen in humans. We demonstrate that ganciclovir suppresses Ad5 replication in the liver of infected hamsters and that it mitigates the consequences of Ad5 infections in these animals when administered prophylactically or therapeutically. We show that ganciclovir inhibits Ad5 DNA synthesis and late gene expression. The mechanism of action for the drug is not clear; preliminary data suggest that it exerts its antiadenoviral effect by directly inhibiting the adenoviral DNA polymerase. While more extensive studies are required, we believe that ganciclovir is a promising drug candidate to treat adenovirus infections. Brincidofovir, a drug with proven activity against Ad5, was used as a positive control in the prophylactic experiment.

Herpesviruses are large double-stranded DNA viruses that cause serious human diseases. Herpesvirus DNA replication depends on multiple processes typically catalyzed by nucleotidyltransferase superfamily (NTS) enzymes. Therefore, we investigated whether inhibitors of NTS enzymes would suppress replication of herpes simplex virus 1 (HSV-1) and HSV-2. Eight of 42 NTS inhibitors suppressed HSV-1 and/or HSV-2 replication by >10-fold at 5 μM, with suppression at 50 μM reaching ∼1 million-fold. Five compounds in two chemical families inhibited HSV replication in Vero and human foreskin fibroblast cells as well as the approved drug acyclovir did. The compounds had 50% effective concentration values as low as 0.22 μM with negligible cytotoxicity in the assays employed. The inhibitors suppressed accumulation of viral genomes and infectious particles and blocked events in the viral replication cycle before and during viral DNA replication. Acyclovir-resistant mutants of HSV-1 and HSV-2 remained highly sensitive to the NTS inhibitors. Five of six NTS inhibitors of the HSVs also blocked replication of another herpesvirus pathogen, human cytomegalovirus. Therefore, NTS enzyme inhibitors are promising candidates for new herpesvirus treatments that may have broad efficacy against members of the herpesvirus family.

There are no drugs approved specifically to treat disseminated adenovirus (Ad) infections in humans. Cidofovir is active against Ad in cell culture, and it is used frequently in the clinic with disseminated infection in pediatric transplant patients; however, controlled clinical studies have not been conducted to prove the anti-Ad efficacy of cidofovir. Brincidofovir, a lipid-linked derivative of cidofovir, which has strong activity against Ad in cell culture and in animal models, is a promising new drug currently in clinical trials. Ribavirin, which has modest activity against some Ad types in cell culture, has been used in the clinic against disseminated Ad, but the efficacy of ribavirin is unknown. In the current study, we have examined the activity of cidofovir, brincidofovir, and ribavirin against disseminated Ad5 infection in the immunosuppressed Syrian hamster model. Hamsters are immunosuppressed by treatment with cyclophosphamide, then infected intravenously with Ad5, leading to disseminated Ad5 infection, especially in the liver. We found that cidofovir and brincidofovir have excellent activity against Ad5 pathology and replication in the liver, even when administered therapeutically starting at 3 days post-challenge with Ad5. Ribavirin did not have anti-Ad5 activity in our model. Our data support the use of cidofovir and brincidofovir in humans for the treatment of disseminated Ad infections in humans.

Human adenovirus type 4 (Ad4), the sole member of subgroup E, contains an open reading frame in the E3 region predicted to encode a unique 30-kDa protein (named Ad4E3-30K). Ad4E3-30K is predicted to be an integral membrane protein containing an N-terminal signal sequence, a lumenal domain, a transmembrane domain near the C-terminus, and a 37-amino-acid cytoplasmic tail. To determine whether this protein is expressed, rabbit polyclonal antisera were raised against 30K-containing fusion proteins expressed in bacteria. A 30K protein was detected by immunoprecipitation from cell-free translation products and from Ad4-infected A549 cells radiolabeled in the presence of tunicamycin. The protein was detected at only low levels in infected cells. It was not synthesized by a mutant with a large E3 deletion that includes the Ad4E3-30K gene. This mutant grows as well as wild-type Ad4 in culture. Features of Ad4E3-30K were characterized in different transient expression systems. The protein underwent glycosylation by addition of approximately six asparagine-linked oligosaccharides. These glycans were sensitive to endoglycosidase H, indicating that they were either high-mannose or hybrid types, but not complex types, and that the protein did not pass through the Golgi apparatus. Immunofluorescence staining of transfected cells revealed that Ad4E3-30K was localized primarily in the endoplasmic reticulum and nuclear envelope.