The small molecule AZD6244 inhibits dengue virus replication in vitro and protects against lethal challenge in a mouse model.

Dengue virus (DENV) is the most common mosquito-borne viral disease. The World Health Organization estimates that 400 million new cases of dengue fever occur every year. Approximately 500,000 individuals develop severe and life-threatening complications from dengue fever, such as dengue shock syndrome (DSS) and dengue hemorrhagic fever (DHF), which cause 22,000 deaths yearly. Currently, there are no specific licensed therapeutics to treat DENV illness. We have previously shown that the MEK/ERK inhibitor U0126 inhibits the replication of the flavivirus yellow fever virus. In this study, we demonstrate that the MEK/ERK inhibitor AZD6244 has potent antiviral efficacy in vitro against DENV-2, DENV-3, and Saint Louis encephalitis virus (SLEV). We also show that it is able to protect AG129 mice from a lethal challenge with DENV-2 (D2S20). The molecule is currently undergoing phase III clinical trials for the treatment of non-small-cell lung cancer. The effect of AZD6244 on the DENV life cycle was attributed to a blockade of morphogenesis. Treatment of AG129 mice twice daily with oral doses of AZD6244 (100 mg/kg/day) prevented the animals from contracting dengue hemorrhagic fever (DHF)-like lethal disease upon intravenous infection with 1 × 105 PFU of D2S20. The effectiveness of AZD6244 was observed even when the treatment of infected animals was initiated 1-2 days postinfection. This was also followed by a reduction in viral copy number in both the serum and the spleen. There was also an increase in IL-1ß and TNF-α levels in mice that were infected with D2S20 and treated with AZD6244 in comparison to infected mice that were treated with the vehicle only. These data demonstrate the potential of AZD6244 as a new therapeutic agent to treat DENV infection and possibly other flavivirus diseases.

Structural and Functional Characterization of Anti-A33 Antibodies Reveal a Potent Cross-Species Orthopoxviruses Neutralizer.

Vaccinia virus A33 is an extracellular enveloped virus (EEV)-specific type II membrane glycoprotein that is essential for efficient EEV formation and long-range viral spread within the host. A33 is a target for neutralizing antibody responses against EEV. In this study, we produced seven murine anti-A33 monoclonal antibodies (MAbs) by immunizing mice with live VACV, followed by boosting with the soluble A33 homodimeric ectodomain. Five A33 specific MAbs were capable of neutralizing EEV in the presence of complement. All MAbs bind to conformational epitopes on A33 but not to linear peptides. To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33. We have further determined the binding kinetics for each of the three antibodies to wild-type A33, as well as to engineered A33 that contained single alanine substitutions within the epitopes of the three crystallized antibodies. While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously. A27D7 binding is resistant to single alanine substitutions within the A33 epitope. A27D7 also demonstrated high-affinity binding with recombinant A33 protein that mimics other orthopoxvirus strains in the A27D7 epitope, such as ectromelia, monkeypox, and cowpox virus, suggesting that A27D7 is a potent cross-neutralizer. Finally, we confirmed that A27D7 protects mice against a lethal challenge with ectromelia virus.

Coagulation factor binding orientation and dimerization may influence infectivity of adenovirus-coagulation factor complexes.

Adenoviruses (Ads) are promising vectors for therapeutic interventions in humans. When injected into the bloodstream, Ad vectors can bind several vitamin K-dependent blood coagulation factors, which contributes to virus sequestration in the liver by facilitating transduction of hepatocytes. Although both coagulation factors FVII and FX bind the hexon protein of human Ad serotype 5 (HAdv5) with a very high affinity, only FX appears to play a role in mediating Ad-hepatocyte transduction in vivo. To understand the discrepancy between efficacy of FVII binding to hexon and its apparently poor capacity for supporting virus cell entry, we analyzed the HAdv5-FVII complex by using high-resolution cryo-electron microscopy (cryo-EM) followed by molecular dynamic flexible fitting (MDFF) simulations. The results indicate that although hexon amino acids T423, E424, and T425, identified earlier as critical for FX binding, are also involved in mediating binding of FVII, the FVII GLA domain sits within the surface-exposed hexon trimer depression in a different orientation from that found for FX. Furthermore, we found that when bound to hexon, two proximal FVII molecules interact via their serine protease (SP) domains and bury potential heparan sulfate proteoglycan (HSPG) receptor binding residues within the dimer interface. In contrast, earlier cryo-EM studies of the Ad-FX interaction showed no evidence of dimer formation. Dimerization of FVII bound to Ad may be a contributing mechanistic factor for the differential infectivity of Ad-FX and Ad-FVII complexes, despite high-affinity binding of both these coagulation factors to the virus.

Expanded anticancer therapeutic window of hexon-modified oncolytic adenovirus.

One of the significant hurdles toward safe and efficacious systemic treatment of cancer with oncolytic adenoviruses (Ads) is dose-limiting hepatotoxicity that prevents the increase of a therapeutic dose. In this study, we expanded the therapeutic window of oncolytic serotype 5 Ad (Ad5) by a genetic modification of hypervariable loop 5 (HVR5) in the capsid protein hexon that prevented infection of hepatocytes due to ablation of binding to blood factors. This oncolytic virus, Ad-GL-HB, had significantly reduced levels of hepatocyte transduction in immunocompetent and immunodeficient mice as compared to parental virus Ad-GL. The hepatocyte detargeting decreased liver damage and increased the maximum tolerated dose of Ad-GL-HB tenfold relative to that of Ad-GL. Intravenous (i.v.) injection of Ad-GL or Ad-GL-HB into tumor-bearing mice produced equally increased survival rates demonstrating that while Ad-GL-HB detargeted hepatocytes, it sustained tumor cell infection after systemic administration. The significantly improved safety of the virus allowed it to be used at increased doses for improved systemic antitumor efficacy. Our results suggest that hexon modifications provide valuable strategies for systemic oncolytic Ad therapy.

Identification of a new human adenovirus protein encoded by a novel late l-strand transcription unit.

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 approximately 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.

Targeting interferon-alpha increases antitumor efficacy and reduces hepatotoxicity of E1A-mutated spread-enhanced oncolytic adenovirus.

Novel approaches are needed to improve the antitumor potency and to increase the cancer specificity of oncolytic adenoviruses (Ad). We hypothesized that the combination of interferon-alpha (IFN-alpha) expression with a specific mutation in the e1a gene of Ad could target vector replication to genetic defects in the IFN-alpha pathway resulting in both improved antitumor efficacy and reduced toxicity. The conditionally replicative Ad vector KD3-IFN carries the dl1101/1107 mutation in the e1a gene that eliminates binding of E1A proteins to p300/CBP and pRb. KD3-IFN expresses human IFN-alpha in concurrence with vector replication and overexpresses the adenovirus death protein (ADP; E3-11.6K). The antitumor activity of KD3-IFN was significantly higher than that of a control vector in established human hepatocellular carcinoma tumors in immunodeficient mice and in hamster kidney cancer tumors in immunocompetent Syrian hamsters. The dl1101/1107 mutation rendered Ad replication sensitive to the antiviral effect of IFN-alpha in normal as opposed to cancer cells. These results translated to reduced vector toxicity upon systemic administration to C57BL/6 mice. The combination of Ad oncolysis, ADP overexpression, and IFN-alpha-mediated immunotherapy represents a three-pronged approach for increasing the anticancer efficacy of replicative Ads. Exploiting the dl1101/1107 mutation provides a mechanism for additional selectivity of IFN-alpha-expressing replication-competent Ads.

Targeting Interferon-α Increases Antitumor Efficacy and Reduces Hepatotoxicity of E1A-mutated Spread-enhanced Oncolytic Adenovirus.

Novel approaches are needed to improve the antitumor potency and to increase the cancer specificity of oncolytic adenoviruses (Ad). We hypothesized that the combination of interferon-alpha (IFN-α) expression with a specific mutation in the e1a gene of Ad could target vector replication to genetic defects in the IFN-α pathway resulting in both improved antitumor efficacy and reduced toxicity. The conditionally replicative Ad vector KD3-IFN carries the dl1101/1107 mutation in the e1a gene that eliminates binding of E1A proteins to p300/CBP and pRb. KD3-IFN expresses human IFN-α in concurrence with vector replication and overexpresses the adenovirus death protein (ADP; E3-11.6K). The antitumor activity of KD3-IFN was significantly higher than that of a control vector in established human hepatocellular carcinoma tumors in immunodeficient mice and in hamster kidney cancer tumors in immunocompetent Syrian hamsters. The dl1101/1107 mutation rendered Ad replication sensitive to the antiviral effect of IFN-α in normal as opposed to cancer cells. These results translated to reduced vector toxicity upon systemic administration to C57BL/6 mice. The combination of Ad oncolysis, ADP overexpression, and IFN-α-mediated immunotherapy represents a three-pronged approach for increasing the anticancer efficacy of replicative Ads. Exploiting the dl1101/1107 mutation provides a mechanism for additional selectivity of IFN-α-expressing replication-competent Ads.

Preparation and titration of CsCl-banded adenovirus stocks.

Adenovirus research often requires purified high-titer virus stocks and accurate virus titers for use in experiments. Accurate titers are important for quantitative, interpretable, and reproducible results. This is especially true when there are comparisons of different mutant viruses following infection. This chapter details the large-scale preparation of adenovirus (either replication-competent or replication-defective) in spinner cultures (e.g., KB, HeLa, or 293 cells). Protocols for harvesting cells and isolation of adenovirus by CsCl banding are presented. Methods for titering adenovirus by plaque assay are presented along with a discussion of how plaque assays can be used to determine the kinetics of cell killing and cytolysis by adenoviruses.

An oncolytic adenovirus vector combining enhanced cell-to-cell spreading, mediated by the ADP cytolytic protein, with selective replication in cancer cells with deregulated wnt signaling.

We have constructed a novel oncolytic adenovirus (Ad) vector named VRX-009 that combines enhanced cell spread with tumor-specific replication. Enhanced spread, which could significantly increase antitumor efficacy, is mediated by overexpression of the Ad cytolytic protein named ADP (also known as E3-11.6K). Replication of VRX-009 is restricted to cells with a deregulated wnt signal transduction pathway by replacement of the wild-type Ad E4 promoter with a synthetic promoter consisting of five consensus binding sites for the T-cell factor transcription factor. Tumor-selective replication is indicated by several lines of evidence. VRX-009 expresses E4ORF3, a representative Ad E4 protein, only in colon cancer cell lines. Furthermore, VRX-009 replicates preferentially in colon cancer cell lines as evidenced by virus productivity 2 orders of magnitude higher in SW480 colon cancer cells than in A549 lung cancer cells. Replication in primary human bronchial epithelial cells and human umbilical vein endothelial cells was also significantly lower than in SW480 cells. When tested in human tumor xenografts in nude mice, VRX-009 effectively suppressed the growth of SW480 colon tumors but not of A549 lung tumors. VRX-009 may provide greater level of antitumor efficacy than standard oncolytic Ad vectors in tumors in which a defect in wnt signaling increases the level of nuclear beta-catenin.

Cotton rat tumor model for the evaluation of oncolytic adenoviruses.

Oncolytic human adenovirus (Ad) vectors exert their antitumor effect by replicating in and lysing tumor cells. These vectors are commonly evaluated in immunodeficient mice bearing human tumor xenografts. However, this model suffers because the mice are immunodeficient and are not permissive for human Ads. We have developed a cotton rat model to test the selectivity, immunogenicity, and efficacy of oncolytic Ad vectors. The cotton rat is a rodent species that is semipermissive for human Ads. We show that the cotton cancer rat cell line LCRT supports the replication of human Ad in tissue culture and that the cells are destroyed on virus replication. When injected subcutaneously, LCRT cells formed tumors in immunocompetent cotton rats, and the growth of these tumors was delayed by the injection of an oncolytic Ad vector. Replication of the Ad vector in the tumor was demonstrated by sampling tumor tissue and isolating infectious virus particles at various times after intratumoral injection of the virus. We propose that the cotton rat can be used as an animal model to evaluate oncolytic Ad vectors.

Avian adenovirus vector CELO-TK displays anticancer activity in human cancer cells and suppresses established murine melanoma tumors.

Avian adenovirus CELO is a novel adenovirus vector system with the advantages of efficient production, high virion stability, and the absence of crossreactivity with Ad5-neutralizing antibodies. In this study, we evaluated the anticancer efficacy of a CELO vector encoding the herpes simplex virus type 1 thymidine kinase, a prodrug-activating therapeutic gene. Vectors carrying the gene for HSV-tk or EGFP under the control of the HCMV promoter in place of the "nonessential" region of the CELO genome were constructed. Anticancer activity of the CELO-TK vector was studied in vitro, in human and murine tumor cells in cell culture, and in vivo, in established subcutaneous murine B16 melanoma tumors in C57BL/6 mice. The CELO-TK vector mediated delivery of functional HSV-tk to tumor cell lines in cell culture. Comparison of the CELO-TK vector to a first-generation human adenovirus type 5 vector Ad5-TK in cultured H1299 cells showed equal levels of functional activity at increasing multiplicities of infection with CELO-based vector. CELO vectors allowed for transduction and expression of EGFP and HSV-tk genes in subcutaneous melanoma tumors in C57BL/6 mice. Intratumoral injections of CELO-TK followed by ganciclovir administration resulted in suppression of tumor growth and significantly increased the median of survival. The results of the study demonstrated the efficacy of CELO vector as a vehicle for the delivery of prodrug-activating genes such as HSV-tk to tumor cells in vitro and in vivo.

Adenovirus immunoregulatory E3 proteins prolong transplants of human cells in immunocompetent mice.

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.

BoHV-4-Based Vector Single Heterologous Antigen Delivery Protects STAT1(-/-) Mice from Monkeypoxvirus Lethal Challenge.

Monkeypox virus (MPXV) is the etiological agent of human (MPX). It is an emerging orthopoxvirus zoonosis in the tropical rain forest of Africa and is endemic in the Congo-basin and sporadic in West Africa; it remains a tropical neglected disease of persons in impoverished rural areas. Interaction of the human population with wildlife increases human infection with MPX virus (MPXV), and infection from human to human is possible. Smallpox vaccination provides good cross-protection against MPX; however, the vaccination campaign ended in Africa in 1980, meaning that a large proportion of the population is currently unprotected against MPXV infection. Disease control hinges on deterring zoonotic exposure to the virus and, barring that, interrupting person-to-person spread. However, there are no FDA-approved therapies against MPX, and current vaccines are limited due to safety concerns. For this reason, new studies on pathogenesis, prophylaxis and therapeutics are still of great interest, not only for the scientific community but also for the governments concerned that MPXV could be used as a bioterror agent. In the present study, a new vaccination strategy approach based on three recombinant bovine herpesvirus 4 (BoHV-4) vectors, each expressing different MPXV glycoproteins, A29L, M1R and B6R were investigated in terms of protection from a lethal MPXV challenge in STAT1 knockout mice. BoHV-4-A-CMV-A29LgD106ΔTK, BoHV-4-A-EF1α-M1RgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK were successfully constructed by recombineering, and their capacity to express their transgene was demonstrated. A small challenge study was performed, and all three recombinant BoHV-4 appeared safe (no weight-loss or obvious adverse events) following intraperitoneal administration. Further, BoHV-4-A-EF1α-M1RgD106ΔTK alone or in combination with BoHV-4-A-CMV-A29LgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK, was shown to be able to protect, 100% alone and 80% in combination, STAT1(-/-) mice against mortality and morbidity. This work demonstrated the efficacy of BoHV-4 based vectors and the use of BoHV-4 as a vaccine-vector platform.

Radiation increases the activity of oncolytic adenovirus cancer gene therapy vectors that overexpress the ADP (E3-11.6K) protein.

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.

Adenovirus replication-competent vectors (KD1, KD3) complement the cytotoxicity and transgene expression from replication-defective vectors (Ad-GFP, Ad-Luc).

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.

Functions and mechanisms of action of the adenovirus E3 proteins.

In the evolutionary battle between viruses and their hosts, viruses have armed themselves with weapons to defeat the host's attacks on infected cells. Various proteins encoded in the adenovirus (Ad) E3 transcription unit protect cells from killing mediated by cytotoxic T cells and death-inducing cytokines such as tumor necrosis factor (TNF), Fas ligand, and TNF-related apoptosis-inducing ligand (TRAIL). The viral protein E3-gp19 K blocks MHC class-I-restricted antigen presentation, which diminishes killing by cytotoxic T cells. The receptor internalization and degradation (RID) complex (formerly E3-10.4 K/14.5 K) stimulates the clearance from the cell surface and subsequent degradation of the receptors for Fas ligand and TRAIL, thereby preventing the action of these important immune mediators. RID also downmodulates the epidermal growth factor receptor (EGFR), although what role, if any, this function has in immune regulation is uncertain. In addition, RID antagonizes TNF-mediated apoptosis and inflammation through a mechanism that does not primarily involve receptor downregulation. E3-6.7 K functions together with RID in downregulating some TRAIL receptors and may block apoptosis independently of other E3 proteins. Furthermore, E3-14.7 K functions as a general inhibitor of TNF-mediated apoptosis and blocks TRAIL-induced apoptosis. Finally, after expending great effort to maintain cell viability during the early part of the virus replication cycle, Ads lyse the cell to allow efficient virus release and dissemination. To perform this task subgroup C Ads synthesize a protein late in infection named ADP (formerly E3-11.6 K) that is required for efficient virus release. This review focuses on recent experiments aimed at discovering the mechanism of action of these critically important viral proteins.

The transcription factor IRF3 triggers "defensive suicide" necrosis in response to viral and bacterial pathogens.

Although molecular components that execute noninflammatory apoptotic cell death are well defined, molecular pathways that trigger necrotic cell death remain poorly characterized. Here, we show that in response to infection with adenovirus or Listeria monocytogenes, macrophages in vivo undergo rapid proinflammatory necrotic death that is controlled by interferon-regulatory factor 3 (IRF3). The transcriptional activity of IRF3 is, surprisingly, not required for the induction of necrosis, and it proceeds normally in mice deficient in all known regulators of necrotic death or IRF3 activation, including RIPK3, caspases 1, 8, or 11, STING, and IPS1/MAVS. Although L. monocytogenes triggers necrosis to promote the infection, IRF3-dependent necrosis is required for reducing pathogen burden in the models of disseminated infection with adenovirus. Therefore, our studies implicate IRF3 as a principal and nonredundant component of a physiologically regulated necrotic cell-death pathway that operates as an effective innate immune mechanism of host protection against disseminated virus infection.

Construction of targeted and armed oncolytic adenoviruses.

Oncolytic (replication-competent) adenoviruses (Ads) represent the most advanced platform for cancer gene therapy. These viral vectors ablate tumors by killing tumor cells in the process of virus replication. As progeny virions are released, they infect remaining cancer cells, generating a bystander effect. Ads engineered for increased cancer specificity produce less damage to normal tissues. First-generation oncolytic Ads have demonstrated acceptable levels of safety while the efficacy was observed only in combination with chemotherapy and/or radiation. Second-generation oncolytic Ads are armed with therapeutic transgenes to increase release, spread, and bystander effect for enhancing the efficacy. Third-generation oncolytic Ads are armed vectors with capsid modifications for transductional detargeting from normal tissues and targeting to cancer cells. Chemical modification of the capsid additionally improves therapeutic window. Here, we describe methods for generation and characterization of advanced-generation oncolytic Ads.

Coagulation factor X activates innate immunity to human species C adenovirus.

Although coagulation factors play a role in host defense for "living fossils" such as horseshoe crabs, the role of the coagulation system in immunity in higher organisms remains unclear. We modeled the interface of human species C adenovirus (HAdv) interaction with coagulation factor X (FX) and introduced a mutation that abrogated formation of the HAdv-FX complex. In vivo genome-wide transcriptional profiling revealed that FX-binding-ablated virus failed to activate a distinct network of nuclear factor κB-dependent early-response genes that are activated by HAdv-FX complex downstream of TLR4/MyD88/TRIF/TRAF6 signaling. Our study implicates host factor "decoration" of the virus as a mechanism to trigger an innate immune sensor that responds to a misplacement of coagulation FX from the blood into intracellular macrophage compartments upon virus entry into the cell.

Infection and killing of multiple myeloma by adenoviruses.

Oncolytic virotherapy makes use of the natural ability of viruses to infect and kill cancer cells. Adenovirus serotype 5 (Ad5) has been approved for use in humans as a therapy for solid cancers. In this study, we have tested whether Ad5 and low-seroprevalence adenoviruses can be used as oncolytics for multiple myeloma (MM). We show that Ad5 productively infects most myeloma cell lines, replicates to various degrees, and mediates oncolytic cell killing in vitro and in vivo. Comparison of Ad5 with low-seroprevalence Ads on primary marrow samples from MM patients revealed striking differences in the abilities of different adenoviral serotypes to kill normal CD138(-) cells and CD138(+) MM cells. Ad5 and Ad6 from species C and Ad26 and Ad48 from species D all mediated killing of CD138(+) cells with low-level killing of CD138(-) cells. In contrast, Ad11, Ad35, Ad40, and Ad41 mediated weak oncolytic effects in all of the cells. Comparison of cell binding, cell entry, and replication revealed that Ad11 and Ad35 bound MM cells 10 to 100 times better than other serotypes. However, after this efficient interaction, Ad11 and Ad35 viral DNA was not replicated and cell killing did not occur. In contrast, Ad5, Ad6, Ad26, and Ad48 all replicated 10- to 100-fold in MM cells and this correlated with cell killing. These data suggest that Ad5 and other low-seroprevalence adenoviruses may have utility as oncolytic agents against MM and other hematologic malignancies.