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PubMed2004

Combination therapy with conditionally replicating adenovirus and replication defective adenovirus.

Low gene transfer rate is the most substantial hurdle in the practical application of gene therapy. One strategy to improve transfer efficiency is the use of a conditionally replicating adenovirus (CRAD) that can selectively replicate in tumor cells. We hypothesized that conventional E1-deleted adenoviruses (ad) can become replication-competent when cotransduced with a CRAD to selectively supply E1 in trans in tumors. The resulting selective production of large numbers of the E1-deleted ad within the tumor mass will increase the transduction efficiency. We used a CRAD (Delta24RGD) that produces a mutant E1 without the ability to bind retinoblastoma but retaining viral replication competence in cancer cells with a defective pRb/p16. Ad-lacZ, adenovirus-luciferase (ad-luc), and adenovirus insulin-like growth factor-1R/dominant-negative (ad-IGF-1R/dn; 482, 950) are E1-deleted replication-defective adenoviruses. The combination of CRAD and ad-lacZ increased the transduction efficiency of lacZ to 100% from 15% observed with ad-lacZ alone. Transfer of media of CRAD and ad-lacZ cotransduced cells induced the transfer of lacZ (media transferable bystander effect). Combination of CRAD and ad-IGF-1R/dn increased the production of truncated IGF-1R or soluble IGF-1R > 10 times compared with transduction with ad-IGF-1R/dn alone. Combined intratumoral injection of CRAD and ad-luc increased the luciferase expression about 70 times compared with ad-luc alone without substantial systemic spread. Combined intratumoral injection of CRAD and ad-IGF-1R/482 induced stronger growth suppression of established lung cancer xenografts than single injections. The combination of CRAD and E1-deleted ad induced tumor-specific replication of CRAD and E1-deleted ad and increased the transduction rate and therapeutic efficacy of these viruses in model tumors.

Adenoviridae↗
PubMed2001

Alternate adenovirus type-pairs for a possible circumvention of host immune response to recombinant adenovirus vectors.

With the help of monoclonal antibodies the existence of at least 18 different earlier not known intertype (IT) specific epitopes were demonstrated in different numbers and combinations on the hexons of different adenovirus serotypes. The IT specific epitopes play an important role in the experimental gene therapy and in the recombinant adenovirus vaccination because of the harmful immune response of the recipient organisms directed against the many different epitopes of the adenovirus vector. For the elimination of harmful effect the authors suggest the use of multiple vectors, each prepared from different adenovirus serotypes showing the loosest antigenic relationship to each other. The vectors would be used sequentially when second or multiple administration is needed. For this purpose the authors determined and described 31 such adenovirus type-pairs, which are probably the best alternates for sequential use in experimental gene therapy.

Adenoviruses, Human↗
PubMed1995

Modulation of gene expression after replication-deficient, recombinant adenovirus-mediated gene transfer by the product of a second adenovirus vector.

To regulate gene expression following adenovirus-mediated gene transfer, a strategy was devised utilizing co-infection with two separate adenovirus vectors designed such that the product of one vector modulated the promoter of the second vector. To evaluate this strategy, AdEGR1.TNF, an adenovirus expressing tumor necrosis factor-alpha (TNF) under the control of the early growth response 1 (EGR1) promoter, was used to regulate a transcription unit in AdIL8.beta gal, an adenovirus vector in which the TNF sensitive interleukin-8 (IL-8) promoter drives the expression of beta-galactosidase (beta-gal). Following infection of HS24 cells with AdIL8.beta gal, addition of TNF to the culture induced the expression of beta-gal. Infection of HS24 cells with AdEGR1.TNF resulted in a dose-dependent secretion of TNF. Little beta-gal was produced following co-infection of the cells with the control vector AdCMV.Null (expressing no specific gene) and AdIL8.beta gal. In contrast, co-infection with AdIL8.beta gal and AdEGR1.TNF demonstrated, for a given dose of AdIL8.beta gal, increasing amounts of beta-gal expression dependent on the dose of AdEGR1.TNF. This model suggests control of gene expression in adenovirus-mediated gene transfer can be regulated by utilizing a promoter-gene expression cassette in one vector that modulates the expression of a promoter-gene expression cassette in a second vector.

Adenoviruses, Human↗
PubMed2002

Chimpanzee adenovirus CV-68 adapted as a gene delivery vector interacts with the coxsackievirus and adenovirus receptor.

A replication-defective form of chimpanzee adenovirus type 68 (C68) has been developed to circumvent problems posed by widespread preexisting immunity to common human adenovirus vectors. To investigate the determinants of C68 tropism, its interaction with the coxsackievirus and adenovirus receptor (CAR) was studied. Although CHO cells were resistant to transduction by C68 as well as by adenovirus type 5 (Ad5), CHO cells expressing either human or murine CAR were transduced readily. C68 transduction, like Ad5 transduction, was blocked when cells were exposed to anti-CAR antibody or when virus was exposed to a soluble form of the CAR extracellular domain. These results indicate that gene delivery by C68 occurs by a CAR-dependent mechanism.

Adaptation, Physiological↗
PubMed1999

The adenovirus that causes hemorrhagic disease of black-tailed deer is closely related to bovine adenovirus-3.

DNA sequence data was obtained from an adenovirus previously shown to be the cause of a distinctive, fatal hemorrhagic disease of black-tailed deer in California. A 256 base fragment of the viral hexon gene was amplified by PCR from purified adenovirus preparations. The amplicon then was cloned and sequenced. Phylogenetic relationships with other mammalian adenoviruses were also determined. Although sequence analysis of this portion of the hexon gene indicates that the black-tailed deer adenovirus is closely related to bovine adenovirus-3, the biologic properties of the two viruses are clearly distinct.

Adenoviridae Infections↗
PubMed2002

Engineered expression of the Coxsackie B and adenovirus receptor (CAR) in human dendritic cells enhances recombinant adenovirus-mediated gene transfer.

Dendritic cells (DCs) are key antigen-presenting cells (APCs) that act as central modulators of cellular immune responses. Genetic modification of DCs has considerable therapeutic potential in the treatment of a wide spectrum of diseases, including cancer and persistent viral infection. In this report, we show that pre-treatment of DCs with a recombinant adenovirus encoding the major adenovirus receptor, Coxsackie B and adenovirus receptor (CAR), significantly increased the uptake of recombinant adenoviruses (Ads) by primary immature monocyte-derived DCs. This could be correlated with CAR mRNA and surface protein expression. Transduction of DCs by recombinant adenoviruses did not significantly alter cellular viability. Therefore, we propose that pre-treatment of DCs with Ad5-CAR is one strategy to increase the susceptibility of DCs to transduction by recombinant Ads.

Antigen Presentation↗
PubMed2001

Generation of adenoviruses encoding the herpes simplex virus vhs gene: a novel strategy to generate adenoviruses expressing genes toxic to producer cells.

Adenovirus vectors expressing suicide genes represent a promising approach for cancer gene therapy. We wanted to determine whether the virion host shutoff (vhs) gene of herpes simplex virus could be used as a suicide gene for gene therapy of glioblastomas. The vhs gene was cloned downstream of the glial fibrillary acidic protein promoter to direct tissue-specific expression, and recombinant adenoviruses were generated in 293 cells. Viruses, which contained the vhs gene but did not express it, could readily be isolated; however, we were unable to plaque purify viruses that expressed vhs protein. We constructed a derivative of 293 cells expressing an antisense RNA to the vhs gene and used them to generate adenovirus vectors that express vhs protein. These cells were used to complement vhs-expressing adenoviruses through three rounds of plaque purification. This approach could be generalized to produce adenovirus vectors expressing other toxic transgenes.

Adenoviridae↗
PubMed1970

Adenovirus type 2-simian virus 40 hybrid population: evidence for a hybrid deoxyribonucleic acid molecule and the absence of adenovirus-encapsidated circular simian virus 40 deoxyribonucleic acid.

The deoxyribonucleic acid (DNA) from the adenovirus-encapsidated particles of the adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid population plaque variant (Ad2(++) HEY), known to yield SV40 virus with high efficiency, was studied by equilibrium density centrifugation followed by ribonucleic acid-DNA hybridization employing virus-specific complementary ribonucleic acids synthesized in vitro. These techniques establish linkage between the Ad2 and SV40 components in the adenovirus-encapsidated particles of this population. The linkage is alkali-resistant and presumably covalent; thus, the Ad2 DNA and SV40 DNA are present in a hybrid molecule. Velocity centrifugation studies in alkaline sucrose gradients eliminated the possibility that supercoiled circular SV40 DNA is present in the adenovirus capsids. The DNA obtained from the adenovirus-encapsidated particles of the Ad2(++) HEY population appears to consist of nonhybrid Ad2 DNA and Ad2-SV40 hybrid DNA molecules.

Adenoviridae↗
PubMed2002

Adenovirus binding to the coxsackievirus and adenovirus receptor or integrins is not required to elicit brain inflammation but is necessary to transduce specific neural cell types.

Intracranial administration of adenovirus vectors elicits rapid, capsid-mediated dose-dependent brain inflammation. The mechanisms through which adenovirus capsids trigger inflammation in the brain remain unknown. We determined whether adenovirus interaction with the primary and secondary cell surface receptors for infection (CAR and alphav integrins) was necessary to trigger acute adenovirus-mediated brain inflammation, and, furthermore, whether capsid mutations that abrogated CAR and integrin binding altered vector tropism in the brain. Vectors ablated for CAR binding, but retaining integrin binding function, transduced equivalent areas of brain compared to vectors with wild-type capsids; however, vector tropsim was dramatically altered. Vectors with wild-type capsids predominantly transduced oligodendrocytes, whereas mutation of the fiber protein to ablate CAR binding resulted in a loss of oligodendrocyte transduction and a consequent redirection of transduction to neurons and other types of glial cells. Combined mutations of fiber and penton base that ablate both CAR and integrin binding almost abolished brain transduction. Thus, doubly-ablated capsids engineered to express new ligands should allow complete vector retargeting in the central nervous system. Although transduction by the doubly-ablated vectors was reduced by greater than 95%, inflammation was not reduced compared to wild-type vectors, demonstrating that brain inflammation occurs independently of adenovirus binding and infection of cells via CAR and integrin receptors.

Adenoviridae↗
PubMed2006

[Construction of a recombinant adenovirus vector for interferon-gamma-inducible protein 10 and the adenovirus preparation].

OBJECTIVE: To construct a recombinant adenovirus vector for expressing interferon-gamma-inducible protein 10 (IP-10) by homogenous bacterial recombination. METHODS: IP-10 gene was cloned into the shuttle plasmid pAdTrack-CMV that contained the coding sequence of enhanced green fluorescent protein (EGFP). The shuttle plasmid was then transformed into E. coli BJ5183 with pAdEasy-1 vector by chemical transformation. The recombinant adenovirus vector pAd/IP-10 was identified by enzyme digestion with Pac I and the linearized plasmid was transfected into HEK293 cells. RESULTS: The positive clones were identified with enzyme digestion and polymerase chain reaction (PCR) and were further verified by DNA sequencing. The recombinant adenovirus of high titration was obtained after transfection and packaging in HEK293 cells. CONCLUSION: A recombinant adenovirus vector for expression of IP-10 has been constructed successfully and high-titer active adenovirus is obtained for functional study of IP-10 protein.

Adenoviridae↗
PubMed1979

Inhibition and enhancement of avian adenovirus plaque production by heavy and light avian adenovirus-associated viral particles.

The effect of heavy and light avian adenovirus-associated viral (A-AV) particles on the replication of several adenovirus serotypes was studied in chicken embryo kidney cells. There was a significant decrease (P less than 0.05) in the number and size of adenovirus-induced plaques at A-AV multiplicities of infection greater than 40. Enhancement of plaque production was observed when A-AV multiplicities of infection were 1 to 40. There was a significant increase in the number and size of infective centers. Analysis of cellular yields indicated an increase in the number of adenoviruses produced per cell. Heavy A-AV particles of buoyant density 1.42 g/cm3 in CsCl were found to enhance plaque production more than light particles (1.38 g/cm3). Conversely, light particles showed greater inhibition of plaque production. Adenovirus serotypes varied in their response to enhancement or inhibition by A-AV particles of different density.

Adenoviridae↗
PubMed1983

Candidate adenoviruses 40 and 41: fastidious adenoviruses from human infant stool.

About 200 antigenically related adenoviruses were isolated from cases of infantile diarrhoea in the Netherlands and North-West Germany. The viruses were fastidious and failed to replicate serially in human diploid fibroblasts and in primary human embryonic kidney cells. A number of strains were established in HeLa, HEp-2, Graham (293), cynomolgus monkey kidney, and Chang conjunctival cells. The viruses were mammalian adenoviruses by the usual criteria. No relationship to the 39 known human adenovirus species was found, either by neutralization tests or by haemagglutination inhibition tests. Neutralization tests showed two distinct variants, represented by strains Tak and Dugan. The variants were identical in haemagglutination inhibition tests. DNA restriction enzyme analysis showed Tak and Dugan to have considerably different genomes, indicating that these variants should be classified as different species (Wadell et al, 1983). It is proposed that the variants should be called Mastadenovirus h 40 (with reference strains Dugan and Hovi X) and Mastadenovirus h 41 (with reference strain Tak). Neutralization and haemagglutination inhibition tests demonstrated that the viruses from Glasgow and Helsinki (Hovi X) described by Johansson et al [1980] and by Kidd and Madeley [1981] belong to these two adenovirus species.

Adenoviridae Infections↗
PubMed1995

Adenovirus protease expressed in insect cells cleaves adenovirus proteins, ovalbumin and baculovirus protease in the absence of activating peptide.

The adenovirus type 2 protease (EP) was expressed by infecting insect cells with a recombinant baculovirus. Immunoblot and activity analysis showed EP to be present in both the nucleus and cytoplasm. While the insect cell expressed EP was more soluble than the Escherichia coli expressed EP, its activity was one quarter of the latter, suggesting that eukaryotic postsynthetic modifications are not essential for enzyme activity. EP inactivated a cytoplasmic cathepsin-like baculovirus-encoded cysteine protease which carries a single EP cleavage site and which was capable of digesting most adenovirus structural proteins in vitro. In addition to cleavage of the baculovirus protease, the adenovirus EP was also able to cleave ovalbumin and canine adenovirus protein pre-VII, in the absence of activating peptide. EP activation therefore may occur by means of factors other than the specific activating peptide.

Adenoviruses, Canine↗
PubMed2006

Efficacy of severe acute respiratory syndrome vaccine based on a nonhuman primate adenovirus in the presence of immunity against human adenovirus.

Replication-deficient human adenovirus type 5 (AdH5) vectors can induce strong transgene product-specific cellular and humoral responses. However, many adult humans have neutralizing antibodies (NAbs) against AdH5 as a result of natural infection with this virus. Therefore, a chimpanzee adenovirus C7 (AdC7) vector was developed to circumvent interference by preexisting immunity to AdH5. This study evaluated the impact of preexisting immunity to human adenovirus on the efficacy of adenovirus-based vaccines against the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). Efficacy was assessed after intramuscular injection of the vector into mice and was measured as the frequency of SARS-CoV-specific T cells and NAbs against SARS-CoV. Immunogenicity of the AdH5-based vaccine was significantly attenuated or completely abolished when the preexisting anti-AdH5 NAb titer was higher than 40. Because 27% of human serum samples from the United States tested so far have an anti-AdH5 NAb titer higher than 40, our results suggested that a significant percentage of humans with preexisting anti-AdH5 immunity would not be candidates for vaccination with an AdH5-based genetic vaccine. In contrast, preexisting anti-AdH5 NAbs have a minimal effect on the potency of the AdC7-based genetic vaccine. Taken together, our studies warrant the further development of AdC7 as a vaccine carrier for human trials.

Adenoviruses, Human↗
PubMed1990

Topoisomerase I and II cleavage of adenovirus DNA in vivo: both topoisomerase activities appear to be required for adenovirus DNA replication.

Sites of topoisomerase I and II cleavage across large portions of the adenovirus type 5 genome were mapped by using the drugs camptothecin and VM26, respectively. These drugs prolong the half-lives of the covalent DNA-protein intermediates in which the DNA is transiently cleaved, and so treatment with protein denaturants after exposure to the drugs leads to DNA strand scission at the site of topoisomerase cleavage. Strong topoisomerase II cleavage sites occurred in clusters throughout the regions examined, including both transcribed regions and transcriptional control regions. The efficiency of topoisomerase II cleavage increased as the rate of adenovirus DNA replication increased and then decreased with the decreasing rate of replication late in the infection cycle. The increase was not dependent on expression of the E1A gene, whose products activate transcription of the early viral genes. Positions of topoisomerase II cleavage sites did not vary during the infection. Topoisomerase I cleavage sites were also found throughout the examined regions, with the strongest sites occurring near the ends of the transcription units. Topoisomerase I cleavage in the E1 region occurred much more frequently than topoisomerase II cleavage, was not dependent on E1A gene expression, and remained at a similar level from the early viral phase into the late viral phase. Treatment of infected cells with either drug prevented efficient replication of adenovirus DNA. Inhibition of topoisomerase I activity led to an immediate cessation of adenovirus DNA replication, while inhibition of topoisomerase II blocked replication only after completion of approximately one additional round.

Adenovirus Early Proteins↗
PubMed1990

The adenovirus DNA-binding protein stimulates the rate of transcription directed by adenovirus and adeno-associated virus promoters.

The DNA-binding protein (DBP) encoded by the E2A region of adenovirus type 5 was found to enhance the expression of a reporter gene controlled by several different promoters within transfected cells. The rate of synthesis of correctly initiated transcripts was increased by the DBP. The adeno-associated virus P5 promoter and the adenovirus E1A and E2A early and major late promoters responded to the DBP by increases in expression ranging from 6- to 27-fold, while the adenovirus E4 promoter was slightly inhibited by DBP. The adenovirus major late promoter showed a greater response to DBP than to the E1A transactivator protein, suggesting that the DBP plays a central role in activation of the late promoter.

Adenovirus Early Proteins↗
PubMed1994

Constructing chimeric type 12/type 5 adenovirus E1A genes and using them to identify an oncogenic determinant of adenovirus type 12.

The E1A gene of highly oncogenic type 12 adenovirus (Ad12) possesses a segment unique to this serotype and comprising 60 base pairs contiguous with and separating conserved regions 2 and 3 in the gene. A similar but slightly longer segment is also present in the E1A gene of highly oncogenic simian adenovirus type 7 (D. Kimelman, J. S. Miller, D. Porter, and B. E. Roberts, J. Virol. 53:399-409, 1985). This segment is missing entirely from the E1A gene of type 5 adenovirus, which is nononcogenic. To test the hypothesis that this unique separating or "spacer" region influences the oncogenicity of Ad12, we constructed ClaI and SmaI restriction sites on either side of it, which allowed reciprocal exchange between this and the equivalent cassette from type 5 adenovirus E1A, bounded by the same restriction sites intrinsic to that gene. The resultant Ad12-based chimeric viruses, ch702 and ch704, in which the spacer region is replaced with (in-frame) type 5 sequence, grow normally on human A549 cells and display wild-type transformation frequencies on baby rat and mouse kidney cells. In contrast, the oncogenic capacity of these chimeric viruses, as measured by tumor induction following virus inoculation in Hooded Lister rats, is greatly reduced. Likewise, cells transformed by ch702 and ch704 display reduced tumorigenicity compared with wild-type transformants in syngeneic rats. These results, coupled with recent preliminary tests using a mutant with a point mutation in this region, support the view that the unique spacer region of type 12 is an oncogenic determinant of this virus.

Adenovirus E1A Proteins↗
PubMed2000

Retargeting the coxsackievirus and adenovirus receptor to the apical surface of polarized epithelial cells reveals the glycocalyx as a barrier to adenovirus-mediated gene transfer.

Lumenal delivery of adenovirus vectors (AdV) results in inefficient gene transfer to human airway epithelium. The human coxsackievirus and adenovirus receptor (hCAR) was detected by immunofluorescence selectively at the basolateral surfaces of freshly excised human airway epithelial cells, suggesting that the absence of apical hCAR constitutes a barrier to adenovirus-mediated gene delivery in vivo. In transfected polarized Madin-Darby canine kidney cells, wild-type hCAR was expressed selectively at the basolateral membrane, whereas hCAR lacking the transmembrane and/or cytoplasmic domains was expressed on both the basolateral and apical membranes. Cells expressing apical hCAR still were not efficiently transduced by AdV applied to the apical surface. However, after the cells were treated with agents that remove components of the apical surface glycocalyx, AdV transduction occurred. These results indicate that adenovirus can infect via receptors located at the apical cell membrane but that the glycocalyx impedes interaction of AdV with apical receptors.

Adenoviruses, Human↗