Replication-Deficient Human Adenovirus Type 35 Vectors for Gene Transfer and Vaccination: Efficient Human Cell Infection and Bypass of Preexisting Adenovirus Immunity

ABSTRACT Replication-deficient human adenovirus type 5 (Ad5) can be produced to high titers in complementing cell lines, such as PER.C6, and is widely used as a vaccine and gene therapy vector. However, preexisting immunity against Ad5 hampers consistency of gene transfer, immunological responses, and vector-mediated toxicities. We report the identification of human Ad35 as a virus with low global prevalence and the generation of an Ad35 vector plasmid system for easy insertion of heterologous genes. In addition, we have identified the minimal sequence of the Ad35-E1B region (molecular weight, 55,000 [55K]), pivotal for complementation of fully E1-lacking Ad35 vector on PER.C6 cells. After stable insertion of the 55K sequence into PER.C6 cells a cell line was obtained (PER.C6/55K) that efficiently transcomplements both Ad5 and Ad35 vectors. We further demonstrate that transduction with Ad35 is not hampered by preexisting Ad5 immunity and that Ad35 efficiently infects dendritic cells, smooth muscle cells, and synoviocytes, in contrast to Ad5.

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Transduction Enhancers Enable Efficient Human Adenovirus Type 5-Mediated Gene Transfer into Human Multipotent Mesenchymal Stromal Cells

Viruses ◽

10.3390/v13061136 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1136

Author(s):

Robin Nilson ◽

Olivia Lübbers ◽

Linus Weiß ◽

Karmveer Singh ◽

Karin Scharffetter-Kochanek ◽

...

Keyword(s):

Gene Transfer ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Coagulation Factor ◽

Human Adenovirus ◽

Adenovirus Type ◽

Multipotent Mesenchymal Stromal Cells ◽

Egfp Expression ◽

Migration Behavior ◽

Adenovirus Type 5

Human multipotent mesenchymal stromal cells (hMSCs) are currently developed as cell therapeutics for different applications, including regenerative medicine, immune modulation, and cancer treatment. The biological properties of hMSCs can be further modulated by genetic engineering. Viral vectors based on human adenovirus type 5 (HAdV-5) belong to the most frequently used vector types for genetic modification of human cells in vitro and in vivo. However, due to a lack of the primary attachment receptor coxsackievirus and adenovirus receptor (CAR) in hMSCs, HAdV-5 vectors are currently not suitable for transduction of this cell type without capsid modification. Here we present several transduction enhancers that strongly enhance HAdV-5-mediated gene transfer into both bone marrow- and adipose tissue-derived hMSCs. Polybrene, poly-l-lysine, human lactoferrin, human blood coagulation factor X, spermine, and spermidine enabled high eGFP expression levels in hMSCs. Importantly, hMSCs treated with enhancers were not affected in their migration behavior, which is a key requisite for many therapeutic applications. Exemplary, strongly increased expression of tumor necrosis factor (TNF)-stimulated gene 6 (TSG-6) (a secreted model therapeutic protein) was achieved by enhancer-facilitated HAdV-5 transduction. Thus, enhancer-mediated HAdV-5 vector transduction is a valuable method for the engineering of hMSCs, which can be further exploited for the development of innovative hMSC therapeutics.

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Degradation and inactivation of adenovirus in water by photo-electro-oxidation

Brazilian Journal of Biology ◽

10.1590/1519-6984.00813suppl ◽

2015 ◽

Vol 75 (4 suppl 2) ◽

pp. 37-42 ◽

Cited By ~ 1

Author(s):

G. S. Monteiro ◽

R. Staggemeier ◽

C. R. Klauck ◽

A. M. Bernardes ◽

M. A. S. Rodrigues ◽

...

Keyword(s):

Cell Culture ◽

Viral Load ◽

Human Adenovirus ◽

Adenovirus Type ◽

Viral Particles ◽

Dnase Treatment ◽

A Cell ◽

Electro Oxidation ◽

Adenovirus Type 5 ◽

Treatment Step

The present study analyzed the efficiency of the photo-electro-oxidation process as a method for degradation and inactivation of adenovirus in water. The experimental design employed a solution prepared from sterile water containing 5.107 genomic copies/L (gc/L) of a standard strain of human adenovirus type 5 (HAdV-5) divided into two equal parts, one to serve as control and one treated by photo-electro-oxidation (PEO) for 3 hours and with a 5A current. Samples collected throughout the exposure process were analyzed by real-time polymerase chain reaction (qPCR) for viral genome identification and quantitation. Prior to gene extraction, a parallel DNAse treatment step was carried out to assess the integrity of viral particles. Integrated cell culture (ICC) analyses assessed the viability of infection in a cell culture. The tested process proved effective for viral degradation, with a 7 log10 reduction in viral load after 60 minutes of treatment. The DNAse-treated samples exhibited complete reduction of viral load after a 75 minute exposure to the process, and ICC analyses showed completely non-viable viral particles at 30 minutes of treatment.

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Canine Adenovirus Vectors: an Alternative for Adenovirus-Mediated Gene Transfer

Journal of Virology ◽

10.1128/jvi.74.1.505-512.2000 ◽

2000 ◽

Vol 74 (1) ◽

pp. 505-512 ◽

Cited By ~ 188

Author(s):

Eric J. Kremer ◽

Sylvie Boutin ◽

Miguel Chillon ◽

Olivier Danos

Keyword(s):

Gene Transfer ◽

Neutralizing Antibodies ◽

Viral Vectors ◽

Human Adenovirus ◽

Adenovirus Type ◽

Serum Samples ◽

Humoral And Cellular Immunity ◽

Adenovirus Vectors ◽

Adenovirus Type 5

ABSTRACT Preclinical studies have shown that gene transfer following readministration of viral vectors is often inefficient due to the presence of neutralizing antibodies. Vectors derived from ubiquitous human adenoviruses may have limited clinical use because preexisting humoral and cellular immunity is found in 90% of the population. Furthermore, risks associated with the use of human adenovirus vectors, such as the need to immunosuppress or tolerize patients to a potentially debilitating virus, are avoidable if efficient nonhuman adenovirus vectors are feasible. Plasmids containing recombinant canine adenovirus (CAV) vectors from which the E1 region had been deleted were generated and transfected into a CAV E1-transcomplementing cell line. Vector stocks, with titers greater than or equal to those obtained with human adenovirus vectors, were free of detectable levels of replication-competent CAV and had a low particle-to-transduction unit ratio. CAV vectors were replication defective in all cell lines tested, transduced human-derived cells at an efficiency similar to that of a comparable human adenovirus type 5 vector, and are amenable to in vivo use. Importantly, 49 of 50 serum samples from healthy individuals did not contain detectable levels of neutralizing CAV antibodies.

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In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

Viruses ◽

10.3390/v13081483 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1483

Author(s):

Emily A. Bates ◽

John R. Counsell ◽

Sophie Alizert ◽

Alexander T. Baker ◽

Natalie Suff ◽

...

Keyword(s):

Viral Vector ◽

Ex Vivo ◽

Human Adenovirus ◽

Adenovirus Type ◽

Cell Types ◽

In Vivo Evaluation ◽

In Vivo Biodistribution ◽

Adenovirus Type 5

The human adenovirus phylogenetic tree is split across seven species (A–G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D—such as their cellular tropism, receptor usage, and in vivo biodistribution profile—remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)—a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.

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The endosomal epsilon-coatomer protein is involved in human adenovirus type 5 internalisation

Acta Veterinaria Hungarica ◽

10.1556/avet.50.2002.4.10 ◽

2002 ◽

Vol 50 (4) ◽

pp. 481-489 ◽

Cited By ~ 1

Author(s):

Cs. Jeney ◽

Boglárka Banizs ◽

Orsolya Dobay ◽

Keyword(s):

Chinese Hamster ◽

Human Adenovirus ◽

Adenovirus Type ◽

Inhibitory Effect ◽

Bafilomycin A1 ◽

Cho Cell ◽

Coxsackie Adenovirus Receptor ◽

Downstream Effector ◽

Adenovirus Receptor ◽

Adenovirus Type 5

The effects of bafilomycin A1 and of the reduced level of endosomal epsilon-COP (coatomer protein) on the infectivity of human adenovirus type 5 were investigated in Coxsackie adenovirus receptor- (CAR-) transfected Chinese hamster ovary (CHO) cells. The endosomal proton pump inhibitor bafilomycin A1 was able to cause only partial inhibition. Using ldlF cells (an epsilon-COP thermosensitive mutant CHO cell line) the reduction of epsilon-COP level also had partial inhibitory effect. Based on these results and comparing them to existing models of the adenovirus entry, we propose a refined model in which there are two pathways of adenoviral entry: the first one involves the epsilon-COP as the downstream effector of the acidification and can be blocked by bafilomycin A1 and the second one is a pH-independent pathway.

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

Journal of General Virology ◽

10.1099/0022-1317-83-1-151 ◽

2002 ◽

Vol 83 (1) ◽

pp. 151-155 ◽

Cited By ~ 43

Author(s):

Christopher J. Cohen ◽

Zhi Quan Xiang ◽

Guang-Ping Gao ◽

Hildegund C. J. Ertl ◽

James M. Wilson ◽

...

Keyword(s):

Gene Delivery ◽

Cho Cells ◽

Human Adenovirus ◽

Adenovirus Type ◽

Soluble Form ◽

Delivery Vector ◽

Coxsackievirus And Adenovirus Receptor ◽

Adenovirus Receptor ◽

Adenovirus Type 5 ◽

Dependent Mechanism

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.

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Bovine herpesvirus 1 glycoprotein D expression in bovine upper respiratory tract mediated by a human adenovirus type 5

Veterinary Research ◽

10.1051/vetres:2004045 ◽

2004 ◽

Vol 35 (6) ◽

pp. 715-721 ◽

Cited By ~ 6

Author(s):

Sacha Gogev ◽

Jean-Pierre Georgin ◽

Fr�d�ric Schynts ◽

Alain Vanderplasschen ◽

Etienne Thiry

Keyword(s):

Respiratory Tract ◽

Bovine Herpesvirus ◽

Human Adenovirus ◽

Adenovirus Type ◽

Upper Respiratory Tract ◽

Glycoprotein D ◽

Bovine Herpesvirus 1 ◽

Upper Respiratory ◽

Herpesvirus 1 ◽

Adenovirus Type 5

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Comparative Sequence Analysis of the Largest E1A Proteins of Human and Simian Adenoviruses

Journal of Virology ◽

10.1128/jvi.76.16.7968-7975.2002 ◽

2002 ◽

Vol 76 (16) ◽

pp. 7968-7975 ◽

Cited By ~ 48

Author(s):

Nikita Avvakumov ◽

Russ Wheeler ◽

Jean Claude D'Halluin ◽

Joe S. Mymryk

Keyword(s):

Gene Expression ◽

Human Adenovirus ◽

Detailed Comparison ◽

Adenovirus Type ◽

Comparative Sequence Analysis ◽

Regulatory Proteins ◽

Carboxyl Terminus ◽

Adenovirus E1a ◽

Simian Adenovirus ◽

Adenovirus Type 5

ABSTRACT The early region 1A (E1A) gene is the first gene expressed after infection with adenovirus and has been most extensively characterized in human adenovirus type 5 (hAd5). The E1A proteins interact with numerous cellular regulatory proteins, influencing a variety of transcriptional and cell cycle events. For this reason, these multifunctional proteins have been useful as tools for dissecting pathways regulating cell growth and gene expression. Despite the large number of studies using hAd5 E1A, relatively little is known about the function of the E1A proteins of other adenoviruses. In 1985, a comparison of E1A sequences from three human and one simian adenovirus identified three regions with higher overall levels of sequence conservation designated conserved regions (CR) 1, 2, and 3. As expected, these regions are critical for a variety of E1A functions. Since that time, the sequences of several other human and simian adenovirus E1A proteins have been determined. Using these, and two additional sequences that we determined, we report here a detailed comparison of the sequences of 15 E1A proteins representing each of the six hAd subgroups and several simian adenoviruses. These analyses refine the positioning of CR1, 2, and 3; define a fourth CR located near the carboxyl terminus of E1A; and suggest several new functions for E1A.

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Mapping of cellular protein-binding sites on the products of early-region 1A of human adenovirus type 5

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3955-3959.1988 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3955-3959 ◽

Cited By ~ 2

Author(s):

C Egan ◽

T N Jelsma ◽

J A Howe ◽

S T Bayley ◽

B Ferguson ◽

...

Keyword(s):

Biological Activity ◽

Binding Sites ◽

Human Adenovirus ◽

Adenovirus Type ◽

Cellular Protein ◽

Cellular Proteins ◽

Deletion Mutants ◽

Protein Binding Sites ◽

Amino Terminal ◽

Adenovirus Type 5

The binding sites for the 300-, 107-, and 105-kilodalton cellular proteins which associate with human adenovirus type 5 E1A products were studied with E1A deletion mutants. All appeared to bind to the amino-terminal half of E1A products in regions necessary for oncogenic transformation. These results suggest that these cellular species may be important for the biological activity of E1A products.

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