Rapid, Efficient, and Modular Generation of Adenoviral Vectors via Isothermal Assembly

Yang, Yong; Chi, Yudan; Tang, Xin‐Ying; Ertl, Hildegund C. J.; Zhou, Dongming

doi:10.1002/0471142727.mb1626s113

Cited by 15 publications

(17 citation statements)

References 46 publications

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“…Some recent publications have described the construction of adenoviral vectors through direct DNA assembly of several PCR products. [29][30][31] The strategy of direct assembly is brief, practical, and easy to understand. For the strategy followed in this study, it takes some effort to find the proper restriction sites and to program the whole procedure before construction begins.…”

Section: Discussionmentioning

confidence: 99%

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Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems

Liu¹,

Zhang

et al. 2019

Human Gene Therapy

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Existing adenoviral vector systems have two drawbacks. It is labor-intensive and time-consuming to load a transgene in these systems, and transgene-harboring vectors are dead ends: they cannot be reused to construct a vector carrying another transgene or achieving new characteristics. To conquer these shortcomings, single plasmid-based adenoviral vector systems were constructed where a unique PmeI site was located at the position for insertion of the exogenous gene. The polymerase chain reaction (PCR) amplified transgene could be cloned into PmeI-linearized starting plasmids using one step of Gibson assembly to generate target adenoviral plasmids, which were then ready for virus rescue. This procedure was termed restriction assembly. To expand the application of these systems, two ClaI sites were created upstream and downstream of the fiber gene to generate an upgraded starting plasmid pKAd5f11pABR-EPG. The modified fiber gene, amplified by overlap extension PCR, could be used to substitute the original fiber in pKAd5f11pABR-EPG to generate an adenoviral plasmid with a new fiber by restriction assembly. On the other hand, pKAd5f11pABR-EPG was also a starting adenoviral plasmid for expressing other transgenes.In conclusion, easy-to-use and upgradable adenoviral vector systems are introduced here, which offer extensive versatility and can serve as a basic platform and functional component library for the synthetic biology of adenoviral vectors.

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Section: Discussionmentioning

confidence: 99%

“…35 Adenoviral vector has been synthesized by assembling several parts of the virus genome. [29][30][31] However, these parts are physical modules. Here, two functional modules (one for transgene cloning and the other one for fiber modification) and the strategy of restriction assembly for vector construction are provided.…”

Section: Discussionmentioning

confidence: 99%

Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems

Liu¹,

Zhang

et al. 2019

Human Gene Therapy

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“…Moreover, nearly any Ad with a known genome sequence can be modified by this method. 28 This method relies on isothermal assembly and is a seamless cloning method that provides direct assembly of multiple DNA fragments in a single isothermal reaction. It can be performed without the use of restriction sites, ligases, additional plasmid vectors, or infrequently used E. coli strains.…”

Section: Methods For Generating Chimpanzee Ad Vectorsmentioning

confidence: 99%

“…Other possible issues and troubleshooting approaches were described in a previous study. 28 Typically, any foreign genes of interest in which the size of the inserted expression cassette is less than 7.5 kb can be cloned into E1and E3-deleted Ad vectors for generating recombinant viruses. In addition to cloning foreign genes of interest into the E1-or E3-deleted area, the Ad capsid is always used to incorporate antigen epitopes for display of antigens on the virus surface to elicit high immunity.…”

Section: Methods For Generating Chimpanzee Ad Vectorsmentioning

confidence: 99%

Development of novel vaccine vectors: Chimpanzee adenoviral vectors

Guo

Mondal

Zhou

2018

Human Vaccines & Immunotherapeutics

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Adenoviral vector has been employed as one of the most efficient means against infectious diseases and cancer. It can be genetically modified and armed with foreign antigens to elicit specific antibody responses and T cell responses in hosts as well as engineered to induce apoptosis in cancer cells. The chimpanzee adenovirus-based vector is one kind of novel vaccine carriers whose unique features and non-reactivity to pre-existing human adenovirus neutralizing antibodies makes it an outstanding candidate for vaccine research and development. Here, we review the different strategies for constructing chimpanzee adenoviral vectors and their applications in recent clinical trials and also discuss the oncolytic virotherapy and immunotherapy based on chimpanzee adenoviral vectors.

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“…The construction of fiber-modified AdV vector was adopted from an optimized isothermal assembly protocol (35,36). Specifically, the AdC68 molecular clone was modified by inserting two PmeI restriction sites into the HI loop (fiber positions 1161 to 1162 and 1170 to 1171) for linearization.…”

Section: Methodsmentioning

confidence: 99%

Recombinant Adenoviruses Displaying Matrix 2 Ectodomain Epitopes on Their Fiber Proteins as Universal Influenza Vaccines

Tang

Yang

Xia

et al. 2017

J Virol

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Influenza is a zoonotic disease that poses severe threats to public health and the global economy. Reemerging influenza pandemics highlight the demand for universal influenza vaccines. We developed a novel virus platform using extracellular domain IV of the matrix 2 protein (M2e), AdC68-F3M2e, by introducing three conserved M2e epitopes into the HI loop of the chimpanzee adenovirus (AdV) fiber protein. The M2e epitopes were expressed sufficiently on the AdV virion surface without affecting fiber trimerization. Additionally, one recombinant adenovirus, AdC68-F3M2e(H1-H5-H7), induced robust M2e-specific antibody responses in BALB/c mice after two sequential vaccinations and conferred efficient protection against homologous and heterologous influenza virus (IV) challenges. We found that the use of AdV with tandem M2e epitopes in fiber is a potential strategy for influenza prevention.IMPORTANCE Influenza epidemics and pandemics severely threaten public health. Universal influenza vaccines have increasingly attracted interest in recent years. Here, we describe a new strategy that incorporates triple M2e epitopes into the fiber protein of chimpanzee adenovirus 68. We optimized the process of inserting foreign genes into the AdC68 structural protein by one-step isothermal assembly and demonstrated that this 225-bp HI loop insertion could be well tolerated. Furthermore, two doses of adjuvant-free fiber-modified AdC68 could confer sufficient protection against homologous and heterologous influenza virus infections in mice. Our results show that AdC68-F3M2e could be pursued as a novel universal influenza vaccine.

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Rapid, Efficient, and Modular Generation of Adenoviral Vectors via Isothermal Assembly

Cited by 15 publications

References 46 publications

Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems

Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems

Development of novel vaccine vectors: Chimpanzee adenoviral vectors

Recombinant Adenoviruses Displaying Matrix 2 Ectodomain Epitopes on Their Fiber Proteins as Universal Influenza Vaccines

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