Vaccine Design: Replication-Defective Adenovirus Vectors
Replication-defective adenovirus (Ad) vectors were initially developed for gene transfer for correction of genetic diseases. Although Ad vectors achieved high levels of transgene product expression in a variety of target cells, expression of therapeutic p
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Introduction Replication-defective adenovirus (Ad) vectors based on human or simian serotypes are being developed as vaccine carriers for a large number on pathogens or cancers [1–14]. Several features render Ad vectors highly attractive as vaccine carriers. They induce potent adaptive immune responses to transgene products [15]. Ad vectors similar to wild-type Ads persist at low levels in activated T cells in a transcriptionally active form, which results in very sustained immune responses [16]. Through deletions of the E1 domain Ad vectors can be rendered replication-defective. In addition, deletion of E1 reduces transcription of Ad antigens without affecting transcription of the transgene, which is typically under the control of a ubiquitously active, strong promoter such as the cytomegalovirus (CMV) early promoter. This in turn allows immune response to focus on the transgene product rather than on Ad antigens. Ad vectors are relatively easy to generate [16], packaging cell lines are available for many serotypes, yields upon propagation suffice for clinical development, and procedures for production and release testing under good manufacturing practice (GMP) are available. Clinical experience has shown that E1-deleted Ad vectors given at immunogenic doses are well tolerated by humans [4, 14]. The 35–40 kb genome of several serotypes of Ad has been cloned into plasmid vectors, which allows for modifications of the genome including excision of domains that are essential for viral replication, such as the E1 domain, or regions that are nonessential such as E3. Replacing the deleted E1 domain with sites for rare restriction enzymes then allows for easy insertion of an for expression of a foreign antigen. The permitted size for inserted sequences depends on the type of deletion. E1-only deleted adenovirus vectors can accommodate ~4 kp, and additional deletion in E3 allows for insertion of ~7.5 kb of foreign sequences.
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_23, © Springer Science+Business Media New York 2016
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Xiangyang Zhou et al.
We previously described procedures to develop viral molecular clones of Ad genomes [16]. This chapter offers a description of generating and quality controlling recombinant Ad vectors from already available viral molecular clones. The focus is on generation of E1-deleted Ad vectors, in which the foreign expression cassette is inserted into E1. Specifically, generation of recombinant pShuttle vectors for E1 is described followed by procedures to clone the expression cassette from pShuttle into the viral molecular clone. Virus is then rescued, expanded, and purified. Methods are provided for quality control of Ad vectors including titration, testing for genetic integrity and stability, assessing potential contaminations with replication-competent Ad, and levels of transgene product expression. Additional processes needed to release clinical lot
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