**1. Introduction to adenoviral vectors**

### **1.1. Adenovirus**

Adenoviruses (Ad) are large (90-100 nm), nonenveloped, not segmented, and linear doublestranded DNA viruses belonging to the viral family *Adenoviridae* that infect a broad range of vertebrate hosts, from fish to humans. They replicate in the nucleus of the infected cells. These viruses have an icosahedral nucleocapsid consisting of three major proteins called hexon (or protein II),

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

penton base (or protein III), and a nodulated fiber (or protein IV) together with a number of other minor proteins, VI, VIII, IX, IIIa, and Iva2. This capsid contains 26–48 Kbp double-stranded DNA genome (**Figure 1A**), which has a terminal protein (TP) attached to one of its ends. They were first isolated in 1953 from a culture of human adenoid cells, hence their name [1]. Of the more than 100 Ad described since then, 57 infect humans causing conjunctivitis, hemorrhagic cystitis, gastroenteritis, and respiratory diseases. The *Adenoviridae* family contains five genera based on DNA composition and host species: *Aviadenovirus*, *Atadenovirus*, *Mastadenovirus*, *Siadenovirus*, and *Ichtadenovirus* [2]. Within the genera, the viruses are grouped into species, and named from the host followed by letters of the alphabet. For example, the human adenoviruses (HuAd) are classified within the *Mastadenovirus* genus and divided into seven subgroups, from A to G [3, 4]. Classification questions remain, however, unresolved for many nonhuman adenoviruses.

Ad possess most of the qualities required to be a successful viral vector. They infect a large variety of mitotic and postmitotic cells replicating episomally without chromosomal integration, thus reducing the risk of insertion mutations and oncogenesis. They have high cloning capacity, high transduction efficiency, and high transgene expression. They are relatively easy to prepare and purify, which permits the obtention of high viral titer with low toxicity. HuAd serotypes 2 and 5 are the best characterized and most used for creating recombinant vectors [6]. The RDAd used as vectors can be divided into three classes, schematized in **Figure 1B** [7, 8], according to the size of the deletions made in their genome, which directly impacts on the size

From a safety point of view, it is preferable to work with replication-defective (RD) Ad [9], and this chapter will mainly focus on RDAd. There are nonetheless several studies that use replication-competent (RC) Ad in veterinary vaccination, for instance to improve mucosal

RDAd vectors induce humoral, cellular, and mucosal protective immune responses in a variety of animal models [12]. They are particularly suited to produce potent cellular immune response to the encoded antigens [13]. Vector innate immunogenicity and antigen expression

Innate immune responses are essential for triggering an effective adaptive response. RDAd activate nucleotide-binding oligomerization domain-like receptor (NLR) and toll-like receptor (TLR) signaling pathways and induce several cytokines such as IL-1, IL-12, IL-6, TNF, and interferon (IFN)-α. Myeloid differentiation protein-88 (MyD88) signaling contributes to the induction of RDAd adaptive immune response since systemic and mucosal immunity was

after RDAd vaccination are, however, not dependent on TLRs or IL1-R family member since T-cell responses are not significantly diminished in mice lacking different TLRs, IL-1R, or IL-8R [15]. Type I IFN production and signaling probably participate to transgene immunity. Type I IFN levels correlate with transgene neutralizing antibody titers [16] and IFN-β promoter stimulator-1 (IPS-1) and type I IFN signaling are required for the induction of antigen-specific


The magnitude and quality of the T cell immune response elicited by RDAd is influenced *in vivo* by the vector cellular tropism, which alters the source of cytokines and chemokines produced during vaccination. After intravenous inoculation, Kupffer cells in liver [20] and macrophages in the marginal zone of the spleen [21] are infected by RDAd, whereas after subcutaneous or intramuscular inoculation (the most commonly used vaccination routes), CD11c<sup>+</sup>


Adenovirus as Tools in Animal Health http://dx.doi.org/10.5772/intechopen.79132 31

affect and shape the adaptive immune response triggered by RDAd infection.

reduced in MyD88-deficient mice after RDAd vaccination [14]. CD8<sup>+</sup>

of the exogenous DNA they can harbor.

CD8<sup>+</sup>

innate immune response to RDAd.

immunity or override maternal-derived immunity [10, 11].

**2. Immunogenicity of adenoviral vectors**

### **1.2. Adenoviral vectors**

Viral vectors are modified viruses used to introduce exogenous DNA into host cells, and their construction uses similar principles. Virus functions can be divided into elements that act in *cis* such as the origins of replication or the encapsidation sequence that must be found in the genome of the viral vector, or act in *trans* such as structural proteins and/or envelope or the machinery necessary for viral replication that do not need to be encoded by the viral genome itself. These *trans* elements can be supplied by stably transfected cells (packaging cells), or through transient transfections with plasmids or helper virus. The general method for viral vector construction consists in substituting the *trans* elements, essential for replication, by the gene of interest. The most popular technique developed for constructing replication-defective (RD) recombinant adenoviral vectors is that described by Dr. F. Graham and known as the "twoplasmid method" (available in commercial kits) [5]. Nonreplicative (defective) particles thus obtained maintain the infectivity of the parental virus, but are unable to produce new infective viral particles, and possess the ability to transfer the therapeutic gene material introduced into their genome. The viruses most commonly used as vectors are poxviruses, retroviruses, and Ad.

**Figure 1.** (A) Schematic representation of the adenoviral genome organization. E, early genes; L, late genes; and ITR, inverted terminal repeat sequences. (B) Diagram of the evolution of the different adenoviral vectors. Deletions (∆) from different areas of the adenoviral genome have improved these vectors in terms of capacity to house an exogenous gene and in terms of safety, avoiding reversions. ψ, cis packaging signal.

Ad possess most of the qualities required to be a successful viral vector. They infect a large variety of mitotic and postmitotic cells replicating episomally without chromosomal integration, thus reducing the risk of insertion mutations and oncogenesis. They have high cloning capacity, high transduction efficiency, and high transgene expression. They are relatively easy to prepare and purify, which permits the obtention of high viral titer with low toxicity. HuAd serotypes 2 and 5 are the best characterized and most used for creating recombinant vectors [6]. The RDAd used as vectors can be divided into three classes, schematized in **Figure 1B** [7, 8], according to the size of the deletions made in their genome, which directly impacts on the size of the exogenous DNA they can harbor.

From a safety point of view, it is preferable to work with replication-defective (RD) Ad [9], and this chapter will mainly focus on RDAd. There are nonetheless several studies that use replication-competent (RC) Ad in veterinary vaccination, for instance to improve mucosal immunity or override maternal-derived immunity [10, 11].
