**2. Immunogenicity of adenoviral vectors**

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.

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.

**1.2. Adenoviral vectors**

30 Adenoviruses

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

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 reduced in MyD88-deficient mice after RDAd vaccination [14]. CD8<sup>+</sup> -T cell responses elicited 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 CD8<sup>+</sup> -T cells in the gut mucosal compartment [17]. Besides TLRs, cells detect cytosolic viral DNA through NLRs, which are at the core of the inflammasome that triggers inflammatory responses producing IL-1β, IL-18, and IL-6 (reviewed in [18]). NF-κB-dependent inflammatory gene expression (IL-1β, IL-6, and MIP-1β) was significantly reduced in NALP3-deficient mice after RDAd inoculation [19], indicating that the NALP3 inflammasome mediates the innate immune response to RDAd.

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>

dendritic cells (DCs) are transduced in the draining lymph node. The CD11c<sup>+</sup> CD8− B220− compartment showed enhanced RDAd uptake and transgene expression [22], but in spite of being less frequently transduced, the CD11c<sup>+</sup> CD8<sup>+</sup> B220− DC subset was more potent at inducing T cell proliferation against the transgene. CD11c<sup>+</sup> DCs are, therefore, critical for eliciting T cell responses against RDAd-encoded transgenes.

of HuAd5, most commonly used in human trials, in animal health can be advantageous, as no previous immunity to this adenoviral vector should exist in animals. Recombinant Ad strongly activate the immune system [32] and generate immunity toward both the vector and the expressed transgene. These strong humoral and cell-mediated antigen-specific responses [12, 13] are a prerequisite for a good vaccine candidate that can even preclude for adjuvant need. But it may also present a problem, since immunity to the vector could be generated in vaccinated animals, which would limit efficacy if a second immunization was needed. Several approaches can be undertaken to solve this problem, from using a single inoculation to induce protection, to using heterologous prime-boost systems or using different Ad serotypes for

Adenovirus as Tools in Animal Health http://dx.doi.org/10.5772/intechopen.79132 33

RDAd recombinant vectors can be produced in large scale with a high titer [34] and lyophilized, or produced in thermostabilized forms [35] so that they can be easily stored and transported, even in conditions in which the maintenance of a cold chain can be problematic as in case of distribution to remote locations in hot climate countries. For veterinary medicine, vaccines need to be particularly inexpensive. As part of the One Health strategy, vaccination also offers the added benefit of limiting antibiotic use in animal production, either through direct vaccination effects or by limiting viral diseases that can lead to opportunistic bacterial infections.

Most veterinary vaccines do not allow infected-recovered animals to be distinguished from vaccinated animals, the so-called differentiating infected from vaccinated animals (DIVA) approach. DIVA vaccines can be used as control tools for disease outbreaks, limiting animal culling in the eradication process. They, thus, have a great economic importance as they facilitate animal health status monitoring and grant disease-free status more quickly to countries affected by an outbreak. RDAd expressing antigenic proteins are suitable DIVA vaccines as vaccinated animals that only respond to proteins encoded by the vaccine can be differentiated from infected animals that also respond to viral proteins not encoded by the RDAd vaccine. An adenovirus-based vaccine was shown to be successful as foot and mouth disease (FMDV) DIVA vaccine [36]. RDHuAd5 that express peste des petits ruminants virus (PPRV)-F or -H proteins are another example of DIVA veterinary vaccines [37–39]. While vaccinated animals developed antibodies against F and H, infected animals also developed antibodies against N, and due to validated commercially available tests for anti-N and anti-H antibodies, infected animals could be differentiated from vaccinated animals. RDAd-based vaccines appear, thus,

When Ad are engineered to be RD and express a transgene, most of the immune response they trigger can be biased toward this transgene since transgene expression replaces early adenoviral gene expression, thus limiting adenoviral protein synthesis [24]. Ad can also be engineered to express transgene while remaining replication competent (RC). In these cases, immune responses to the transgene can be enhanced [9, 31, 40], but the immune system is also more prone to react to the vector than in the case of RD vectors since infective lytic cycles occur. This can result in sero-neutralization of the vector over time that limits vaccine

consecutive inoculations [33].

**3.2. Adenoviral vectors as DIVA vaccines**

particularly suited to implement DIVA strategies.

**3.3. Replication-competent vs. replication-defective adenoviral vectors**

High transgene antigen-specific responses after infection with Ad serotypes, such as HuAd5, are associated with high transgene expression levels *in vivo* [23]. The amount and duration of the antigen expression is thus one of the most relevant parameters that shape the immune response induced by RDAd. In mice, HuAd5 and chimpanzee-derived ChAd3 produce high and persistent antigen expression with low innate immunity activation resulting in strong T cell response induction, whereas RDAds that express less antigen and trigger a robust innate immunity are less potent inducers of T cell responses [23].

Pre-existing vector-specific humoral and cellular immunity limits the duration of transgene expression and is one of the main problems for RDAd uses as vaccines [24]. Vector-specific neutralizing-antibodies suppress the immunogenicity of adenoviral vector vaccines [25]. Although neutralizing antibodies are serotype specific and mainly directed against the hypervariable loops of the viral hexon, non-neutralizing antibodies to more conserved regions of the adenoviral particle cross-react between Ad serotypes [26]. Passive antibody transfer from RDAd-immunized animals to naïve animals demonstrated that adeno-specific neutralizing antibodies reduced the induction of transgene-specific CD8<sup>+</sup> T cells after homologous challenge. Nonetheless, these neutralizing antibodies change the fate of the CD8<sup>+</sup> T cells and promote their transition into the memory cell pool [27]. This could be highly relevant for vaccine design, since enhanced CD8<sup>+</sup> cell expansion to the transgene can be detected when boost inoculation was given with a heterologous RDAd.

It, thus, appears that the balance between immunity to the vector and the transgene defines successful RDAd vaccination strategies. Recognition of the vector is necessary for Ad adjuvancy to take place, while high transgene expression and immunogenicity are also required to drive the immune response toward the antigen of interest.
