**5. Safeties and risks of adenoviral vectors**

Different issues such as the oncogenic or mutagenic risk of the modified vector, its origin, its tropism, or its pathogenicity are some of the potential concerns around adenoviral vector use, not only for the host but also for the environment [84]. Adenoviral vectors are classified as Risk Group 2 (RG2) agents, defined as pathogens causing infrequent serious human diseases with available prevention therapies. This group of agents has to be manipulated in a biosafety level 2 containment facility (BSL2) [85]. Gloves, eye, nose, and mouth protection and laboratory coat are required to prevent mucous membrane contact, inhalation of aerosolized droplets, ingestion, or parenteral inoculation.

Ad cause usually mild illnesses, except in immunocompromised individuals. Potential toxicity is documented *in vitro* and in *in vivo* mouse models for the first-generation RDAds, which contain a great proportion of the Ad genome [86]. These vectors also have the risk of reversion to replication competence because of recombination or complementation between the left terminus end of the vector and the partially overlapping E1 sequence present in HEK293 cell genome or in adenoviral sequences previously acquired by the host (due to the general distribution of the Ad) [87]. Packaging cell lines with nonhomologous sequences with the vector or testing viral vector stocks for RC virus can be employed to reduce this risk [88]. Deletion of the E2A, E2B or E4 regions in the second-generation vectors reduces this risk but complicates the packaging of the recombinant adenoviral particle since specific packaging cells have to be designed to complement the missing adenoviral genome. Obtaining high titer stocks with these systems is more difficult, which often leads to reduced immunogenicity as only lower vaccine doses can be obtained [89]. These issues are even more pronounced with "gutless" adenoviral vectors, which are perfect in terms of safety, but can be problematic in terms of immunogenicity and ease of production.

antibiotic needs, and public health. That vaccination is an integral part of global disease prevention, which can even eradicate diseases is a fact. We have examples of this in both human and animal health with the eradication of smallpox and rinderpest. However, there are still many animal diseases without vaccines or for which treatment needs improvement. Numerous studies constructing, testing, characterizing, optimizing, and identifying adenoviral-based vaccines as optimal against different animal diseases appeared in the last decades. They elicit potent cellular and humoral immunity and can be implemented along DIVA diagnostic tests. RDAd can also be used to deliver immunomodulation to improve disease treatment. Transference to the veterinary market is, however, lagging behind laboratory advances, and no adenoviral vector-based vaccine has yet obtained a veterinary license for systematic use in the field. This nonetheless appears nowadays closer with the recent publication of a positive safety report on an RDHuAd5 FMDV vaccine [43]. Recombinant RDAd reagents could, therefore, have great economic relevance in the future in veterinary medicine. Regulatory committees both in the EU and in the US should favor the approval of these reagents, based on the increasing scientific evidence for their efficacy and safety so that recombinant RDAd can make the leap from laboratory to the field. At the moment, the regulatory bases (EMEA/CVMP/004/04) for the use of adenoviral vector-based vaccines in farms are not well defined, although there are bases established in the EU by the European Medicine Agency (EMEA) and its Committee for Veterinary medicinal Products (CVMP) and in the US by the Animal and Plant Health Inspection Service (APHIS) from The United States Department of Agriculture (USDA). A global cooperation between the veterinary industry and governments is needed in the future for adenoviral vector-based vaccines to reach the market.

Adenovirus as Tools in Animal Health http://dx.doi.org/10.5772/intechopen.79132 41

The work in the lab was funded by Grants RyC2010-06516, AGL2011-25025, AGL2012-33289, AGL2015-64290R, ADENONET-Redes de Excelencia (BIO2015-68990-REDT) from the Spanish Ministerio de Economía y Competitividad, and Grant S2013/ABI-2906-PLATESA from the Comunidad de Madrid and the European Union (Fondo Europeo de Desarrollo Regional,

FEDER funds) and 731014-VetBioNet Project from European Union.

APHIS animal and plant health inspection service

**Acknowledgements**

**Conflict of interest**

**Abbreviations**

Ab antibody

Ad adenovirus

atm atmosphere

The authors declare no conflict of interest.

The route of administration is also relevant in RDAd shedding. Intravenous (or systemic) administration results predominantly in liver adenovirus localization with minimal or no shedding to biologic fluids [6, 90]. When administered subcutaneously or intramuscularly, the point of inoculation should be disinfected to minimize the risk of vector propagation to the environment as leaks can sometimes be detected at the site. Nonetheless, no vector was detected in rodents 72 h after injection in tail swab, and the vectors were cleared from blood within 24 h [90]. As previously mentioned, RDAd vectors do not integrate efficiently into the host cell genome, the transgene expression is only transient and they do not produce infective particles, which inherently improves their biosafety [8]. Adenoviral vector survival in bedding or caging is also reduced compared to parent Ad [8]. Adenoviral vectors can, however, trigger episodes of inflammatory responses. This includes one death after a high dose direct injection of an adenoviral vector into the hepatic artery [91], which produced a fulminant immune reaction probably due to pre-existing vector immunity. It is, however, very difficult to detect vertical or germline transmission of adenovirus vectors in experimental animal models [92].

All measures (autoclave treatments for 30 min at 121°C under 1 atm pressure, 0.5% sodium hypochlorite, 5% phenol, or 2% glutaraldehyde) sufficient to eliminate the peril of adenoviral transmission have to be met to minimize risks (alcohol is not a good decontaminant for Ad), but we must not forget that RDAd do not replicate and should not, unless recombination and complementation occur, be able to shed from inoculated animals, and are thus even less likely to infect another organism. In the case of an RCAd, the risk is reduced to the range and tropism of the Ad; for example, human adenovirus is only known to replicate in two nonhuman species: cotton rat and hamster [93].

It is necessary to deepen in the knowledge of the biodistribution, dissemination, and *in vivo* transgene expression duration of these vectors in veterinary medicine to assess their risk more thoroughly. No standard procedures to monitor these risks exist, and thus, each independent study analyses arbitrarily which biosafety parameters are evaluated.
