**3.1. Considerations for veterinary vaccines and adenoviral vector vaccines**

The use of vaccines to fight animal diseases is one of the most efficient strategies of preventive medicine regarding cost-effect ratio. It helps reduce disease, minimizes long-term healthcare costs, and ultimately reduces inequity in health [28]. Maladies such as rinder pest have been eradicated thanks to vaccine campaigns. Multiple parameters need to be considered for a potential vaccine to become successful, such as its efficacy, safety and immunogenicity, and the possibility of large-scale production at low cost while maintaining genetic stability. Ideally, a vaccine should also be single dose and provide long-term systemic and mucosal immunity [29].

In veterinary medicine, adenoviral vectors that express immunogenic pathogen proteins have been used as vaccine to activate a protective immune response to the pathogen [30, 31]. The use 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 consecutive inoculations [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.

### **3.2. Adenoviral vectors as DIVA vaccines**

dendritic cells (DCs) are transduced in the draining lymph node. The CD11c<sup>+</sup>

CD8<sup>+</sup>

less frequently transduced, the CD11c<sup>+</sup>

32 Adenoviruses

cell proliferation against the transgene. CD11c<sup>+</sup>

immunity are less potent inducers of T cell responses [23].

antibodies reduced the induction of transgene-specific CD8<sup>+</sup>

boost inoculation was given with a heterologous RDAd.

to drive the immune response toward the antigen of interest.

**3. Recombinant adenoviral vectors in veterinary medicine**

**3.1. Considerations for veterinary vaccines and adenoviral vector vaccines**

vaccine design, since enhanced CD8<sup>+</sup>

lenge. Nonetheless, these neutralizing antibodies change the fate of the CD8<sup>+</sup>

responses against RDAd-encoded transgenes.

partment showed enhanced RDAd uptake and transgene expression [22], but in spite of being

B220−

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

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

promote their transition into the memory cell pool [27]. This could be highly relevant for

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

The use of vaccines to fight animal diseases is one of the most efficient strategies of preventive medicine regarding cost-effect ratio. It helps reduce disease, minimizes long-term healthcare costs, and ultimately reduces inequity in health [28]. Maladies such as rinder pest have been eradicated thanks to vaccine campaigns. Multiple parameters need to be considered for a potential vaccine to become successful, such as its efficacy, safety and immunogenicity, and the possibility of large-scale production at low cost while maintaining genetic stability. Ideally, a vaccine should also be single dose and provide long-term systemic and mucosal immunity [29]. In veterinary medicine, adenoviral vectors that express immunogenic pathogen proteins have been used as vaccine to activate a protective immune response to the pathogen [30, 31]. The use

CD8−

T cells after homologous chal-

cell expansion to the transgene can be detected when

T cells and

DC subset was more potent at inducing T

DCs are, therefore, critical for eliciting T cell

B220−

com-

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, particularly suited to implement DIVA strategies.

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

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 efficacy if booster immunizations are required. Care should also be taken when immunizing immunocompromised individuals with RCAd vectors as vaccine-derived pathology could be induced. Importantly, RCAd could potentially escape the vaccinated host, which limits their application and hinders their approval by legislative bodies. RCAd can nonetheless have applications in veterinary science as demonstrated by the effective campaigns for rabies control in Canada with RC adenoviral vectors expressing the rabies virus glycoprotein delivered to wildlife through baiting [41]. The vaccine was safe in a number of species and showed minimal risk of horizontal transmission [42].

suitable vaccine strains or FoAd could be manipulated to become vectors that express recombinant immunogenic proteins [52]. Because of the dissemination risks posed by RCAd, RDAd

Adenovirus as Tools in Animal Health http://dx.doi.org/10.5772/intechopen.79132 35

One of the main barriers for the development of nonhuman RDAd vectors is the necessity to construct cell lines capable of complementing the viral genome so that these vaccines can be propagated. The production of RD vectors has nonetheless been achieved for several nonprimate species [48, 53], and RDCaAd2 vectors expressing immunogenic viral subunits have shown potential for vaccination against rabies [54], bluetongue virus (BTV) [55], or FMDV [56]. Because Ad infect a wide range of mammalian cells from different species, these nonhuman RDAd vectors could also be used to circumvent pre-existing immunity. Ultimately, this could help broaden the range of adenoviral vectors available for vaccine design. Understanding nonhuman adenovirus biology and advancing in their manipulation can, therefore, help vaccinologist design novel strategies in veterinary medicine and in human medicine where pre-

Typically, RDAd are engineered to express an immunogenic antigen from the pathogen and used as vaccine. However, since RDAd can accommodate fairly large inserts, they can encode for multiple genes and produce virus-like particles. RDAd can also be used to boost adjuvancy in vaccine preparations by expressing cytokines or co-stimulatory molecules, or even

RDAd encoding for immunogenic determinants showed promising vaccination results in a range of relevant veterinary diseases (**Table 1**). In PPRV, which is the next disease targeted by the World Organization for Animal Health (OIE) for eradication, RDHuAd5 vectors expressing PPRV fusion protein (F) or hemagglutinin (H) induced strong cellular and humoral immunity and protected goats and sheep against virulent challenge [38, 39]. In BTV, immunizations with RDHuAd5 expressing the VP2 and/or VP7 proteins are protected from homologous challenge [57]. RDHuAd5 expressing the FMDV P1 region and the 3Cpro protease can protect swine and cattle from the disease [58]. RDAd vaccines can protect multiple mammalian hosts (sheep, goats, and cattle) from Rift Valley fever virus (RVFV) challenge, and induce immunity in camels [47]. RDAd vaccines can also protect across animal classes as an RDHuAd5 vector vaccine expressing the influenza A virus (IAV) H protected chicken from viral challenge [59]. This broad spectrum of hosts makes RDAd vaccines particularly attractive for vaccine design against zoonotic diseases. The choice of antigen is of prime importance for RDAd vaccine clinical efficiency. The immunogenicity of the transgene influences the immunity triggered to the vector [24, 31]. Strongly, immunogenic transgene products skew the immune response toward these proteins, whereas weakly immunogenic transgene products favor anti-vector immunity that eliminates transduced cells and shortens antigen exposure [60]. For instance, RDAd vaccine expressing only the

appear nonetheless as the way forward even for nonhuman Ad.

existing immunity to these vectors will be minimal.

**4.1. Antigen-encoding RDAd as vaccines**

**4. Applications of RDAd in veterinary medicine**

impair viral replication by encoding for interfering RNA sequences.

The present chapter will mainly focus on RD adenoviral vectors as veterinary tools since RDAd genetic stability makes them particularly suited for the design of safe and legislatively acceptable vaccines. Despite being one of the most studied recombinant vectors in veterinary medicine, no RDAd vaccine is currently licensed for veterinary use. An RDHuAd5 vector expressing the FMDV P1 region and the 3Cpro protease has nonetheless received a conditional US veterinary biological product license. An exhaustive safety study for the issue of a US veterinary biological license product for this vaccine was recently completed [43]. No evidence of reversion to virulence, shedding from vaccinees or presence in milk products was detected indicating that RDAd vaccines are safe and recombinant vaccine particles are unlikely to be found in animal products used for human consumption.

### **3.4. Human vs. nonhuman adenovirus for veterinary use**

HuAd5 vector is the most extensively used adenoviral vector for vaccine design and gene therapy. However, pre-existing adenoviral immunity complicates its use in human therapy since this drastically decreases efficacy [44], but in veterinary medicine, no immunity to HuAd should be present. Indeed pre-existing neutralizing antibodies and cell-mediated immunity to the veterinary specie Ad usually do not cross-react with human adenoviral vectors [45]. This implicates that human adenoviral vectors can trigger strong immune response in the veterinary host. There are nonetheless risks that need assessment prior to commercial release like reversion to virulence. Importantly for livestock animals, it is essential to demonstrate that the recombinant vaccine is absent from the animal products consumed by the human population (e.g., meat and milk) so that veterinary use of RDHuAd vaccines is not perceived as a health risk by legislative bodies and the public in general.

To circumvent pre-existing immunity, nonhuman adenoviral vectors can be used. These are often studied for gene therapy as they improve gene delivery and expression [46], but they could still hold veterinary vaccine applications. For instance, in the cases of zoonosis like Rift Valley fever (RVF) that affect human populations, it could prove advantageous to develop adenoviralbased vaccines on the backbone of nonhuman species to avoid HuAd pre-existing immunity [47]. Since most nonprimate adenoviral vectors produce abortive infections in human cells [48], the risk of virulence reversion and recombinant vector spreading in humans is further minimized. These nonhuman vectors also produce strong immune responses in the veterinary host, although most studies thus far have used RCAd constructs [9, 49, 50]. Nonhuman RCAd could have applications in veterinary vaccination when the Ad itself is pathogenic [51]. Recombinant technology could be used to attenuate pathogenic fowl adenoviruses (FoAd) strains to produce suitable vaccine strains or FoAd could be manipulated to become vectors that express recombinant immunogenic proteins [52]. Because of the dissemination risks posed by RCAd, RDAd appear nonetheless as the way forward even for nonhuman Ad.

One of the main barriers for the development of nonhuman RDAd vectors is the necessity to construct cell lines capable of complementing the viral genome so that these vaccines can be propagated. The production of RD vectors has nonetheless been achieved for several nonprimate species [48, 53], and RDCaAd2 vectors expressing immunogenic viral subunits have shown potential for vaccination against rabies [54], bluetongue virus (BTV) [55], or FMDV [56]. Because Ad infect a wide range of mammalian cells from different species, these nonhuman RDAd vectors could also be used to circumvent pre-existing immunity. Ultimately, this could help broaden the range of adenoviral vectors available for vaccine design. Understanding nonhuman adenovirus biology and advancing in their manipulation can, therefore, help vaccinologist design novel strategies in veterinary medicine and in human medicine where preexisting immunity to these vectors will be minimal.
