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

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

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

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

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

minimal risk of horizontal transmission [42].

34 Adenoviruses

found in animal products used for human consumption.

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

as a health risk by legislative bodies and the public in general.

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


**Adenovirus\*** **RNA interference**

HuAd5 HuAd5 **Immunomodulation**

HuAd5 HuAd5 HuAd5 HuAd5 HuAd5 HuAd5 HuAd5

PRRSV \*All adenoviral vectors are replication deficient unless otherwise stated (i.e., RC).

Examples of adenoviral vector use in veterinary medicine.

**Table 1.**

GP3 GP5 fusion protein, HSP70

Swine

PRRSV

PCV2

poGM-CSF

poCD40L PCV Capsid protein

Gp3 GP5 fusion protein poCD40L

Swine

Partial protection

[97]

Ab and CMI

CD40L improve efficacy

IFN-γ and IL-4

HSP70 improves efficacy

in sera VNA

[83]

Adenovirus as Tools in Animal Health http://dx.doi.org/10.5772/intechopen.79132 37

IAV Salmonella

ovIFN-τ poGCSF

FMDV FMDV

poIFN-α +

poIFN-α

poIFN-γ

Swine Swine Mouse

Swine Swine

Synergistic protection

Protection vs. several FMDV

serotypes

Protection Protection against Salmonella

shedding and colonization

Reduced viremia

[81]

[73]

[72]

[74]

[76]

FMDV

shRNA

FMDV

poIFN-α +

poIFN-γ + siRNA

Mouse

Protection

[79]

Guinea pigs

Swine

Guinea pigs

Partial protection

[78]

Swine

against NS proteins

**Disease**

**Transgene**

**Model/natural host**

**Efficacy/findings**

**References**


**Adenovirus\*** **Antigen encoding**

HuAd5 HuAd5 ChAdY25

HuAd5 HuAd5 HuAd5 HuAd5 **VLP encoding**

RC CaAd2

CaAd2 HuAd5 HuAd5

FMDV

P1/3Cpro

RHDV FMDV FMDV

VP60 P1/3C PPV-VP2 expressing FMDV VP1

epitopes

Rabbit Guinea pigs

Mouse

Swine

Swine

Cattle

Protection Ab production

Ab production and protection

Protection

VNA production

Protection

[58, 62, 63]

[56]

[96]

[50]

CSFV

E2 protein

FMDV

poGMCSF, VP1, VP1 epitopes

Mouse

Guinea pigs

Swine

Swine

Complete protection in DNA-Ad

[67]

prime boost

BTV

VP2, VP7

Mouse

Sheep

PPRV

F, H

RVFV

Gn, Gc

Sheep

Cattle

Goats

Camels

Sheep

Protection

[38]

VNA, Ab production, CMI

VNA, Ab production CMI

[57]

and protection

Protection

[95]

IAV

HA

Mouse poultry

Protection

[59]

Ab + CMI

s.c. vaccinated chicken protected

Multispecies protection

[47]

VNA induction in camels

IAV

HA

Swine

Protection in homol-challenge

[94]

Partial in heterol-challenge

**Disease**

**Transgene**

**Model/natural host**

**Efficacy/findings**

**References**

36 Adenoviruses

**Table 1.** Examples of adenoviral vector use in veterinary medicine. FMDV VP1 capsid protein can only induce low levels of neutralizing antibodies [61], whereas RDAd vaccines expressing the complete P1-encoded capsid polypeptide of FMDV and the 3Cpro protease can fully protect swine and cattle [58, 62, 63]. Protection was also achieved in animal models with this FMDV antigen formulation expressed in an RDCaAd2 vector instead of the "traditional" RDHuAd5 vector [56], highlighting the efficacy of this antigen construct. The choice of antigen for vaccination should, therefore, be based on the knowledge of host-pathogen interactions and the characterization of the protective immunity that arises during infection.

off-the-shelf antiviral agents in the early stages of an outbreak in a disease-free country that could control disease spread for highly contagious viral pathogens like FMDV. They can also help bridge the gap in immunity in naïve herds, while the adaptive immune response to the vaccine is being triggered. Cytokine expression by RDAd could also have applications for the treatment of bacterial infections, since for instance, RDAd-expressed porcine G-CSF was

Adenovirus as Tools in Animal Health http://dx.doi.org/10.5772/intechopen.79132 39

RNA interference can be an effective mean to impair viral replication [77], and its delivery through an RDAd vector could be attractive to treat some viral diseases. Expression of small hairpin RNAs specific for the FMDV 3D polymerase and the structural 1D protein could partially protect pigs against challenge [78]. RDAd delivering small interfering RNA, IFN-α, and IFN-γ enhanced anti-FMDV effects and was effective against multiple FMDV serotypes [79]. RNA interference delivered by RDAd could, therefore, be used as a fast-acting antiviral. This strategy could complement the efficacy of IFN-expressing RDAd, as these antiviral effects act

Antigen expression on RDAd can be engineered to promote antigen presentation. This has been achieved for instance by linking the antigen to the invariant chain to promote antigen presentation and thus enhances cell-mediated immunity [80]. Inclusion of GM-CSF or CD40L expression in the RDAd vectors probably favors antigen presentation and improves vaccine effectiveness [81]. Antigen delivery can also be improved by expressing the antigen of interest linked to heat shock proteins. Expression of the HSP70 C-terminal gene linked to the hantavirus glycoprotein Gn can augment cellular and humoral immunity and protects mice from a virulent challenge [82]. Co-expression of HSP70 and PRRSV gp3 and gp5 glycoproteins in an RDAd vector also enhances immunity to the antigens and improves vaccine efficacy [83]. Strategies that boost transgene antigen presentation can, therefore, become a valuable tool to

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

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

successful at reducing Salmonella shedding and colonization in challenged pigs [76].

**4.3. RNA interference of viral replication and enhanced antigen presentation**

through different pathways.

improve RDAd vaccine immunogenicity.

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

droplets, ingestion, or parenteral inoculation.

Typically, RDAd are very effective at triggering cell-mediated immunity, since transduction allows for prolonged presentation of intracellular antigen encoded by the transgene. This can be very useful for vaccine design, and inclusion of genes targeted by cell-mediated immunity could improve immunogenicity [57, 64]. Cell-mediated immunity can target epitopes encoded by conserved genes and thereby recognize infected cells independently of the virus serotype [65]. This could potentially provide some degree of protection against heterologous serotypes [66] in diseases like FMDV, IAV, or BTV in which cross-protection between serotypes is very limited. Inclusion of immunogenic antigens for cell immunity will likely improve RDAd vaccine efficacy.

RDAd vector expressing antigens are nonetheless fully protective in only few cases. Ideally, a veterinary vaccine should consist of a single-dose immunization that provides long-term protection so that costs are maintained low. Some RDAd vaccines can achieve this [58, 66], but experimental vaccination protocols often employ prime-boost strategies for RDAd vaccines to trigger protective immunity. In some cases, prime-boost strategies appear necessary to RDAd vaccine activity [67]. Administration route can also affect RDAd vaccine efficacy [68], and induction of mucosal immunity can be limited. Oral/nasal RDAd administration can nonetheless trigger the mucosal immunity necessary for protection against influenza for instance [66, 69]. RDAd administration route should, therefore, be given careful attention when designing vaccination protocol.

### **4.2. Immunomodulation through RDAd vectors**

Enhancing the immunogenicity of RDAd vaccine candidates so that efficacy is improved is a continuous goal for researchers. This could be achieved through addition of external adjuvant [70], or by making the adenoviral vector encode for immunomodulatory molecules that would favor immune response to the antigen (**Table 1**).

The antiviral activity of the IFN system is well documented [71]. IFNs induce an antiviral state in cells that help the host control viral infections. Systemic administration of recombinant IFNs is nonetheless toxic and too expensive for veterinary medicine. As an alternative, inclusion of IFNs as RDAd transgenes could boost vaccine efficacy and/or provide early protection when highly contagious virus outbreaks occur. Recombinant expression of IFN-α with FMDV VP1 protein or epitopes enhanced the RDAd vaccine activity [61]. IFN-expressing RDAd have nonetheless shown their potential as antiviral agents when administered on their own. RDAd expressing porcine IFN-α can protect against multiple FMDV serotypes [72] and work synergistically with IFN-γ to protect against FMDV challenge [73]. Ovine IFN-τ expression in RDAd demonstrated antiviral efficacy in influenza virus murine model [74]. This ruminant IFN displays many of the antiviral activities of IFN-α in a wide range of mammalian hosts but with reduced toxicity [75]. IFN-expressing RDAd have, therefore, the potential to be used as off-the-shelf antiviral agents in the early stages of an outbreak in a disease-free country that could control disease spread for highly contagious viral pathogens like FMDV. They can also help bridge the gap in immunity in naïve herds, while the adaptive immune response to the vaccine is being triggered. Cytokine expression by RDAd could also have applications for the treatment of bacterial infections, since for instance, RDAd-expressed porcine G-CSF was successful at reducing Salmonella shedding and colonization in challenged pigs [76].
