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

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.

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.

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

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

**4.2. Immunomodulation through RDAd vectors**

38 Adenoviruses

would favor immune response to the antigen (**Table 1**).

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 through different pathways.

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 improve RDAd vaccine immunogenicity.
