**4. Exploiting innate responses for antiviral, therapeutic and adjuvation strategies**

The example described above illustrates the potential of RNA regions in the viral genome, known to elicit innate responses, for antiviral and therapeutic applications.

Viral pattern recognition system may offer unique translational implications in medical approaches, taking advantage of the innate immune function of PRRs to trigger cell autono‐ mous responses in tumour cells along with cytotoxic T-cell responses that target tumour cells. Tumour cells are much more sensitive to cytotoxic effects after RLR ligation than are untrans‐ formed cells, allowing for tumour-specific effects despite systemic application of the ligands in mouse models [50, 51]. The concept of using targeted application of PAMPs to mimic a situation of viral infection for clinical application like immunotherapy is being extensively explored [52]. In addition to being interesting targets for the immunotherapy of cancer, RLRs have been found to play a role in other disease conditions like type-I diabetes and other autoimmune diseases like psoriasis or selective IgA-deficiency [53-55]. RLRs ligands have been shown to have a therapeutic effect for autoimmune inflammatory disease of the central nervous system in mice [56].

The antiviral effect exerted in vivo by these small synthetic non-infectious RNA molecules was analyzed extensively, using a wide range of viral doses and different serotype isolates [49]. The time course of resistance to FMDV of the RNA-transfected mice was also studied. Inoculation with all RNAs remarkably increased the 50% lethal dose (LD50) of the virus, determined for the control group. Mice inoculated with IRES or S transcripts 24 h before challenge became at least 10000-foldless susceptible tothevirus thanPBS-inoculatedmice.Interestingly, 90%ofthe IRES-

(undiluted viral stock), showing the outstanding protective effect of these RNA molecules. The level of protection against viral infection was dose-dependent. Complete or very high protec‐ tion was achieved when IRES RNA was inoculated 8 or 24 h prior to FMDV infection with 7 x

 PFU, with 100 and 86% survival, respectively. Inoculation of the transcripts at longer times pre-infectionstronglydecreasedtheirprotective effect against viralinfection. Co-inoculationof S or IRES transcripts and the virus induced high levels of protection (about 90%), and the IRES RNA had a higher protective effect inoculated at 8 h than at 4 h before infection, suggesting that a fine balance between the routes activating the innate immune response by the RNAs and the viral replication kinetics or antagonistic mechanisms triggered by the virus, might determine eithertheoutcomeofdiseaseortheviralclearance.Additionally,highsurvivalpercentageswere observed for those groups inoculated with the RNAs at short times after infection (89 and 87% ofmiceinoculatedwiththeIRESat4hand8hpost-infectionsurvived,respectively),andcomplete protection (100% survival) was achieved when mice were inoculated with the S transcripts at 4 or 8 h post-infection [49]. No protective effect was observed for mice inoculated with the RNAs 24 h after viral infection. These results suggest that the antiviral response induced by the RNAs is rapidly established and effective to counteract the viral replication if administered shortly afterinfection,while24hlateritwastoolatetorestraintheprogressofinfection.Ourdatasupport the potential use of this RNAs as both prophylactic as well as therapeutic molecules in a certain time window. These small non-infectious RNAs could be useful to induce a rapid antiviral state in combination with effective FMD vaccines to overcome the problem of the susceptibility window until protective levels of antibodies are produced by vaccinated animals. These results provide, as well, a new insightinto broad-spectrumantiviraldevelopment strategies (Figure 3).

**4. Exploiting innate responses for antiviral, therapeutic and adjuvation**

The example described above illustrates the potential of RNA regions in the viral genome,

Viral pattern recognition system may offer unique translational implications in medical approaches, taking advantage of the innate immune function of PRRs to trigger cell autono‐ mous responses in tumour cells along with cytotoxic T-cell responses that target tumour cells. Tumour cells are much more sensitive to cytotoxic effects after RLR ligation than are untrans‐ formed cells, allowing for tumour-specific effects despite systemic application of the ligands in mouse models [50, 51]. The concept of using targeted application of PAMPs to mimic a situation of viral infection for clinical application like immunotherapy is being extensively explored [52]. In addition to being interesting targets for the immunotherapy of cancer, RLRs

known to elicit innate responses, for antiviral and therapeutic applications.

plaque forming units (PFU)

transfected mice survived after infection with a viral dose of 7 x 106

200 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

104

**strategies**

**Figure 3.** Synthetic RNA transcripts derived from structural domains present in the 5´and 3´NCRs of the FMDV genome as innate immunity elicitors **Figure 3.** Synthetic RNA transcripts derived from structural domains present in the 5' and 3'NCRs of the FMDV ge‐ nome as innate immunity elicitors

Other than its role in driving innate immune defenses, IFN plays a major role in modulating the adaptive immune response [57]. IFN is required to promote T cell survival and clonal expansion after antigen presentation. IFN also induces the cytolytic activity of NK cells and cytotoxic lymphocytes and promotes B cell differentiation and antibody production, as well as expression of MHC class I molecules [58, 59]. The specific role of RLR signaling in regulating IFN production and its regulation of the adaptive immune response is less clear and appears to vary from virus to virus [60].

Luke et al. showed that the potency of a DNA vaccine against influenza virus could be augmented by the incorporation of a RIG-I activating immunostimulatory RNA into the vector backbone [61]. Mice receiving the vaccine exhibited increased virus-specific serum antibody response as compared to those receiving the DNA vaccine alone. These results suggest that RLR signaling can enhance antibody development after vaccination by activation of innate immunity and improved adaptive immune responses. However, in a previous study Koyama et al. found a defect in antigen-specific B and T cell activation in MyD88-defficient mice, unlike MAVS-deficient mice, suggesting that adaptive immune responses against influenza A virus are governed by the TLR pathway [62]. On the contrary, MAVS-deficient mice infected with WNV displayed uncontrolled inflammation including elevated systemic IFN, proinflamma‐ tory cytokine and chemokine levels, and enhanced humoral responses marked by complete loss of virus neutralization activity with a failure to protect against WNV infection [63]. This work defined an innate/adaptive immune interface mediated through MAVS-dependent RLR signaling that regulates the quantity, quality and balance of the immune response to WNV. Using MDA5-defficient mice, Anz et al. showed that the loss of T regulatory cell function on infection with encephalomyocarditis virus (EMCV) is strictly dependent on RLR signaling [64]. In a different study, a normal and effective adaptive immune response against respiratory syncytial virus (RSV) infection was reported in the absence of both MAVS and MyD88, RLRand TLR-adaptor molecules, respectively [65]. All together, the results suggest that, in the case of flavivirus and picornavirus infections, RLR signaling is important in the control of the quantity, quality and balance of the adaptive immune response, with specific and differential mechanisms of regulation operating depending on the specific virus infections. The under‐ standing of the molecular features underlying these processes could offer new strategies for immune and antiviral therapy targeting the RLR pathway for therapeutic control of the viral infection and enhancement of the induced immune response.

advanced in adjuvant development, but require additional considerations, as variation in TLR expression and influence of age in responsiveness or the risk of autoimmunity by induction of excessive inflammatory responses [68]. Other TLR agonists currently in clinical trials of a

Use of RNA Domains in the Viral Genome as Innate Immunity Inducers for Antiviral Strategies and Vaccine

Improvement

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http://dx.doi.org/10.5772/56099

In addition to recognition of viral ssRNA like that derived from HIV (Human immunodefi‐ ciency virus) or influrenza viruses, TLR7/8 can also be activated by certain synthetic agonists such as the imidazoquinoline derivatives imiquimod and resiquimod (R-848), as well as the guanine nucleotide analog loxoribine. TLR7/8 imidazoquinolines can activate appropriate immune cells and modulate cellular and humoral immunity and have been found to be excellent vaccine adjuvants [70]. Most TLR agonists induce antibody and Th1 responses, although some can induce Th2 and possibly Th17 responses. Knowledge of the response outcomes in terms of cytokines, chemokines and T-cell subtypes generated by activation of combination of PRRs would help in the design of vaccine formulations including the appro‐ priate combination of adjuvants in the future that can contribute to develop new vaccines

In the case of FMDV, many efforts are being invested on development of new vaccine formula‐ tions aimedtoimprove currentlyusedvaccines [71].AlthoughFMDvaccines are available since the early 1900s, the disease still affects millions of animals around the globe and remains the main sanitary barrier to the commerce of animals and animal products. Among the limitations of the currently available inactivated antigen vaccines are the short duration of immunity and the lack of serotype-cross protection. Administration of this vaccine or an experimental vaccine basedona replication-defectivehumanadenovirus (Ad5)vectorthatdelivers the FMDVcapsid and 3C proteins requires ~7 days to induce protective immunity in animals [72, 73]. New approaches aimed to shorten this susceptibility window and induce a more robust and longlasting adaptive immune response are being developed, such as expression of type-I,-II and – IIIIFNswithAd5vectorswithgoodbutdifferentialresultsdependingonthehostspeciesassayed [74, 75]. Treatment with IFN has proved, so far, to be the best biotherapeutic approach tested against FMDV. Recent data show that poly I:C stabilized with poly-L-lysine and carboxymeth‐ yl cellulose (poly ICLC), a TLR-3 and MDA5 agonist, is a potent stimulator of IFN and ISGs in swine and at an adequate dose is sufficient to induce complete protection against FMD [77]. A different study shows that the combined application of recombinant adenoviruses expressing IFN-α or siRNA and other antiviral agents such as ribavirin may enhance their inhibitory effect on FMDV [77]. Our own data, discussed above, support the use of the FMDV NCR RNAs, mimicking structural domains in the viral genome acting as potent type-I IFN inducers, as promising non-infectious and synthetic molecules in future antiviral and vaccine develop‐

Understanding how the innate immune system senses the infection of different viruses with a variety of genome structures and signals and the crosstalk between different PRRs will help to understand the complex regulation of immunity to infection. The increasing knowledge on

malaria vaccine are the TLR-9 agonists CpG oligodeoxynucleotides (ODNs).

against infectious diseases.

**5. Conclusions**

ments against FMDV and likely other viral infections.

The vaccine development field is evolving from traditional whole cell vaccines to more defined and safer subunit vaccines. In the idea of exploiting the innate responses in vaccine adjuvant design, a growing demand for the use of immunopotentiators in poorly immunogenic subunit vaccines is arising with the development of a new generation of vaccine adjuvants. New vaccine adjuvants are designed to improve the recruitment and activation of dendritic cells, then enabling transition from the innate to adaptive immune system for priming of B- and Tcell responses. Endogenous or therapeutically induced early type-I IFN responses may confer protection until adaptive immunity is activated to an extent that the pathogen can be elimi‐ nated. In that context, PRRs come into sight as targets of new vaccine adjuvants beside their role as sentinels in innate immunity.

Evidence is now emerging that many empiric vaccines and adjuvants inherently stimulate PRRs, like the yellow fever vaccine 17D, one of the most effective vaccines available, shown to activate multiple DC subsets through stimulation of several TLRs (including TLR-7, -8 and -9) [66], highlighting the potential of vaccination strategies that use combinations of different PRRs ligands to stimulate polyvalent immune responses.

The current vaccine adjuvants licensed for use in human vaccines are limited [67], but other PRR agonists in clinical stages of development are emerging as potential vaccine adjuvant candidates [68], such as the TLR3 and MDA5 agonist poly I:C, a promising mucosal adjuvant for intranasal H5N1 influenza vaccination [69]. Clearly, TLR agonists are the most clinically advanced in adjuvant development, but require additional considerations, as variation in TLR expression and influence of age in responsiveness or the risk of autoimmunity by induction of excessive inflammatory responses [68]. Other TLR agonists currently in clinical trials of a malaria vaccine are the TLR-9 agonists CpG oligodeoxynucleotides (ODNs).

In addition to recognition of viral ssRNA like that derived from HIV (Human immunodefi‐ ciency virus) or influrenza viruses, TLR7/8 can also be activated by certain synthetic agonists such as the imidazoquinoline derivatives imiquimod and resiquimod (R-848), as well as the guanine nucleotide analog loxoribine. TLR7/8 imidazoquinolines can activate appropriate immune cells and modulate cellular and humoral immunity and have been found to be excellent vaccine adjuvants [70]. Most TLR agonists induce antibody and Th1 responses, although some can induce Th2 and possibly Th17 responses. Knowledge of the response outcomes in terms of cytokines, chemokines and T-cell subtypes generated by activation of combination of PRRs would help in the design of vaccine formulations including the appro‐ priate combination of adjuvants in the future that can contribute to develop new vaccines against infectious diseases.

In the case of FMDV, many efforts are being invested on development of new vaccine formula‐ tions aimedtoimprove currentlyusedvaccines [71].AlthoughFMDvaccines are available since the early 1900s, the disease still affects millions of animals around the globe and remains the main sanitary barrier to the commerce of animals and animal products. Among the limitations of the currently available inactivated antigen vaccines are the short duration of immunity and the lack of serotype-cross protection. Administration of this vaccine or an experimental vaccine basedona replication-defectivehumanadenovirus (Ad5)vectorthatdelivers the FMDVcapsid and 3C proteins requires ~7 days to induce protective immunity in animals [72, 73]. New approaches aimed to shorten this susceptibility window and induce a more robust and longlasting adaptive immune response are being developed, such as expression of type-I,-II and – IIIIFNswithAd5vectorswithgoodbutdifferentialresultsdependingonthehostspeciesassayed [74, 75]. Treatment with IFN has proved, so far, to be the best biotherapeutic approach tested against FMDV. Recent data show that poly I:C stabilized with poly-L-lysine and carboxymeth‐ yl cellulose (poly ICLC), a TLR-3 and MDA5 agonist, is a potent stimulator of IFN and ISGs in swine and at an adequate dose is sufficient to induce complete protection against FMD [77]. A different study shows that the combined application of recombinant adenoviruses expressing IFN-α or siRNA and other antiviral agents such as ribavirin may enhance their inhibitory effect on FMDV [77]. Our own data, discussed above, support the use of the FMDV NCR RNAs, mimicking structural domains in the viral genome acting as potent type-I IFN inducers, as promising non-infectious and synthetic molecules in future antiviral and vaccine develop‐ ments against FMDV and likely other viral infections.
