Foot-and-mouth disease (FMD) is one of the most economically devastating agricultural illnesses globally. The etiologic agent of this disease is FMD virus (FMDV), a positive sense, single stranded RNA virus of the family Picornaviridae. FMDV is the most rapidly replicating virus known. Its incubation period is short, levels of viral shedding are exceptionally high—particularly in pigs—and the rate of transmission between affected animals is rapid (Grubman and Baxt, 2004). There are seven FMDV serotypes [A, O, C, Asia 1 and South African Territories (SAT) 1, 2, and 3] and many subtypes (reviewed in Domingo et al., 2002 and Grubman and Baxt, 2004). Clinical disease is characterized by fever, depression, anorexia, lameness, salivation, and development of vesicular lesions on the hooves, mouth, snout, and teats of cloven-hoofed animals. International trade of FMDV-susceptible animals or derived products from enzootic countries is prohibited by international policies (World Organisation for Animal Health, 2015). This has significant economic impacts for countries with ongoing outbreaks and is a significant biosecurity risk to FMD-free countries. The currently approved chemically inactivated whole virus FMD vaccine (Doel, 2003), and an adenoviral vectored subunit FMD vaccine (Ad5-FMDV) approved for emergency use in the United States (Grubman et al., 2010; Moraes et al., 2011), take at least 7 days to confer protection to vaccinated animals, during which time they are still susceptible to infection. There is a significant focus on developing biotherapeutics (Medina et al., 2020b; Kim et al., 2022) or antiviral agents (Li et al., 2019; Mei-Jiao et al., 2019; Naeem et al., 2021; Zhang et al., 2023) to prevent the occurrence of FMDV infections during the vulnerable period prior to vaccine-induced humoral immunity, thereby closing this window of susceptibility.

Interferons (IFNs) are among the first line of defense against viral pathogens. During a typical viral infection, common pathogen-associated molecular patterns (PAMPs) bind cellular pattern recognition receptors (PRRs) in infected host cells and trigger signaling cascades, leading to the upregulation and secretion of Types I and III IFNs (reviewed in Lazear et al., 2019, Mesev et al., 2019, and Carty et al., 2021). Interaction of these IFNs with their cellular receptors triggers signal transduction cascades that initiate the upregulation of a battery of IFN stimulated genes (ISGs) responsible for limiting viral replication through a variety of mechanisms (reviewed in Schoggins, 2019). As a result of these selective pressures, viruses develop adaptations to evade host immune responses. Certainly, FMDV is one of the most notorious immune-evading viruses (reviewed in Medina et al., 2018). It earned this reputation through its ability to: shut down cap-dependent translation; cleave important innate immune signaling proteins; induce multiple cellular membrane rearrangements; upregulate autophagy; and more (Devaney et al., 1988; Monaghan et al., 2004; Teterina et al., 2006; de Los Santos et al., 2007; Odonnell et al., 2011; Wang et al., 2011a; Gladue et al., 2012). Over 20 years of FMDV research has demonstrated how acutely sensitive the virus is to pre-exposure prophylactic treatment with Types I, II and III IFNs. In the early 2000s, Chinsangaram et al. demonstrated that treatment of bovine cells with recombinant IFNα or IFNβ—two type I IFNs—suppressed FMDV replication at the level of translation (Chinsangaram et al., 1999, 2001). Similar results were observed in porcine cells and, most importantly, in swine pre-treated with a replication defective human adenoviral 5 (Ad5)-vector expressing the porcine IFNα gene (Chinsangaram et al., 2003). Moraes et al. (2007) found that treating with an Ad5-vectored porcine IFNγ—the only member of the type II IFN family—in combination with the Ad5-vectored porcine IFNα, at 1 day prior to challenge with FMDV, induced enhanced antiviral activity and fully protected swine against disease. Similar results were observed in mice by using a single Ad5 vector co-expressing porcine IFNs-α and-γ (Kim et al., 2014). Recombinant porcine IFNδ8 and ovine IFNζ, both members of the type I IFN family, have also been shown to effectively upregulate ISGs and protect against two FMDV serotypes in cells (Usharani et al., 2017; Li et al., 2018). However, treatment of cattle with Ad5-vectored type I or type II IFN only had limited efficacy (Wu et al., 2003).

Type III IFNs, the latest family of IFNs described, have also been a subject of study for application as a prophylactic treatment against FMD. Currently there are three subtypes of bovine type III IFN also known as IFNλs or interleukin (IL) 28/29: IFNλ1 or IL29; IFNλ2 or IL28A; and IFNλ3 or IL28B (Kotenko et al., 2003; Sheppard et al., 2003). We have previously demonstrated that bovine and porcine IFNλ3 effectively block FMDV infection in vitro and in vivo in cattle and swine. One attractive characteristic of IFNλ is that—due to the tissue restriction of its receptor (a heterodimer of IL-28Rα and IL-10Rβ) to epithelial cells—there is low potential for off-target effects and inflammatory pathology related to the therapeutic itself (Diaz-San Segundo et al., 2011). In 2011, replication of FMDV was shown to be inhibited by treatment with an Ad5-vector system-secreted IFNλ3 in bovine EBK and MDBK cells (Diaz-San Segundo et al., 2011) or by treatment with recombinant porcine IFNλ1 in porcine IBRS-2 cells (Wang et al., 2011b). Moreover, systemic antiviral activity and induction of ISGs were detected in cattle treated with the Ad5-vectored bovine IFNλ3 (Ad5-boIFNλ3) (Diaz-San Segundo et al., 2011, 2016). Perez-Martin et al. (2012) demonstrated that inoculation of cattle with Ad5-boIFNλ3 significantly upregulated selected ISGs in the upper airways, protecting them against FMDV challenge. The same was demonstrated in swine using a similar Ad5porIFNλ3 (Perez-Martin et al., 2014). In 2016, the Ad5-boIFNλ3 platform was used in combination with an Ad5-FMDV O1Manisa vaccine in cattle, inducing a strong CD4 and CD8 IFNγ response within 2 days of treatment. Interestingly, the combination was 100% effective at preventing clinical disease in cattle challenged with FMDV at 3 days post-vaccination, despite a lack of detectable neutralizing antibody response at that time, suggesting Ad5-boIFNλ3 might have an adjuvant effect on cellular immunity (Diaz-San Segundo et al., 2016). The combination treatment also more robustly upregulated the expression of key immune receptors, including CD40L, CD80/CD86, and CCR7, which play crucial roles in promoting adaptive immune responses and memory T cell polarization.

Still, however, the therapeutic window for application of these antiviral treatments is limited and the production costs of the adenovirus vectors as a method of delivery are high. To overcome the relatively limited half-life of these IFN prophylactic treatments in vivo and reduce the cost-per-dose, synthesis of recombinant proteins with modifications such as polyethylene glycol conjugation (PEGylation), immunoglobulin Fc fragment or albumin fusion, among others, have been applied to some of the most promising IFN prophylactic therapies, including porcine IFNα (Miyakawa et al., 2011; Vallee et al., 2012; Lazear et al., 2019; Diaz-San Segundo et al., 2021). It is well known that PEGylation can be used to modulate the biophysical properties and/or biological activity of a biotherapeutic protein. Indeed, Diaz-San Segundo et al. (2021) used a pegylated porcine IFNα (PEGpoIFNα) as a successful strategy to prevent clinical FMD in swine challenged 5 days post treatment. However, high doses of protein were required, causing pleiotropic side effects (i.e., jaundice) in some cases, likely due to the ubiquitous distribution of the Type I IFN receptor.

Typically, PEG moieties are covalently linked to a target biotherapeutic via its naturally occurring amino acid residues, such as lysine or cysteine, or the N-terminus, which contain reactive moieties. However, the reactive sites of these naturally-occurring amino acids, which may seem suitable for PEGylation, may play a significant role in receptor binding. Thus, indiscriminate attachment of polymer chains such as PEG to such reactive sites on a biotherapeutic protein can lead to a significant reduction or even total loss of its biological activity (Clark et al., 1996). PEG derivatives can also undergo side reactions with residues other than those targeted for modification, which can create complex and poorly defined heterogeneous mixtures of PEG-derivatized biotherapeutics with reduced biological activity. One technology which promises to overcome many of these limitations is the incorporation of synthetic amino acids into proteins (see, e.g., Wang et al., 2001; Wang and Schultz, 2002; Chin et al., 2003; Tian et al., 2014). These and other studies have demonstrated that it is possible to site-specifically introduce into a protein a synthetic amino acid containing a chemical functional group that is not found in the 20 common amino acids. These synthetic amino acids can be used to react efficiently and selectively form stable covalent linkages with moieties, such as water-soluble polymer moieties, that are chosen for conjugation with the protein.

The objectives of the current study were: to determine whether a novel, site-specific PEGylated boIFNλ3 (PEGboIFNλ3) could extend its half-life in cattle; to determine if administration of the PEGboIFNλ3 alone or in combination with an FMD vaccine prior to FMDV exposure could provide improved efficacy prior to the onset of protective antibody titers; and to determine whether PEGboIFNλ3 could act as a vaccine adjuvant when administered in combination with an FMD vaccine. Our results demonstrate that this molecule exhibits extended biological activity and that it fully protects against FMDV challenge in cattle, both alone and in combination with an FMD vaccine, when administered 3–5 days prior to challenge. PEGboIFNλ3, therefore, shows promise as both a biotherapeutic and adjuvant capable of both effectively bridging the immunity gap following vaccination and boosting adaptive immunity against FMD in cattle.

Through site-specific PEGylation of boIFNλ3, we have demonstrated that we can: (1) achieve complete protection against FMD using recombinant PEGboIFNλ3 alone prophylactically; (2) extend the pre-exposure prophylactic window, effectively preventing clinical disease in FMDV-exposed cattle from 3 to 5 days pre-challenge; and (3) attain adjuvant effect of PEGboIFNλ3 when combined with an Ad5-FMDV vaccine, increasing the immunogenicity of the vaccine. These results highlight the exceptional versatility of PEGboIFNλ3 and its potential application during an emergency FMD outbreak response.

To the best of our knowledge, the present study is the first to report full clinical efficacy of an IFN therapy in cattle against FMD, within 3 and up to 5 days prior to challenge. Protection against clinical disease development among our PEGboIFNλ3 only treatment groups seems to be due largely to the extended antiviral activity afforded by site-specific PEGylation of boIFNλ3 via a stable oxime linkage to the synthetic amino acid pAF. Overall, similar clinical results were obtained in swine by treating them with large doses of PEGpoIFNα (Diaz-San Segundo et al., 2021), though this IFN only ever demonstrated partial protection when applied in cattle (Wu et al., 2003). Conversely, our previous Ad5-vectored boIFNλ3 study found that serum antiviral activity could only be detected until 2, but not 3 dpt (which was the day of challenge), consistent with the reduced protection observed among those animals administered the Ad5-boIFNλ3 treatment without concurrent FMD vaccine administration (Diaz-San Segundo et al., 2016). In the present study, we observed detectable antiviral activity in PEGboIFNλ3-treated cattle out to 4 days post-administration. Interestingly, we observed generation of serum neutralizing antibody levels in our PEGboIFNλ3 only treated animals comparable to those administered the combination therapy, indicating that this therapy does not prevent viral replication to a degree that would stop antibody formation (i.e., sterile protection). However, the induction of IFNγ+ T cells among PEGboIFNλ3 only treated cattle was suppressed in comparison to cattle given combination therapy. This data suggests that treatment with IFNλ3 therapy alone suppresses viral replication enough to shunt formation of a T cell response, while inducing an antibody response. However, further testing with greater sample sizes would be needed to support this hypothesis.

Previous studies from our lab have demonstrated tissue-specific upregulation of a variety of ISGs following treatment with an Ad5-boIFNλ3, particularly in the nasopharynx and palatine tonsil, and to a lesser extent in circulating PBMCs (Diaz-San Segundo et al., 2011). Given the kinetics of adenoviral vector clearance and the limited temporal range of translation of the boIFNλ3 gene within, the systemic antiviral activity afforded by this therapy was short-lived and animals that received only Ad5-boIFNλ3 all became clinically sick when challenged with FMDV at 3 dpt (Diaz-San Segundo et al., 2016). In the current study, while serum antiviral activity was reduced to near the limit of detection by 0 dpc in groups that were administered PEGboIFNλ3 at −5 dpc, a variety of ISGs were highly upregulated in circulating PBMCs in a sustained manner in all PEGboIFNλ3-treated groups. Among the most highly upregulated genes is ISG15, a potent antiviral (Perng and Lenschow, 2018). ISG15, a ubiquitin-like protein that serves a dual role in innate immunity, acts as both an intracellular protein modifier and an extracellular signaling molecule that boosts IFNγ secretion and has been reported to induce NK cell proliferation (D'Cunha et al., 1996), subsequently boosting the CD8+ CTL response (Iglesias-Guimarais et al., 2020), as we observe in our study in those cattle that were given combination therapies. Additionally, ISG15 induces DC cell maturation (Padovan et al., 2012), which may explain the upregulation of IFNγ in the assumed DC population CD3-CD8 + CD335-observed in this study. In a typical WT FMDV infection, Leader protease (L^pro) inhibits several antiviral pathways in the host cell through cleavage of a variety of targets, including those modified by ISG15. However, our research group has previously demonstrated that overexpression of ISG15 in porcine cells can reduce WT FMDV replication in vitro (Medina et al., 2020a). This supports the idea that administration of PEGboIFNλ3 inhibits FMDV infection not only through direct antiviral mechanisms, but also by overwhelming the immune evasion strategies that FMDV employs, such as by upregulating ISG15. However, since we have only assessed expression at the transcript level, further studies would be needed to confirm protein levels and enzymatic activity. Other ISGs significantly upregulated by administration of PEGboIFNλ3 include RIG-I and MDA5, two members of the RIG-I-like receptor family of cytosolic RNA helicases that work by binding viral dsRNA. While MDA5 has been demonstrated to bind FMDV RNA, RIG-I has not, though this work was performed in porcine cells (Husser et al., 2011). PKR, Mx-1, and OAS1 were also shown to be upregulated in cattle that received the PEGboIFNλ3 treatment, consistent with past studies in this laboratory. These three genes are understood to play a role in the antiviral response against FMDV (de Los Santos et al., 2006), and while it has not been experimentally established that PKR interacts with FMDV RNA, depletion of PKR by siRNA or gene KO in tissue culture results in significantly higher virus yields (Chinsangaram et al., 2001; de Los Santos et al., 2006). Also, upregulated among PEGboIFNλ3-treated cattle were chemokines CXCL10 and CCL20, which have been shown to play a role in DC maturation, along with chemotaxis of DC and effector/memory T/B cells. Their expression in the context of FMDV vaccination and biotherapeutics is associated with protection against challenge (Diaz-San Segundo et al., 2010) and provides evidence of the adjuvanting effect of PEGboIFNλ3 when administered in conjunction with an FMD vaccine. Our results showed mild upregulation of several of the above-mentioned genes in the control group animals 1 or 2 days after inoculation with Ad5-Blue. Although this is somewhat surprising, it could be associated with the stress the animals were going through during manipulation for sample collection (Dhabhar, 2014), though more testing would be needed to confirm this hypothesis. However, importantly, the level of upregulation in the IFN-treated animals is consistently higher than the animals in the control group. Furthermore, the concerted and sustained systemic expression of these ISGs following treatment is consistent with the observed blockade of local and systemic viral replication among PEGboIFNλ3-treated cattle.

Previous literature demonstrates that FMD protective immunity is largely conferred by neutralizing antibodies and that this can occur in a T cell-dependent or-independent manner, depending upon whether the antigens are nonstructural or capsid-associated, respectively (Juleff et al., 2009; Carr et al., 2013). During the early post-vaccination period before neutralizing antibody titers are detected, strong innate immune activation and chemotaxis (Rigden et al., 2003), along with local antibody production may mediate immunity (Pega et al., 2013). This may serve as a partial explanation of immunity in the present study, even among the Adt-O1M treatment group, in light of the lack of antibodies and only low levels of IFNγ+ T cells. At the time of challenge, none of the cattle in the current study from any treatment group had detectable levels of circulating neutralizing antibodies, consistent with our 2016 study utilizing the Ad5-boIFNλ3. However, in that study, animals treated with both the vaccine and the Ad5-boIFNλ3 displayed an elevated percentage of IFNγ+ CD8+ and CD4+ T cells on the day of challenge. Moreover, the percentage of IFNγ+ CD8+ cells among animals administered only FMD Adt-O1M did not reach comparable levels to the combination treatment group until 5 dpc, indicating that the Ad5-boIFNλ3 may have acted as an adjuvant for the FMD Adt-O1M vaccine. In the current study, while robust T cell and neutralizing antibody responses developed in all cattle administered both PEGboIFNλ3 and Adt-O1M vaccine, this largely did not occur until after challenge. Importantly, animals receiving the combination treatment, Adt-O1M + PEGboIFNλ3, at either −3 or −5 dpc, developed significant levels of IFNγ+ T cells at 3 dpc. The group receiving vaccine alone demonstrated modest elevations in the levels of IFNγ+ T cells, but did not show significant upregulation in either of these cell populations until 14 dpc (CD4+). In general, the induction of IFNγ following restimulation was variable and even negative on certain days, despite small positive populations of these single-positive T cells among unstimulated cells (data not shown). This may be related to immune checkpoint pathways in cattle immunology. While there is a lack of research into T cell checkpoint controls in the context of FMDV, it has been established that cattle are capable of experiencing T cell dysregulation and exhaustion in the context of chronic or persistent infection (as reviewed in Konnai et al., 2017), as FMD often becomes in cattle. On a shorter time scale, checkpoint cell surface markers such as CTLA-4 (CD152) are shuttled to the immune synapse in pre-formed vesicles at a rate proportional to the strength of TCR stimulation and this increased cell surface presentation could lead to a dampening of IFNγ expression (Sansom et al., 2003; Watari et al., 2019). However, this deserves further exploration and clearly was not an impediment to the establishment of protective immunity in the current study.

Attending the T cell IFNγ response observed among cattle receiving the combination therapy in this study is an upregulation in several genes involved in adaptive immunity, most notably CD40L and CD80, though we found that expression even within groups was highly variable. The cell surface receptor CD40L is a costimulatory marker principally expressed on CD4+ T cells, which binds CD40 on DCs and B cells. The resulting signal transduction cascade increases survival and proliferation responses in both T and B cells, resulting in increased secretion of immunoglobulins from B cells (Hirano et al., 1997; Estes et al., 1998). Though susceptible to only an abortive infection, DCs experimentally infected with FMDV have been shown to downregulate CD40 expression, failing to stimulate T cell proliferation and leading to a dysfunctional T cell response early in FMDV infection (Ostrowski et al., 2005). While CD40 expression was not assessed in the current study, both the 2016 Ad5-boIFNλ3 study (Diaz-San Segundo et al., 2016) and the current study demonstrate that CD40L is significantly upregulated in a synergistic manner by the coadministration of PEGboIFNλ3 and Adt-O1M vaccine, boosting the T cell response. CD80 is a costimulatory molecule that is present on B cells and provides survival and activation signals to T cells (when bound to their CD28 receptor) and monocytes in a coregulatory partnership with CD86 (Dilioglou et al., 2003; Watari et al., 2019). Curiously, we did not see any upregulation of adaptive immunity genes at any of the time points tested among our Adt-O1M vaccine only treatment group, though cattle in this group displayed robust neutralizing antibody and T cell responses to the vaccine by 7 dpc. This lack of upregulation of important adaptive immunity genes in the Adt-O1M group provides further evidence of the adjuvanting capabilities of PEGboIFNλ3.

In conclusion, the current study is the first to demonstrate full protection of cattle against FMD conferred by administration of a recombinant, site-specific PEGylated bovine IFNλ3, and provides compelling rationale for applying this novel biotherapeutic in concert with FMD vaccines, as both an adjuvant as well as a means of bridging the gap in immunity during the first 3–7 days following vaccination.

The original contributions presented in the study are publicly available. This data can be found here: www.ncbi.nlm.nih.gov, accession number: GSE262192.

Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used. The animal study was approved by Institutional Animal Care and Use Committee (IACUC) of the Plum Island Animal Disease Center (USDA/APHIS/AC Certificate number: 21-F-0001; Protocol 244.01-19-R). The study was conducted in accordance with the local legislation and institutional requirements.

SaA: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review & editing. CS: Investigation, Resources, Writing – review & editing. MR-C: Investigation, Resources, Writing – review & editing. AM: Investigation, Writing – review & editing. SoA: Investigation, Writing – review & editing. PA: Investigation, Writing – review & editing. PC: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Writing – review & editing. LS: Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – review & editing. JN: Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – review & editing. NK: Writing – review & editing, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization. GM: Conceptualization, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – review & editing. TS: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Visualization, Writing – review & editing. FD-S: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review & editing.