Inoculation of swine with foot-and-mouth disease SAP-mutant virus induces early protection against disease.

Foot-and-mouth disease virus (FMDV) leader proteinase (L(pro)) cleaves itself from the viral polyprotein and cleaves the translation initiation factor eIF4G. As a result, host cell translation is inhibited, affecting the host innate immune response. We have demonstrated that L(pro) is also associated with degradation of nuclear factor κB (NF-κB), a process that requires L(pro) nuclear localization. Additionally, we reported that disruption of a conserved protein domain within the L(pro) coding sequence, SAP mutation, prevented L(pro) nuclear retention and degradation of NF-κB, resulting in in vitro attenuation. Here we report that inoculation of swine with this SAP-mutant virus does not cause clinical signs of disease, viremia, or virus shedding even when inoculated at doses 100-fold higher than those required to cause disease with wild-type (WT) virus. Remarkably, SAP-mutant virus-inoculated animals developed a strong neutralizing antibody response and were completely protected against challenge with WT FMDV as early as 2 days postinoculation and for at least 21 days postinoculation. Early protection correlated with a distinct pattern in the serum levels of proinflammatory cytokines in comparison to the levels detected in animals inoculated with WT FMDV that developed disease. In addition, animals inoculated with the FMDV SAP mutant displayed a memory T cell response that resembled infection with WT virus. Our results suggest that L(pro) plays a pivotal role in modulating several pathways of the immune response. Furthermore, manipulation of the L(pro) coding region may serve as a viable strategy to derive live attenuated strains with potential for development as effective vaccines against foot-and-mouth disease.

Deoptimization of FMDV P1 Region Results in Robust Serotype-Independent Viral Attenuation.

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is a highly contagious disease of cloven-hoofed livestock that can have severe economic impacts. Control and prevention strategies, including the development of improved vaccines, are urgently needed to effectively control FMD outbreaks in endemic settings. Previously, we employed two distinct strategies (codon pair bias deoptimization (CPD) and codon bias deoptimization (CD)) to deoptimize various regions of the FMDV serotype A subtype A12 genome, which resulted in the development of an attenuated virus in vitro and in vivo, inducing varying levels of humoral responses. In the current study, we examined the versatility of the system by using CPD applied to the P1 capsid coding region of FMDV serotype A subtype, A24, and another serotype, Asia1. Viruses carrying recoded P1 (A24-P1Deopt or Asia1-P1Deopt) exhibited different degrees of attenuation (i.e., delayed viral growth kinetics and replication) in cultured cells. Studies in vivo using a mouse model of FMD demonstrated that inoculation with the A24-P1Deopt and Asia1-P1Deopt strains elicited a strong humoral immune response capable of offering protection against challenge with homologous wildtype (WT) viruses. However, different results were obtained in pigs. While clear attenuation was detected for both the A24-P1Deopt and Asia1-P1Deopt strains, only a limited induction of adaptive immunity and protection against challenge was detected, depending on the inoculated dose and serotype deoptimized. Our work demonstrates that while CPD of the P1 coding region attenuates viral strains of multiple FMDV serotypes/subtypes, a thorough assessment of virulence and induction of adaptive immunity in the natural host is required in each case in order to finely adjust the degree of deoptimization required for attenuation without affecting the induction of protective adaptive immune responses.

A conserved domain in the leader proteinase of foot-and-mouth disease virus is required for proper subcellular localization and function.

The leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) is involved in antagonizing the innate immune response by blocking the expression of interferon (IFN) and by reducing the immediate-early induction of IFN-beta mRNA and IFN-stimulated genes. In addition to its role in shutting off cap-dependent host mRNA translation, L(pro) is associated with the degradation of the p65/RelA subunit of nuclear factor kappaB (NF-kappaB). Bioinformatics analysis suggests that L(pro) contains a SAP (for SAF-A/B, Acinus, and PIAS) domain, a protein structure associated in some cases with the nuclear retention of molecules involved in transcriptional control. We have introduced a single or a double mutation in conserved amino acid residues contained within this domain of L(pro). Although three stable mutant viruses were obtained, only the double mutant displayed an attenuated phenotype in cell culture. Indirect immunofluorescence analysis showed that L(pro) subcellular distribution is altered in cells infected with the double mutant virus. Interestingly, nuclear p65/RelA staining disappeared from wild-type (WT) FMDV-infected cells but not from double mutant virus-infected cells. Consistent with these results, NF-kappaB-dependent transcription was not inhibited in cells infected with double mutant virus in contrast to cells infected with WT virus. However, degradation of the translation initiation factor eIF-4G was very similar for both the WT and the double mutant viruses. Since L(pro) catalytic activity was demonstrated to be a requirement for p65/RelA degradation, our results indicate that mutation of the SAP domain reveals a novel separation-of-function activity for FMDV L(pro).

The Different Tactics of Foot-and-Mouth Disease Virus to Evade Innate Immunity.

Like all pathogens, foot-and-mouth disease virus (FMDV) is recognized by the immune system inducing a heightened immune response mainly mediated by type I and type III IFNs. To overcome the strong antiviral response induced by these cytokines, FMDV has evolved many strategies exploiting each region of its small RNA genome. These include: (a) inhibition of IFN induction at the transcriptional and translational level, (b) inhibition of protein trafficking; (c) blockage of specific post-translational modifications in proteins that regulate innate immune signaling; (d) modulation of autophagy; (e) inhibition of stress granule formation; and (f) in vivo modulation of immune cell function. Here, we summarize and discuss FMDV virulence factors and the host immune footprint that characterize infection in cell culture and in the natural hosts.

Novel 6xHis tagged foot-and-mouth disease virus vaccine bound to nanolipoprotein adjuvant via metal ions provides antigenic distinction and effective protective immunity.

Here, we engineered two FMD viruses with histidine residues inserted into or fused to the FMDV capsid. Both 6xHis viruses exhibited growth kinetics, plaque morphologies and antigenic characteristics similar to wild-type virus. The 6xHis tag allowed one-step purification of the mutant virions by Co(2+) affinity columns. Electron microscopy and biochemical assays showed that the 6xHis FMDVs readily assembled into antigen: adjuvant complexes in solution, by conjugating with Ni(2+)-chelated nanolipoprotein and monophosphoryl lipid A adjuvant (MPLA:NiNLP). Animals Immunized with the inactivated 6xHis-FMDV:MPLA:NiNLP vaccine acquired enhanced protective immunity against FMDV challenge compared to virions alone. Induction of anti-6xHis and anti-FMDV neutralizing antibodies in the immunized animals could be exploited in the differentiation of vaccinated from infected animals needed for the improvement of FMD control measures. The novel marker vaccine/nanolipid technology described here has broad applications for the development of distinctive and effective immune responses to other pathogens of importance.

Subclinical bovine spongiform encephalopathy infection in transgenic mice expressing porcine prion protein.

The bovine-porcine species barrier to bovine spongiform encephalopathy (BSE) infection was explored by generating transgenic mouse lines expressing the porcine prion protein (PrP) gene. All of the porcine transgenic (poTg) mice showed clinical signs of BSE after intracerebral inoculation with a high-titer BSE inoculum. The protease-resistant PrP (PrP(res)) was detected in 14% (3 of 22) of the BSE-infected poTg mice by immunohistochemical or immunoblot analysis. Despite being able to infect 42% (5 of 12) of control mice, a low-dose BSE inoculum failed to penetrate the species barrier in our poTg mouse model. The findings of these infectivity studies suggest that there is a strong species barrier between cows and pigs. However, after second-passage infection of poTg mice using brain homogenates of BSE-inoculated mice scoring negative for the incoming prion protein as inoculum, it was possible to detect the presence of the infectious agent. Thus, porcine-adapted BSE inocula were efficient at infecting poTg mice, giving rise to an incubation period substantially reduced from 300 to 177 d after inoculation and to the presence of PrP(res) in 100% (21 of 21) of the mice. We were therefore able to conclude that initial exposure to the bovine prion may lead to subclinical infection such that brain homogenates from poTg mice classified as uninfected on the basis of the absence of PrP(res) are infectious when used to reinoculate poTg mice. Collectively, our findings suggest that these poTg mice could be used as a sensitive bioassay model for prion detection in pigs.

Porcine type I interferon rapidly protects swine against challenge with multiple serotypes of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. Current inactivated vaccines require approximately 7 days to induce protection, but before this time vaccinated animals remain susceptible to disease. Previously, we demonstrated that intramuscular (IM) inoculation of a replication-defective human adenovirus type 5 (Ad5) vector containing a porcine interferon α gene (pIFNα) can protect swine challenged 1 day later by intradermal (ID) injection with FMDV A24 Cruzeiro from both clinical disease and virus replication. To extend these studies to other FMDV serotypes, we demonstrated the effectiveness of Ad5-pIFNα against ID challenge with O1 Manisa and Asia-1 and against A24 Cruzeiro in a direct contact challenge model. We also showed that an Ad5 vector containing the pIFNß gene can protect swine against ID challenge with A24 Cruzeiro. Further, IM inoculation of a 10-fold lower dose of Ad5-pIFNα at 4 sites in the neck compared with 1 site in the hind limb can protect swine against ID challenge. These studies demonstrate the ability of Ad5-delivered type I IFN to rapidly protect swine against several FMDV serotypes and suggest that various modifications of this approach may enable this strategy to be successfully used in other FMD susceptible species.

Increased efficacy of an adenovirus-vectored foot-and-mouth disease capsid subunit vaccine expressing nonstructural protein 2B is associated with a specific T cell response.

We previously demonstrated that an adenovirus-based foot-and-mouth disease virus (FMDV) serotype A24 capsid subunit vaccine, Ad5-A24, expressed under the control of a cytomegalovirus promoter (CMV) can protect swine and bovines against homologous challenge, but in a similar approach using swine vaccinated with an Ad5-vectored FMDV O1 Campos vaccine, Ad5-O1C, the animals were only partially protected when challenged at 21 days post-vaccination (dpv). Recently, we demonstrated that inclusion of the complete coding region of nonstructural protein 2B in the Ad5-A24 vector resulted in improved immune responses in pigs. We also found that inclusion of a modified CMV promoter (pCI), Ad5-CI-A24-2B, enhanced the efficacy of the vector. To address the limited immunogenicity of Ad5-O1C, we have produced a new set of Ad5 vectors with the complete 2B coding region under the control of either the original or the modified version of the CMV promoter, Ad5-O1C-2B, or Ad5-CI-O1C-2B, respectively. To evaluate the potency and efficacy of the new vectors we performed 2 sets of experiments in cattle. In the first experiment we compared the original vector with vectors containing the pCI promoter and partial or full-length 2B. All groups were challenged, intradermally in the tongue, at 21 dpv with FMDV O1C. We found that in all vaccinated groups 2 of 4 animals were protected from clinical disease. In the second experiment we directly compared the efficacy of vectors with a partial or full-length 2B under the control of the original CMV promoter. While all animals in the control group developed clinical disease, 2 of 4 animals in the group receiving Ad5-O1C vaccine and 3 of 4 animals in the group receiving Ad5-O1C-2B vaccine were completely protected after challenge. We also observed a 100-fold reduction of virus shedding in Ad5-O1C vaccinated animals and the group receiving Ad5-O1C-2B had an additional 10-fold reduction compared with the Ad5-O1C vaccinated group. There was no difference in the level of neutralizing antibodies in the vaccinated groups. However, we detected a significant antigen specific-CD4(+) and CD8(+) T cell response as early as 1 day post-challenge (dpc) in both Ad5-O1C and Ad5-O1C-2B groups. Interestingly, the group receiving Ad5-O1C-2B had a statistically significant higher antigen specific-CD4(+) and CD8(+) T cell response at 5 dpc and 3 and 5 dpc, respectively, as compared to the Ad5-O1C inoculated group. These results indicate that inclusion of the complete 2B coding region improves the efficacy of Ad5 vaccines against FMDV serotype O and induces specific-CD4(+) and CD8(+) T cell responses that correlate with protection.

Delivery of a foot-and-mouth disease virus empty capsid subunit antigen with nonstructural protein 2B improves protection of swine.

To develop a more efficacious human adenovirus (Ad5)-vectored foot-and-mouth disease virus (FMDV) subunit vaccine (Ad5-A24) we have included coding regions for FMDV nonstructural proteins 2B and 2C. These proteins are involved in membrane re-arrangements resulting in the proliferation of cytoplasmic vesicles which serve as the sites of virus replication. Cells infected with a vector containing full-length 2B (Ad5-CI-A24-2B) had a significant increase in the number of cytoplasmic vesicles as compared to cells infected with the original vector or a vector containing full-length 2BC. Swine inoculated with Ad5-CI-A24-2B developed an enhanced FMDV-specific neutralizing antibody response as compared to animals inoculated with the original vector and showed no clinical signs of disease after challenge. In a second experiment animals vaccinated with Ad5-CI-A24-2B were not fully protected but had a more rapid and robust humoral response and two out of three pigs had delayed and less severe disease than animals in the other vaccinated groups. These results suggest that incorporation of the complete coding region of 2B into the vaccine enhances its potency and protective efficacy.