A Vaccine Based on the A/ASIA/G-VII Lineage of Foot-and-Mouth Disease Virus Offers Low Levels of Protection against Circulating Viruses from the A/ASIA/Iran-05 lineage.

The recent emergence and circulation of the A/ASIA/G-VII (A/G-VII) lineage of foot-and-mouth disease virus (FMDV) in the Middle East has resulted in the development of homologous vaccines to ensure susceptible animals are sufficiently protected against clinical disease. However, a second serotype A lineage called A/ASIA/Iran-05 (A/IRN/05) continues to circulate in the region and it is therefore imperative to ensure vaccine strains used will protect against both lineages. In addition, for FMDV vaccine banks that usually hold a limited number of strains, it is necessary to include strains with a broad antigenic coverage. To assess the cross protective ability of an A/G-VII emergency vaccine (formulated at 43 (95% CI 8-230) PD50/dose as determined during homologous challenge), we performed a heterologous potency test according to the European Pharmacopoeia design using a field isolate from the A/IRN/05 lineage as the challenge virus. The estimated heterologous potency in this study was 2.0 (95% CI 0.4-6.0) PD50/dose, which is below the minimum potency recommended by the World Organisation for Animal Health (OIE). Furthermore, the cross-reactive antibody titres against the heterologous challenge virus were poor (≤log10 0.9), even in those cattle that had received the full dose of vaccine. The geometric mean r1-value was 0.2 (95% CI 0.03-0.8), similar to the potency ratio of 0.04 (95% CI 0.004-0.3). Vaccination decreased viraemia and virus excretion compared to the unvaccinated controls. Our results indicate that this A/G-VII vaccine does not provide sufficient protection against viruses belonging to the A/IRN/05 lineage and therefore the A/G-VII vaccine strain cannot replace the A/IRN/05 vaccine strain but could be considered an additional strain for use in vaccines and antigen banks.

Phage T7 as a Potential Platform for Vaccine Development.

Bacteriophages have been explored for their uses in vaccine development, due to the ease of propagation while displaying epitopes in high density. Bacteriophage T7 has been demonstrated to be useful in the production of potential vaccine candidates for various diseases, including influenza A, foot-and mouth disease (FMD), and cancers. In this chapter, we described the use of phage T7 to display potential foot-and-mouth disease virus (FMDV) epitope, from cloning to expression, purification, and immunization in a mouse model.

Mooring Stone-Like Arg114 Pulls Diverse Bulged Peptides: First Insight into African Swine Fever Virus-Derived T Cell Epitopes Presented by Swine Major Histocompatibility Complex Class I.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), which is a devastating pig disease threatening the global pork industry. However, currently, no commercial vaccines are available. During the pig immune response, major histocompatibility complex class I (MHC-I) molecules select viral peptide epitopes and present them to host cytotoxic T lymphocytes, thereby playing critical roles in eliminating viral infections. Here, we screened peptides derived from ASFV and determined the molecular basis of ASFV-derived peptides presented by the swine leukocyte antigen 1*0101 (SLA-1*0101). We found that peptide binding in SLA-1*0101 differs from the traditional mammalian binding patterns. Unlike the typical B and F pockets used by the common MHC-I molecule, SLA-1*0101 uses the D and F pockets as major peptide anchor pockets. Furthermore, the conformationally stable Arg114 residue located in the peptide-binding groove (PBG) was highly selective for the peptides. Arg114 draws negatively charged residues at positions P5 to P7 of the peptides, which led to multiple bulged conformations of different peptides binding to SLA-1*0101 and creating diversity for T cell receptor (TCR) docking. Thus, the solid Arg114 residue acts as a "mooring stone" and pulls the peptides into the PBG of SLA-1*0101. Notably, the T cell recognition and activation of p72-derived peptides were verified by SLA-1*0101 tetramer-based flow cytometry in peripheral blood mononuclear cells (PBMCs) of the donor pigs. These results refresh our understanding of MHC-I molecular anchor peptides and provide new insights into vaccine development for the prevention and control of ASF. IMPORTANCE The spread of African swine fever virus (ASFV) has caused enormous losses to the pork industry worldwide. Here, a series of ASFV-derived peptides were identified, which could bind to swine leukocyte antigen 1*0101 (SLA-1*0101), a prevalent SLA allele among Yorkshire pigs. The crystal structure of four ASFV-derived peptides and one foot-and-mouth disease virus (FMDV)-derived peptide complexed with SLA-1*0101 revealed an unusual peptide anchoring mode of SLA-1*0101 with D and F pockets as anchoring pockets. Negatively charged residues are preferred within the middle portion of SLA-1*0101-binding peptides. Notably, we determined an unexpected role of Arg114 of SLA-1*0101 as a "mooring stone" which pulls the peptide anchoring into the PBG in diverse "M"- or "n"-shaped conformation. Furthermore, T cells from donor pigs could activate through the recognition of ASFV-derived peptides. Our study sheds light on the uncommon presentation of ASFV peptides by swine MHC-I and benefits the development of ASF vaccines.

Interleukin-10-Mediated Lymphopenia Caused by Acute Infection with Foot-and-Mouth Disease Virus in Mice.

Foot-and-mouth disease (FMD) is characterized by a pronounced lymphopenia that is associated with immune suppression. However, the mechanisms leading to lymphopenia remain unclear. In this study, the number of total CD4+, CD8+ T cells, B cells, and NK cells in the peripheral blood were dramatically reduced in C57BL/6 mice infected with foot-and-mouth disease virus (FMDV) serotype O, and it was noted that mice with severe clinical symptoms had expressively lower lymphocyte counts than mice with mild or without clinical symptoms, indicating that lymphopenia was associated with disease severity. A further analysis revealed that lymphocyte apoptosis and trafficking occurred after FMDV infection. In addition, coinhibitory molecules were upregulated in the expression of CD4+ and CD8+ T cells from FMDV-infected mice, including CTLA-4, LAG-3, 2B4, and TIGIT. Interestingly, the elevated IL-10 in the serum was correlated with the appearance of lymphopenia during FMDV infection but not IL-6, IL-2, IL-17, IL-18, IL-1ß, TNF-α, IFN-α/ß, TGF-ß, and CXCL1. Knocking out IL-10 (IL-10-/-) mice or blocking IL-10/IL-10R signaling in vivo was able to prevent lymphopenia via downregulating apoptosis, trafficking, and the coinhibitory expression of lymphocytes in the peripheral blood, which contribute to enhance the survival of mice infected with FMDV. Our findings support that blocking IL-10/IL-10R signaling may represent a novel therapeutic approach for FMD.

Demonstration of Co-Infection and Trans-Encapsidation of Viral RNA In Vitro Using Epitope-Tagged Foot-and-Mouth Disease Viruses.

Foot-and-mouth disease, caused by foot-and-mouth disease virus (FMDV), is an economically devastating disease affecting several important livestock species. FMDV is antigenically diverse and exists as seven serotypes comprised of many strains which are poorly cross-neutralised by antibodies induced by infection or vaccination. Co-infection and recombination are important drivers of antigenic diversity, especially in regions where several serotypes co-circulate at high prevalence, and therefore experimental systems to study these events in vitro would be beneficial. Here we have utilised recombinant FMDVs containing an HA or a FLAG epitope tag within the VP1 capsid protein to investigate the products of co-infection in vitro. Co-infection with viruses from the same and from different serotypes was demonstrated by immunofluorescence microscopy and flow cytometry using anti-tag antibodies. FLAG-tagged VP1 and HA-tagged VP1 could be co-immunoprecipitated from co-infected cells, suggesting that newly synthesised capsids may contain VP1 proteins from both co-infecting viruses. Furthermore, we provide the first demonstration of trans-encapsidation of an FMDV genome into capsids comprised of proteins encoded by a co-infecting heterologous virus. This system provides a useful tool for investigating co-infection dynamics in vitro, particularly between closely related strains, and has the advantage that it does not depend upon the availability of strain-specific FMDV antibodies.

Structures of Foot-and-Mouth Disease Virus with Bovine Neutralizing Antibodies Reveal the Determinant of Intraserotype Cross-Neutralization.

Foot-and-mouth disease virus (FMDV) exhibits broad antigenic diversity with poor intraserotype cross-neutralizing activity. Studies of the determinant involved in this diversity are essential for the development of broadly protective vaccines. In this work, we isolated a bovine antibody, designated R55, that displays cross-reaction with both FMDV A/AF/72 (hereafter named FMDV-AAF) and FMDV A/WH/09 (hereafter named FMDV-AWH) but only has a neutralizing effect on FMDV-AWH. Near-atomic resolution structures of FMDV-AAF-R55 and FMDV-AWH-R55 show that R55 engages the capsids of both FMDV-AAF and FMDV-AWH near the icosahedral 3-fold axis and binds to the ßB and BC/HI-loops of VP2 and to the B-B knob of VP3. The common interaction residues are highly conserved, which is the major determinant for cross-reaction with both FMDV-AAF and FMDV-AWH. In addition, the cryo-EM structure of the FMDV-AWH-R55 complex also shows that R55 binds to VP3E70 located at the VP3 BC-loop in an adjacent pentamer, which enhances the acid and thermal stabilities of the viral capsid. This may prevent capsid dissociation and genome release into host cells, eventually leading to neutralization of the viral infection. In contrast, R55 binds only to the FMDV-AAF capsid within one pentamer due to the VP3E70G variation, which neither enhances capsid stability nor neutralizes FMDV-AAF infection. The VP3E70G mutation is the major determinant involved in the neutralizing differences between FMDV-AWH and FMDV-AAF. The crucial amino acid VP3E70 is a key component of the neutralizing epitopes, which may aid in the development of broadly protective vaccines. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious and economically devastating disease in cloven-hoofed animals, and neutralizing antibodies play critical roles in the defense against viral infections. Here, we isolated a bovine antibody (R55) using the single B cell antibody isolation technique. Enzyme-linked immunosorbent assays (ELISA) and virus neutralization tests (VNT) showed that R55 displays cross-reactions with both FMDV-AWH and FMDV-AAF but only has a neutralizing effect on FMDV-AWH. Cryo-EM structures, fluorescence-based thermal stability assays and acid stability assays showed that R55 engages the capsid of FMDV-AWH near the icosahedral 3-fold axis and informs an interpentamer epitope, which overstabilizes virions to hinder capsid dissociation to release the genome, eventually leading to neutralization of viral infection. The crucial amino acid VP3E70 forms a key component of neutralizing epitopes, and the determination of the VP3E70G mutation involved in the neutralizing differences between FMDV-AWH and FMDV-AAF could aid in the development of broadly protective vaccines.

Targeted FMD Vaccines for Eastern Africa: The AgResults Foot and Mouth Disease Vaccine Challenge Project

As one of the most infectious livestock diseases in the world, foot and mouth disease (FMD) presents a constant global threat to animal trade and national economies. FMD remains a severe constraint on development and poverty reduction throughout the developing world due to many reasons, including the cost of control measures, closure of access to valuable global FMD-free markets for livestock products, production losses through reduced milk yield, reduced live weight gain, and the inability of infected livestock to perform traction. FMD virus infects a variety of cloven-hoofed animals, including cattle, sheep, goats, swine, all wild ruminants, and suidae, with high morbidity in adult animals. High mortality can occur in young animals due to myocarditis. FMD is endemic in Africa, most of Asia, the Middle East, and parts of South America. The global clustering of FMD viruses has been divided into seven virus pools, where multiple serotypes occur but within which are topotypes that remain mostly confined to that pool. Three pools cover Europe, the Middle East, and Asia; three pools cover Africa; and one pool covers the Americas. The highly infectious nature of FMDV, the existence of numerous continually circulating serotypes and associated topotypes, the potential for wildlife reservoirs, and the frequent emergence of new strains that are poorly matched to existing vaccines all serve to compound the difficulties faced by the governments of endemic countries to effectively control and reduce the burden of the disease at the national and regional levels. This clustering of viruses suggests that if vaccination is to be a major tool for control, each pool could benefit from the use of tailored or more specific vaccines relevant to the topotypes present in that pool, rather than a continued reliance on the currently more widely available vaccines. It should also be noted that, currently, there are varying degrees of effort to identify improved vaccines in different regions. There are relatively few targeted for use in Africa, while the developed world's vaccine banks have a good stock of vaccines destined for emergency outbreak use in FMDV-free countries. The AgResults Foot and Mouth Disease (FMD) Vaccine Challenge Project (the "Project") is an eight-year, US $17.68 million prize competition that supports the development and uptake of high-quality quadrivalent FMD vaccines tailored to meet the needs of Eastern Africa (EA). The Project targets the following Pool Four countries: Burundi, Ethiopia, Kenya, Rwanda, Tanzania and Uganda. The Project is being run in two phases: a development phase, which will encourage the production of regionally relevant vaccines, and a cost-share phase, designed to help to reduce the price of these vaccines in the marketplace to the end users, which is hoped will encourage broader uptake. Manufacturers can submit quadrivalent FMD vaccines containing serotypes A, O, SAT1, and SAT2, which will be assessed as relevant for use in the region through a unique component of the Project requiring the screening of vaccines against the Eastern Africa Foot and Mouth Disease Virus Reference Antigen Panel assembled by the World Reference Laboratory for FMD (WRLFMD), at the Pirbright Institute, UK, in collaboration with the OIE/FAO FMD Reference Laboratory Network. To be eligible for the Project, sera from vaccinated cattle will be used to evaluate serological responses of FMD vaccines for their suitability for use in Eastern African countries. If they pass a determined cut-off threshold, they will be confirmed as relevant for use in the region and will be entered into the Project's cost-share phase.

Molecular Basis of Antigenic Drift in Serotype O Foot-and-Mouth Disease Viruses (2013-2018) from Southeast Asia.

Foot and mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals with serious economic consequences. FMD is endemic in Southeast Asia (SEA) and East Asia (EA) with the circulation of multiple serotypes, posing a threat to Australia and other FMD-free countries. Although vaccination is one of the most important control measures to prevent FMD outbreaks, the available vaccines may not be able to provide enough cross-protection against the FMD viruses (FMDVs) circulating in these countries due to the incursion of new lineages and sub-lineages as experienced in South Korea during 2010, a FMD-free country, when a new lineage of serotype O FMDV (Mya-98) spread to the country, resulting in devastating economic consequences. In this study, a total of 62 serotype O (2013-2018) viruses selected from SEA and EA countries were antigenically characterized by virus neutralization tests using three existing (O/HKN/6/83, O/IND/R2/75 and O/PanAsia-2) and one putative (O/MYA/2009) vaccine strains and full capsid sequencing. The Capsid sequence analysis revealed three topotypes, Cathay, SEA and Middle East-South Asia (ME-SA) of FMDVs circulating in the region. The vaccines used in this study showed a good match with the SEA and ME-SA viruses. However, none of the recently circulating Cathay topotype viruses were protected by any of the vaccine strains, including the existing Cathay topotype vaccine (O/HKN/6/83), indicating an antigenic drift and, also the urgency to monitor this topotype in the region and develop a new vaccine strain if necessary, although currently the presence of this topotype is mainly restricted to China, Hong Kong, Taiwan and Vietnam. Further, the capsid sequences of these viruses were analyzed that identified several capsid amino acid substitutions involving neutralizing antigenic sites 1, 2 and 5, which either individually or together could underpin the observed antigenic drift.

Immunogenicity of Foot-and-Mouth Disease Virus Dendrimer Peptides: Need for a T-Cell Epitope and Ability to Elicit Heterotypic Responses.

An approach based on a dendrimer display of B- and T-cell epitopes relevant for antibody induction has been shown to be effective as a foot-and-mouth disease (FMD) vaccine. B2T dendrimers combining two copies of the major FMD virus (FMDV) type O B-cell epitope (capsid proteinVP1 (140-158)) covalently linked to a heterotypic T-cell epitope from non-structural protein 3A (21-35), henceforth B2T-3A, has previously been shown to elicit high neutralizing antibody (nAb) titers and IFN-γ-producing cells in both mice and pigs. Here, we provide evidence that the B- and T-cell epitopes need to be tethered to a single molecular platform for successful T-cell help, leading to efficient nAb induction in mice. In addition, mice immunized with a non-covalent mixture of B2T-3A dendrimers containing the B-cell epitopes of FMDV types O and C induced similarly high nAb levels against both serotypes, opening the way for a multivalent vaccine platform against a variety of serologically different FMDVs. These findings are relevant for the design of vaccine strategies based on B- and T-cell epitope combinations.

Epidemiological study on foot-and-mouth disease in small ruminants: Sero-prevalence and risk factor assessment in Kenya.

Foot-and-mouth disease (FMD) is endemic in Kenya affecting cloven-hoofed ruminants. The epidemiology of the disease in small ruminants (SR) in Kenya is not documented. We carried out a cross-sectional study, the first in Kenya, to estimate the sero-prevalence of FMD in SR and the associated risk factors nationally. Selection of animals to be sampled used a multistage cluster sampling approach. Serum samples totaling 7564 were screened for FMD antibodies of non-structural-proteins using ID Screen® NSP Competition ELISA kit. To identify the risk factors, generalized linear mixed effects (GLMM) logistic regression analysis with county and villages as random effect variables was used. The country animal level sero-prevalence was 22.5% (95% CI: 22.3%-24.3%) while herd level sero-prevalence was 77.6% (95% CI: 73.9%-80.9%). The risk factor that was significantly positively associated with FMD sero-positivity in SR was multipurpose production type (OR = 1.307; p = 0.042). The risk factors that were significantly negatively associated with FMD sero-positivity were male sex (OR = 0.796; p = 0.007), young age (OR = 0.470; p = 0.010), and sedentary production zone (OR = 0.324; p<0.001). There were no statistically significant intra class correlations among the random effect variables but interactions between age and sex variables among the studied animals were statistically significant (p = 0.019). This study showed that there may be widespread undetected virus circulation in SR indicated by the near ubiquitous spatial distribution of significant FMD sero-positivity in the country. Strengthening of risk-based FMD surveillance in small ruminants is recommended. Adjustment of husbandry practices to control FMD in SR and in-contact species is suggested. Cross-transmission of FMD and more risk factors need to be researched.

Two Cross-Protective Antigen Sites on Foot-and-Mouth Disease Virus Serotype O Structurally Revealed by Broadly Neutralizing Antibodies from Cattle.

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that infects cloven-hoofed animals. Neutralizing antibodies play critical roles in antiviral infection. Although five known antigen sites that induce neutralizing antibodies have been defined, studies on cross-protective antigen sites are still scarce. We mapped two cross-protective antigen sites using 13 bovine-derived broadly neutralizing monoclonal antibodies (bnAbs) capable of neutralizing 4 lineages within 3 topotypes of FMDV serotype O. One antigen site was formed by a novel cluster of VP3-focused epitopes recognized by bnAb C4 and C4-like antibodies. The cryo-electron microscopy (cryo-EM) structure of the FMDV-OTi (O/Tibet/99)-C4 complex showed close contact with VP3 and a novel interprotomer antigen epitope around the icosahedral 3-fold axis of the FMDV particle, which is far beyond the known antigen site 4. The key determinants of the neutralizing function of C4 and C4-like antibodies on the capsid were ßB (T65), the B-C loop (T68), the E-F loop (E131 and K134), and the H-I loop (G196), revealing a novel antigen site on VP3. The other antigen site comprised two group epitopes on VP2 recognized by 9 bnAbs (B57, B73, B77, B82, F28, F145, F150, E46, and E54), which belong to the known antigen site 2 of FMDV serotype O. Notably, bnAb C4 potently promoted FMDV RNA release in response to damage to viral particles, suggesting that the targeted epitope contains a trigger mechanism for particle disassembly. This study revealed two cross-protective antigen sites that can elicit cross-reactive neutralizing antibodies in cattle and provided new structural information for the design of a broad-spectrum molecular vaccine against FMDV serotype O. IMPORTANCE FMDV is the causative agent of foot-and-mouth disease (FMD), which is one of the most contagious and economically devastating diseases of domestic animals. The antigenic structure of FMDV serotype O is rather complicated, especially for those sites that can elicit a cross-protective neutralizing antibody response. Monoclonal neutralization antibodies provide both crucial defense components against FMDV infection and valuable tools for fine analysis of the antigenic structure. In this study, we found a cluster of novel VP3-focused epitopes using 13 bnAbs against FMDV serotype O from natural host cattle, which revealed two cross-protective antigen sites on VP2 and VP3. Antibody C4 targeting this novel epitope potently promoted viral particle disassembly and RNA release before infection, which may indicate a vulnerable region of FMDV. This study reveals new structural information about cross-protective antigen sites of FMDV serotype O, providing valuable and strong support for future research on broad-spectrum vaccines against FMD.

In Vivo Sustained Release of Peptide Vaccine Mediated by Dendritic Mesoporous Silica Nanocarriers.

Mesoporous silica nanoparticles have drawn increasing attention as promising candidates in vaccine delivery. Previous studies evaluating silica-based vaccine delivery systems concentrated largely on macromolecular antigens, such as inactivated whole viruses. In this study, we synthesized dendritic mesoporous silica nanoparticles (DMSNs), and we evaluated their effectiveness as delivery platforms for peptide-based subunit vaccines. We encapsulated and tested in vivo an earlier reported foot-and-mouth disease virus (FMDV) peptide vaccine (B2T). The B2T@DMSNs formulation contained the peptide vaccine and the DMSNs without further need of other compounds neither adjuvants nor emulsions. We measured in vitro a sustained release up to 930 h. B2T@DMSNs-57 and B2T@DMSNs-156 released 23.7% (135 µg) and 22.8% (132 µg) of the total B2T. The formation of a corona of serum proteins around the DMSNs increased the B2T release up to 61% (348 µg/mg) and 80% (464 µg/mg) for B2T@DMSNs-57 and B2T@DMSNs-156. In vitro results point out to a longer sustained release, assisted by the formation of a protein corona around DMSNs, compared to the reference formulation (i.e., B2T emulsified in Montanide). We further confirmed in vivo immunogenicity of B2T@DMSNs in a particle size-dependent manner. Since B2T@DMSNs elicited specific immune responses in mice with high IgG production like the reference B2T@Montanide™, self-adjuvant properties of the DMSNs could be ascribed. Our results display DMSNs as efficacious nanocarriers for peptide-based vaccine administration.

Isolation and characterization of porcine monoclonal antibodies revealed two distinct serotype-independent epitopes on VP2 of foot-and-mouth disease virus.

Pigs are susceptible to foot-and-mouth disease virus (FMDV), and the humoral immune response plays an essential role in protection against FMDV infection. However, little information is available about FMDV-specific mAbs derived from single B cells of pigs. This study aimed to determine the antigenic features of FMDV that are recognized by antibodies from pigs. Therefore, a panel of pig-derived mAbs against FMDV were developed using fluorescence-based single B cell antibody technology. Western blotting revealed that three of the antibodies (1C6, P2-7E and P2-8G) recognized conserved antigen epitopes on capsid protein VP2, and exhibited broad reactivity against both FMDV serotypes A and O. An alanine-substitution scanning assay and sequence conservation analysis elucidated that these porcine mAbs recognized two conserved epitopes on VP2: a linear epitope (2KKTEETTLL10) in the N terminus and a conformational epitope involving residues K63, H65, L66, F67, D68 and L81 on two ß-sheets (B-sheet and C-sheet) that depended on the integrity of VP2. Random parings of heavy and light chains of the IgGs confirmed that the heavy chain is predominantly involved in binding to antigen. The light chain of porcine IgG contributes to the binding affinity toward an antigen and may function as a support platform for antibody stability. In summary, this study is the first to reveal the conserved antigenic profile of FMDV recognized by porcine B cells and provides a novel method for analysing the antibody response against FMDV in its natural hosts (i.e. pigs) at the clonal level.

Development and Validation of a Mucosal Antibody (IgA) Test to Identify Persistent Infection with Foot-and-Mouth Disease Virus.

It is well known that approximately 50% of cattle infected with foot-and-mouth disease (FMD) virus (FMDV) may become asymptomatic carrier (persistently infected) animals. Although transmission of FMDV from carrier cattle to naïve cattle has not been demonstrated experimentally, circumstantial evidence from field studies has linked FMDV-carrier cattle to cause subsequent outbreaks. Therefore, the asymptomatic carrier state complicates the control and eradication of FMD. Current serological diagnosis using tests for antibodies to the viral non-structural proteins (NSP-ELISA) are not sensitive enough to detect all carrier animals, if persistently infected after vaccination and do not distinguish between carriers and non-carriers. The specificity of the NSP ELISA may also be reduced after vaccination, in particular after multiple vaccination. FMDV-specific mucosal antibodies (IgA) are not produced in vaccinated cattle but are elevated transiently during the acute phase of infection and can be detected at a high level in cattle persistently infected with FMDV, irrespective of their vaccination status. Therefore, detection of IgA by ELISA may be considered a diagnostic alternative to RT-PCR for assessing FMDV persistent infection in ruminants in both vaccinated and unvaccinated infected populations. This study reports on the development and validation of a new mucosal IgA ELISA for the detection of carrier animals using nasal, saliva, and oro-pharyngeal fluid (OPF) samples. The diagnostic performance of the IgA ELISA using nasal samples from experimentally vaccinated and infected cattle demonstrated a high level of specificity (99%) and an improved level of sensitivity (76.5%). Furthermore, the detection of carrier animals reached 96.9% when parallel testing of samples was carried out using both the IgA-ELISA and NSP-ELISA.

Ribosomal Protein L13 Participates in Innate Immune Response Induced by Foot-and-Mouth Disease Virus.

In addition to ribosomal protein synthesis and protein translation, ribosomal proteins also participate in tumorigenesis and tumor progression, immune responses, and viral replication. Here, we show that ribosomal protein L13 (RPL13) participates in the antiviral immune response induced by foot-and-mouth disease virus (FMDV), inhibiting FMDV replication. The overexpression of RPL13 promoted the induction and activation of the promoters of the nuclear factor-κB (NF-κB) and interferon-ß (IFN-ß) genes, and the expression and protein secretion of the antiviral factor IFN-ß and proinflammatory cytokine interleukin-6 (IL-6). The knockdown of RPL13 had the opposite effects. We also found that the FMDV 3Cpro protease interacts with RPL13, and that its activity reduces the expression of RPL13, thus antagonizing the RPL13-mediated antiviral activity. This study extends our knowledge of the extraribosomal functions of ribosomal proteins and provides new scientific information on cellular antiviral defenses and virus-antagonizing mechanisms.

Development and Validation of Confirmatory Foot-and-Mouth Disease Virus Antibody ELISAs to Identify Infected Animals in Vaccinated Populations.

In foot-and-mouth disease (FMD)-endemic countries, vaccination is commonly used to control the disease, whilst in FMD-free countries, vaccination is considered as an option, in addition to culling the infected and in contact animals. FMD vaccines are mainly comprised of inactivated virions and stimulate protective antibodies to virus structural proteins. In contrast, infection with FMD virus leads to virus replication and additional antibody responses to viral nonstructural proteins (NSP). Therefore, antibodies against NSPs are used to differentiate infection in vaccinated animals (DIVA), in order to estimate the prevalence of infection or its absence. Another advantage of NSP antibody tests is that they detect FMD infection in the field, irrespective of the serotypes of virus in circulation. In cattle, the NSP tests that target the 3ABC polyprotein provides the highest sensitivity, detecting up to 90% of vaccinated animals that become carriers after exposure to infection, with a specificity of around 99%. Due to insufficient diagnostic sensitivity and specificity, detection of a low level of infection is difficult at the population level with a high degree of confidence. The low level of non-specific responses can be overcome by retesting samples scored positive using a second confirmatory test, which should have at least comparable sensitivity to the first test. In this study, six in-house tests were developed incorporating different NSP antigens, and validated using bovine sera from naïve animals, field cases and experimentally vaccinated and/or infected animals. In addition, two (short and long incubation) new commercial NSP tests based on 3ABC competitive blocking ELISAs (ID Screen® FMD NSP Competition, IDvet, France) were validated in this study. The two commercial ELISAs had very similar sensitivities and specificities that were not improved by lengthening the incubation period. Several of the new in-house tests had performance characteristics that were nearly as good as the commercial ELISAs. Finally, the in-house tests were evaluated for use as confirmatory tests following screening with the PrioCHECK® and ID Screen® FMDV NS commercial kits, to assess the diagnostic performance produced by a multiple testing strategy. The in-house tests could be used in series (to confirm) or in parallel (to augment) with the PrioCHECK® and IDvet® FMDV NS commercial kits, in order to improve either the specificity or sensitivity of the overall test system, although this comes at the cost of a reduction in the counterpart (sensitivity/specificity) parameter.

Antigenicity and Immunogenicity Analysis of the E. coli Expressed FMDV Structural Proteins; VP1, VP0, VP3 of the South African Territories Type 2 Virus.

An alternative vaccine design approach and diagnostic kits are highly required against the anticipated pandemicity caused by the South African Territories type 2 (SAT2) Foot and Mouth Disease Virus (FMDV). However, the distinct antigenicity and immunogenicity of VP1, VP0, and VP3 of FMDV serotype SAT2 are poorly understood. Similarly, the particular roles of the three structural proteins in novel vaccine design and development remain unexplained. We therefore constructed VP1, VP0, and VP3 encoding gene (SAT2:JX014256 strain) separately fused with His-SUMO (histidine-small ubiquitin-related modifier) inserted into pET-32a cassette to express the three recombinant proteins and separately evaluated their antigenicity and immunogenicity in mice. The fusion protein was successfully expressed and purified by the Ni-NTA resin chromatography. The level of serum antibody, spleen lymphocyte proliferation, and cytokines against the three distinct recombinant proteins were analyzed. Results showed that the anti-FMDV humoral response was triggered by these proteins, and the fusion proteins did enhance the splenocyte immune response in the separately immunized mice. We observed low variations among the three fusion proteins in terms of the antibody and cytokine production in mice. Hence, in this study, results demonstrated that the structural proteins of SAT2 FMDV could be used for the development of immunodiagnostic kits and subunit vaccine designs.

Immunogenic profiling and designing of a novel vaccine from capsid proteins of FMDV serotype Asia-1 through reverse vaccinology.

Foot-and-mouth disease is one of the devastating transboundary animal diseases causing heavy losses to the livestock industry. Different vaccines based on the inactivated FMD virus are used against this disease, but lack of immunological memory and the need for high biocontainment are the major drawbacks of these vaccines. A novel vaccine comprising recombinant antigenic regions is effective, as they lack viruses for production. Considering the fact, capsid proteins vp4, vp2, vp3, and vp1 with 3C protease of FMDV serotype Asia-1 were analyzed through reverse vaccinology approaches in this study. The sequence and structural analysis of the proteins is carried out through various bioinformatic tools and the sequence analysis has figured out the acidic nature and thermal stability of the proteins, likewise, the phylogenetic analysis helped us to trace the FMDV isolates, elucidating that selected proteins belong to the strain (Group VII), which is currently circulating in Pakistan. Next, the B-cell and MHC Class-I epitopes are identified from the antigenic proteins by immunoinformatic tools. The highly conserved, antigenic, and non-allergenic epitopes are used to design the vaccine. Accordingly, the codon adaptation and in silico cloning of the corresponding genes is performed. Thus, the bacterial expression vector could be used for efficient expression and large-scale production of the vaccine.

The rescue and selection of thermally stable type O vaccine candidate strains of foot-and-mouth disease virus.

Inactivated foot-and-mouth disease virus (FMDV) vaccines have been used widely to control foot-and-mouth disease (FMD). However, the virions (146S) of this virus are easily dissociated into pentamer subunits (12S), which limits the immune protective efficacy of inactivated vaccines when the temperature is higher than 30 °C. A cold-chain system can maintain the quality of the vaccines, but such systems are usually not reliable in limited-resource settings. Thus, it is imperative to improve the thermostability of vaccine strains to guarantee the quality of the vaccines. In this study, four recombinant FMDV strains containing single or multiple amino acid substitutions in the structural proteins were rescued using a previously constructed FMDV type O full-length infectious clone (pO/DY-VP1). We found that single or multiple amino acid substitutions in the structural proteins affected viral replication to different degrees. Furthermore, the heat and acid stability of the recombinant viruses was significantly increased when compared with the parental virus. Three thermally stable recombinant viruses (rHN/DY-VP1Y2098F, rHN/DY-VP1V2090A-S2093H, and rHN/DY-VP1V2090A-S2093H-Y2098F) were prepared as inactivated vaccines to immunize pigs. Blood samples were collected every week to prepare sera, and a virus neutralization test showed that the substitutions S2093H and Y2098F, separately or in combination, did not affect the immunogenicity of the virus, but the Y2098F mutation increased the thermostability significantly (p < 0.05). Therefore, the rHN/DY-VP1Y2098F mutant should be considered for use in future vaccines.

The detection of long-lasting memory foot-and-mouth disease (FMD) virus serotype O-specific CD4+ T cells from FMD-vaccinated cattle by bovine major histocompatibility complex class II tetramer.

Foot-and-mouth disease (FMD) is a highly contagious, economically devastating disease of cloven-hooved animals. The development of long-lasting effective FMD vaccines would greatly benefit the global FMD control programme. Deep analysis of adaptive immunity in cattle vaccinated against FMD is technically challenging due to the lack of species-specific tools. In this study, we aimed to identify CD4+ T-cell epitopes in the FMD virus (FMDV) capsid and to phenotype the CD4+ T cells that recognize them using bovine major histocompatibility complex (BoLA) class II tetramer. A BoLA class II tetramer based on the DRA/DRB3*020:02 allele and FMDV antigen-stimulated PBMCs from bovine vaccinates were used to successfully identify four epitopes in the FMDV capsid, three of which have not been previously reported; two epitopes were identified in the structural protein VP1, one in VP3 and one in VP4. Specificity of the three novel epitopes was confirmed by proliferation assay. All epitope-expanded T-cell populations produced IFN-γ in vitro, indicating a long-lasting Th1 cell phenotype after FMD vaccination. VP3-specific CD4+ T cells exhibited the highest frequency amongst the identified epitopes, comprising >0·004% of the CD4+ T-cell population. CD45RO+ CCR7+ defined central memory CD4+ T-cell subpopulations were present in higher frequency in FMDV-specific CD4+ T-cell populations from FMD-vaccinated cattle ex vivo. This indicates an important role in maintaining cell adaptive immunity after FMD vaccination. Notably, FMDV epitope-loaded tetramers detected the presence of FMDV-specific CD4+ T cells in bovine PBMC more than four years after vaccination. This work contributes to our understanding of vaccine efficacy.