Optimization of a foot-and-mouth disease virus Southern African Territories-specific solid-phase competitive ELISA for small ruminant serum samples.

We optimized and verified a single-spot solid-phase competitive ELISA (ss-SPCE) to detect antibodies against structural proteins of Southern African Territories (SAT) serotypes of foot-and-mouth disease virus (FMDV) in small ruminants. Sera from goats vaccinated and experimentally challenged with a SAT1 FMDV pool were tested in duplicate at 4 dilutions (1:10, 1:15, 1:22.5, 1:33.8) to optimize the assay. To assess the performance of the assay in naturally infected animals, we evaluated 316 goat and sheep field sera collected during active SAT2 outbreaks. Relative to results of the virus neutralization test, the optimal serum dilution and cutoff percentage inhibition (PI) were 1:15 and 50%, respectively. At these values, the Spearman rank correlation coefficient was 0.85 (p < 0.001), and the sensitivity and specificity (95% CI) were 80.3% (72.6, 87.2) and 91.1% (84.1, 95.9), respectively. Relative to the liquid-phase blocking ELISA and the nonstructural protein ELISA, the ss-SPCE exhibited divergent performance characteristics between the goat and sheep field sera. Repeatability was better for goats, but the correlation and agreement among all 3 assays were better for the sheep sera. The prevalence of SAT2 FMDV infection in the sampled sheep was 23.6%; sampled goats were seemingly FMDV-free. The ss-SPCE is an appropriate FMDV detection tool to investigate the role of small ruminants in the epidemiology of FMD in Africa.

Efficacy of SAT2 Foot-and-Mouth Disease Vaccines Formulated with Montanide ISA 206B and Quil-A Saponin Adjuvants.

The effective control of foot-and-mouth disease (FMD) relies strongly on the separation of susceptible and infected livestock or susceptible livestock and persistently infected wildlife, vaccination, and veterinary sanitary measures. Vaccines affording protection against multiple serotypes for longer than six months and that are less reliant on the cold chain during handling are urgently needed for the effective control of FMD in endemic regions. Although much effort has been devoted to improving the immune responses elicited through the use of modern adjuvants, their efficacy is dependent on the formulation recipe, target species and administration route. Here we compared and evaluated the efficacy of two adjuvant formulations in combination with a structurally stabilized SAT2 vaccine antigen, designed to have improved thermostability, antigen shelf-life and longevity of antibody response. Protection mediated by the Montanide ISA 206B-adjuvanted or Quil-A Saponin-adjuvanted SAT2 vaccines were comparable. The Montanide ISA 206B-adjuvanted vaccine elicited a higher SAT2 neutralizing antibody response and three times higher levels of systemic IFN-γ responses at 14- and 28-days post-vaccination (dpv) were observed compared to the Quil-A Saponin-adjuvanted vaccine group. Interestingly, serum antibodies from the immunized animals reacted similarly to the parental vaccine virus and viruses containing mutations in the VP2 protein that simulate antigenic drift in nature.

Genetic Basis of Antigenic Variation of SAT3 Foot-And-Mouth Disease Viruses in Southern Africa.

Foot-and-mouth disease (FMD) continues to be a major burden for livestock owners in endemic countries and a continuous threat to FMD-free countries. The epidemiology and control of FMD in Africa is complicated by the presence of five clinically indistinguishable serotypes. Of these the Southern African Territories (SAT) type 3 has received limited attention, likely due to its restricted distribution and it being less frequently detected. We investigated the intratypic genetic variation of the complete P1 capsid-coding region of 22 SAT3 viruses and confirmed the geographical distribution of five of the six SAT3 topotypes. The antigenic cross-reactivity of 12 SAT3 viruses against reference antisera was assessed by performing virus neutralization assays and calculating the r1-values, which is a ratio of the heterologous neutralizing titer to the homologous neutralizing titer. Interestingly, cross-reactivity between the SAT3 reference antisera and many SAT3 viruses was notably high (r1-values >0.3). Moreover, some of the SAT3 viruses reacted more strongly to the reference sera compared to the homologous virus (r1-values >1). An increase in the avidity of the reference antisera to the heterologous viruses could explain some of the higher neutralization titers observed. Subsequently, we used the antigenic variability data and corresponding genetic and structural data to predict naturally occurring amino acid positions that correlate with antigenic changes. We identified four unique residues within the VP1, VP2, and VP3 proteins, associated with a change in cross-reactivity, with two sites that change simultaneously. The analysis of antigenic variation in the context of sequence differences is critical for both surveillance-informed selection of effective vaccines and the rational design of vaccine antigens tailored for specific geographic localities, using reverse genetics.

Diagnostic and Epitope Mapping Potential of Single-Chain Antibody Fragments Against Foot-and-Mouth Disease Virus Serotypes A, SAT1, and SAT3.

Foot-and-mouth disease (FMD) affects cloven-hoofed domestic and wildlife animals and an outbreak can cause severe losses in milk production, reduction in meat production and death amongst young animals. Several parts of Asia, most of Africa, and the Middle East remain endemic, thus emphasis on improved FMD vaccines, diagnostic assays, and control measures are key research areas. FMD virus (FMDV) populations are quasispecies, which pose serious implications in vaccine design and efficacy where an effective vaccine should include multiple independent neutralizing epitopes to elicit an adequate immune response. Further investigation of the residues that comprise the antigenic determinants of the virus will allow the identification of mutations in outbreak strains that potentially lessen the efficacy of a vaccine. Additionally, of utmost importance in endemic regions, is the accurate diagnosis of FMDV infection for the control and eradication of the disease. To this end, a phage display library was explored to identify FMDV epitopes for recombinant vaccines and for the generation of reagents for improved diagnostic FMD enzyme-linked immunosorbent assays (ELISAs). A naïve semi-synthetic chicken single chain variable fragment (scFv) phage display library i.e., the Nkuku ® library was used for bio-panning against FMD Southern-African Territories (SAT) 1, SAT3, and serotype A viruses. Biopanning yielded one unique scFv against SAT1, two for SAT3, and nine for A22. SAT1 and SAT3 specific scFvs were exploited as capturing and detecting reagents to develop an improved diagnostic ELISA for FMDV. The SAT1 soluble scFv showed potential as a detecting reagent in the liquid phase blocking ELISA (LPBE) as it reacted specifically with a panel of SAT1 viruses, albeit with different ELISA absorbance signals. The SAT1svFv1 had little or no change on its paratope when coated on polystyrene plates whilst the SAT3scFv's paratope may have changed. SAT1 and SAT3 soluble scFvs did not neutralize the SAT1 and SAT3 viruses; however, three of the nine A22 binders i.e., A22scFv1, A22scFv2, and A22scFv8 were able to neutralize A22 virus. Following the generation of virus escape mutants through successive virus passage under scFv pressure, FMDV epitopes were postulated i.e., RGD+3 and +4 positions respectively, proving the epitope mapping potential of scFvs.

Pathogenesis, biophysical stability and phenotypic variance of SAT2 foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease of cloven-hoofed animals, which severely decreases livestock productivity. FMD virus (FMDV), the causative agent, initiates infection by interaction with integrin cellular receptors on pharyngeal epithelium cells, causing clinical signs one to four days after transmission to a susceptible host. However, some Southern African Territories (SAT) viruses have been reported to cause mild or subclinical infections that may go undiagnosed in field conditions and are likely to be more common than previously expected. The studies presented here demonstrate that not all SAT2 viruses are equally virulent in cattle. The two SAT2 viruses, ZIM/5/83 and ZIM/7/83, were both highly attenuated in cattle, as evidenced by the mild clinical signs observed after needle challenge, while two incongruent SAT2 viruses showed significantly different clinical signs in challenged cattle. We then explored the ability of the SAT2 viruses to infect different cell types with defined receptors that are utilised by FMDV and found differences in their ability to lyse cells in culture and to compete in a controlled cell culture environment. The population sequence variation between ZIM/5/83 and ZIM/7/83 revealed multiple sites of single nucleotide variants of low frequency between the predominant virus populations, as could be expected from the genome of an RNA virus. An assessment of the biophysical stability of SAT2 virions during acidification indicated that the SAT2 virus EGY/09/12 was more resilient to acidification than the ZIM/5/83 and ZIM/7/83 viruses; however, whether this difference relates to differences in virulence in vivo is unclear. This study is a consolidated view of the key findings of SAT2 viruses studied over a 14-year period involving many different experiments.

Impact of foot-and-mouth-disease on goat behaviour after experimental infection with serotype SAT1 virus.

Infectious diseases and parasitic infestations can cause a set of non-specific clinical signs, such as increased body temperature and resting, and a decrease in food intake. These physiological and behavioural changes have an adaptive function facilitating defences against the pathogen and to support immune functions. These so-called' sickness behaviours' can also be used as an early detection tool for disease. Foot-and-mouth disease (FMD) still causes great economic losses in endemic countries, especially to smallholder farmers. The aim of this study was to determine if behavioural changes in goats can be used as an early indicator of FMD virus (FMDV) infection. The efficacy of a Southern African Territories (SAT) FMD vaccine was studied on forty South African indigenous goats. Changes in daily activities (resting, feeding, walking), as well as social behaviours (social resting, social feeding, dominance behaviours) were recorded and then compared over time and between clinically affected and unaffected goats. Pedometers were used to estimate average daily steps and to compare between groups of study animals. Eleven goats developed clinical signs of FMD, as well as non-FMD related sicknesses during the course of the study. Overall walking and resting behaviours were not significantly affected by the presence of FMD related clinical signs (p > 0.05). However, during the time of FMDV infection, social resting increased significantly (p < 0.001). Although goats developed FMD lesions on lips and tongues, percentage of time feeding was not affected (p = 0.762), suggesting that the study goats did not perceive the oral lesions as an important disturbance. Similarly, the number of steps did not consistently decrease in the presence of FMD-associated foot lesions. When affected by non-FMD related sicknesses, animals did not have an overall reduction in the time spent feeding (p = 0.867). However, goats affected with non-FMD conditions reduced the amount of social feeding (p = 0.002), potentially avoiding energetically costly competition at the feeding points. Overall, goats affected with FMD did not show more sickness behaviour, suggesting that FMDV infection in goats might not lead to obvious and therefore, easily detectable behavioural changes. This might have implications for farmers and animal health personnel, as individual goats infected with FMDV might be undetected within a flock due to the absence of obvious sickness behaviours, and the virus can therefore be spread more easily between herds through animal movements.

Determining the epitope dominance on the capsid of a serotype SAT2 foot-and-mouth disease virus by mutational analyses.

UNLABELLED: Monoclonal-antibody (MAb)-resistant mutants were used to map antigenic sites on foot-and-mouth disease virus (FMDV), which resulted in the identification of neutralizing epitopes in the flexible ßG-ßH loop in VP1. For FMDV SAT2 viruses, studies have shown that at least two antigenic sites exist. By use of an infectious SAT2 cDNA clone, 10 structurally exposed and highly variable loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of SAT2/Zimbabwe (ZIM)/7/83 (topotype II) and replaced with the corresponding regions of SAT2/Kruger National Park (KNP)/19/89 (topotype I). Virus neutralization assays using convalescent-phase antisera raised against the parental virus, SAT2/ZIM/7/83, indicated that the mutant virus containing the TQQS-to-ETPV mutation in the N-terminal part of the ßG-ßH loop of VP1 showed not only a significant increase in the neutralization titer but also an increase in the index of avidity to the convalescent-phase antisera. Furthermore, antigenic profiling of the epitope-replaced and parental viruses with nonneutralizing SAT2-specific MAbs led to the identification of two nonneutralizing antigenic regions. Both regions were mapped to incorporate residues 71 to 72 of VP2 as the major contact point. The binding footprint of one of the antigenic regions encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 48 to 50 of VP1, and the second antigenic region encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 84 to 86 and 109 to 11 of VP1. This is the first time that antigenic regions encompassing residues 71 to 72 of VP2 have been identified on the capsid of a SAT2 FMDV. IMPORTANCE: Monoclonal-antibody-resistant mutants have traditionally been used to map antigenic sites on foot-and-mouth disease virus (FMDV). However, for SAT2-type viruses, which are responsible for most of the FMD outbreaks in Africa and are the most varied of all seven serotypes, only two antigenic sites have been identified. We have followed a unique approach using an infectious SAT2 cDNA genome-length clone. Ten structurally surface-exposed, highly varied loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of the SAT2/ZIM/7/83 virus. These regions were replaced with the corresponding regions of an antigenically disparate virus, SAT2/KNP/19/89. Antigenic profiling of the epitope-replaced and parental viruses with SAT2-specific MAbs led to the identification of two unique antibody-binding footprints on the SAT2 capsid. In this report, evidence for the structural engineering of antigenic sites of a SAT2 capsid to broaden cross-reactivity with antisera is provided.

Challenges and prospects for the control of foot-and-mouth disease: an African perspective.

The epidemiology of foot-and-mouth disease (FMD) in Africa is unique in the sense that six of the seven serotypes of FMD viruses (Southern African Territories [SAT] 1, SAT2, SAT3, A, O, and C), with the exception of Asia-1, have occurred in the last decade. Due to underreporting of FMD, the current strains circulating throughout sub-Saharan Africa are in many cases unknown. For SAT1, SAT2, and serotype A viruses, the genetic diversity is reflected in antigenic variation, and indications are that vaccine strains may be needed for each topotype. This has serious implications for control using vaccines and for choice of strains to include in regional antigen banks. The epidemiology is further complicated by the fact that SAT1, SAT2, and SAT3 viruses are maintained and spread by wildlife, persistently infecting African buffalo in particular. Although the precise mechanism of transmission of FMD from buffalo to cattle is not well understood, it is facilitated by direct contact between these two species. Once cattle are infected they may maintain SAT infections without the further involvement of buffalo. No single strategy for control of FMD in Africa is applicable. Decision on the most effective regional control strategy should focus on an ecosystem approach, identification of primary endemic areas, animal husbandry practices, climate, and animal movement. Within each ecosystem, human behavior could be integrated in disease control planning. Different regions in sub-Saharan Africa are at different developmental stages and are thus facing unique challenges and priorities in terms of veterinary disease control. Many science-based options targeting improved vaccinology, diagnostics, and other control measures have been described. This review therefore aims to emphasize, on one hand, the progress that has been achieved in the development of new technologies, including research towards improved tailored vaccines, appropriate vaccine strain selection, vaccine potency, and diagnostics, and how it relates to the conditions in Africa. On the other hand, we focus on the unique epidemiological, ecological, livestock farming and marketing, socioeconomic, and governance issues that constrain effective FMD control. Any such new technologies should have the availability of safe livestock products for trade as the ultimate goal.

Mapping of antigenic determinants on a SAT2 foot-and-mouth disease virus using chicken single-chain antibody fragments.

Recombinant single-chain variable fragments (scFvs) of antibodies make it possible to localize antigenic and immunogenic determinants, identify protective epitopes and can be exploited for the design of improved diagnostic tests and vaccines. A neutralizing epitope, as well as other potential antigenic sites of a SAT2 foot-and-mouth disease virus (FMDV) were identified using phage-displayed scFvs. Three unique ZIM/7/83-specific scFvs, designated scFv1, scFv2 and scFv3, were isolated. Further characterization of these scFvs revealed that only scFv2 was capable of neutralizing the ZIM/7/83 virus and was used to generate neutralization-resistant virus variants. Sequence analysis of the P1 region of virus escaping neutralization revealed a residue change from His to Arg at position 159 of the VP1 protein. Residue 159 is not only surface exposed but is also located at the C-terminal base of the G-H loop, a known immunogenic region of FMDV. A synthetic peptide, of which the sequence corresponded to the predicted antigenic site of the VP1 G-H loop of ZIM/7/83, inhibited binding of scFv2 to ZIM/7/83 in a concentration-dependent manner. This region can therefore be considered in the design of SAT2 vaccine seed viruses for the regional control of FMD in Africa.

Sequence-based prediction for vaccine strain selection and identification of antigenic variability in foot-and-mouth disease virus.

Identifying when past exposure to an infectious disease will protect against newly emerging strains is central to understanding the spread and the severity of epidemics, but the prediction of viral cross-protection remains an important unsolved problem. For foot-and-mouth disease virus (FMDV) research in particular, improved methods for predicting this cross-protection are critical for predicting the severity of outbreaks within endemic settings where multiple serotypes and subtypes commonly co-circulate, as well as for deciding whether appropriate vaccine(s) exist and how much they could mitigate the effects of any outbreak. To identify antigenic relationships and their predictors, we used linear mixed effects models to account for variation in pairwise cross-neutralization titres using only viral sequences and structural data. We identified those substitutions in surface-exposed structural proteins that are correlates of loss of cross-reactivity. These allowed prediction of both the best vaccine match for any single virus and the breadth of coverage of new vaccine candidates from their capsid sequences as effectively as or better than serology. Sub-sequences chosen by the model-building process all contained sites that are known epitopes on other serotypes. Furthermore, for the SAT1 serotype, for which epitopes have never previously been identified, we provide strong evidence--by controlling for phylogenetic structure--for the presence of three epitopes across a panel of viruses and quantify the relative significance of some individual residues in determining cross-neutralization. Identifying and quantifying the importance of sites that predict viral strain cross-reactivity not just for single viruses but across entire serotypes can help in the design of vaccines with better targeting and broader coverage. These techniques can be generalized to any infectious agents where cross-reactivity assays have been carried out. As the parameterization uses pre-existing datasets, this approach quickly and cheaply increases both our understanding of antigenic relationships and our power to control disease.