David James Paton

Despite widespread use of inactivated vaccines to control foot-and-mouth disease (FMD), there is no systematic approach to demonstrate the regional relevance of these products against the specific serotypes and strains that circulate in endemic countries in Africa and Asia. Failure to adopt independent testing of FMD vaccines has contributed to poor trust in their quality and a lack of investment in vaccination programmes. Therefore, a reference antigen panel representing four serotypes, tailored for East Africa, has been established and used to measure FMDV-specific antibody responses in cattle after administration of FMD vaccines commercially available in the region. This revealed inconsistencies and gaps in cross-neutralisation responses that are evident for some vaccines even after giving booster doses. It is concluded that the East Africa reference antigen panel can be used to evaluate FMD vaccine potency and drive up vaccine quality. Further panels could be developed and deployed for other endemic regions.

Vaccination is widely used to control foot-and-mouth disease (FMD), but maternal antibodies may interfere with the response to vaccination in calves. This study, conducted on a regularly vaccinated Malaysian dairy farm, aimed to optimise the vaccination regime by measuring the in vitro neutralising virus antibody responses of 51 calves before and after vaccination with a one or two dose vaccination regime starting at 2–7 months old. The presence of maternal antibodies was associated with poor post-vaccination antibody responses after a single dose of vaccine in calves less than 6 months old. However, a second dose of vaccine given three weeks later, improved the antibody responses in all ages of calves. This confirms the view that in regularly vaccinated farms, some combination of delay and revaccination is needed to achieve effective immunization of calves. Sera from cows and pre-vaccinated calves neutralised homologous serotype A vaccine virus more strongly than a heterologous serotype A field virus, but this pattern was reversed in some calves after vaccination. The strength of heterologous responses in calves 49 days after first vaccination correlated to the amount of transferred maternal antibody, suggesting that pre-existing antibodies could have modulated the specificity of these active antibody responses. If confirmed, such an effect by pre-existing antibodies could have wider implications for broadening the coverage of FMD vaccine responses.

The recent introduction of foot-and-mouth disease (FMD) virus serotype O (O/EA-2 topotype) in Southern Africa has changed the epidemiology of the disease and vaccine requirements of the region. Commercial and subsistence cattle herds in Zambia were vaccinated with an FMD virus serotype O Manisa vaccine according to a double- or single-dose vaccination schedule. Heterologous antibody responses induced by this vaccine against a representative O/EA-2 virus from Zambia were determined. Virus neutralisation tests (VNTs) showed double-dosed cattle had a mean reciprocal log virus neutralisation titre of 2.02 (standard error [SE] = 0.16, n = 9) for commercial herds and 1.65 (SE = 0.17, n = 5) for subsistence herds 56 days after the first vaccination (dpv). Significantly lower mean titres were observed for single-dosed commercial herds (0.90, SE = 0.08, n = 9) and subsistence herds (1.15, SE = 0.18, n = 3) 56 dpv. A comparison of these results and those generated by solid-phase competitive ELISA (SPCE) tests showed a statistically significant positive correlation by Cohen’s kappa coefficient. Therefore, SPCE might be used in assessing the immunogenicity of vaccines in place of VNT. Furthermore, for this vaccine and field strain, a vaccination regime employing a two-dose primary course and revaccination after 4–6 months is likely to be appropriate.

The recent introduction of foot-and-mouth disease virus serotype O (O/EA-2 topotype) in Southern Africa region has changed virus dynamics and the FMD vaccine requirements. This study describes an assessment of the heterologous antibody responses induced by O Manisa vaccine (three PD50/dose) against a representative O/EA-2 virus collected from an FMD outbreak in Zambia. Commercial and subsistence cattle herds were monitored following vaccination, where the benefits of employing a double vaccination schedule were compared to single-dose vaccination. Testing using virus neutralisation showed cattle that received two vaccine doses had a mean reciprocal log virus neutralisation titre of 2.02 (standard error [SE] = 0.16, n = 9) for commercial herds and 1.65 (SE = 0.17, n = 5) for subsistence herds , 56 days after first vaccination (dpv). Significantly lower mean titres were observed for single-dosed commercial herds (0.90, SE = 0.08, n = 9) and subsistence herds (1.15, SE = 0.18, n = 3), 56 dpv. Comparison of these results and those generated by solid-phase competitive ELISA by cohen’s kappa coefficient observed a statistically significant (ρ = 0.598 for 1:10 dilution, and ρ = 0.562 for 1:30 dilution of SPCE % inhibition results, p < 0.001 for both dilutions) positive correlation.

Serology is widely used to predict whether vaccinated individuals and populations will be protected against infectious diseases, including foot-and-mouth disease (FMD), which affects cloven-hoofed animals. Neutralising antibody titres to FMD challenge viruses correlate to protection against FMD, for vaccinated cattle that are infected with the same strain as in the vaccine (homologous protection). Similar relationships exist for cross-strain protection between different vaccine and challenge viruses, although much less data are available for these heterologous studies. Poor inter-laboratory reproducibility of the virus neutralisation test (VNT) also hampers comparisons between studies. Therefore, day-of-challenge sera (n = 180) were assembled from 13 previous FMD cross-protection experiments for serotypes O (n = 2), A (n = 10), and SAT 2 (n = 1). These were tested by VNT against the challenge viruses at the FMD FAO World Reference Laboratory (WRLFMD) and the titres were compared to challenge outcomes (protected or not). This dataset was combined with equivalent serology and protection data for 61 sera from four cross-protection experiments carried out at WRLFMD for serotypes O (n = 2), A (n = 1), and Asia 1 (n = 1). VNT results and protection outcomes were also analysed for a serotype O cross-protection experiment involving 39 cattle, where the sera were not available for retesting at WRLFMD. Three categories of association between heterologous neutralising antibody titre and heterologous protection were found (Group 1–3). The log₁₀ reciprocal titres associated on average with 75% protection (with 95% credible limits) were: Group 1: 2.46 (2.11–2.97); Group 2: 1.67 (1.49–1.92); Group 3: 1.17 (1.06–1.30). Further cross-protection data are needed to understand the factors that underpin this variability and to develop more robust antibody thresholds. Establishing cut-off serological titres that can be used to score the adequacy of vaccine-induced immunity will facilitate the monitoring and thereby the performance of FMD vaccination in the field.

Antibodies to the foot-and-mouth disease virus (FMDV) capsid induced by infection or vaccination can provide serotype-specific protection and be measured using virus neutralization tests and viral structural-protein (SP-)ELISAs. Separate tests are needed for each serotype, but cross-serotype reactions complicate serotyping. In this study, inter-serotypic responses were quantified for five SP-ELISA formats by testing 294 monovalent mainly bovine sera collected following infection, vaccination, or vaccination and infection with one of five serotypes of FMDV. Over half of the samples, representing all three immunization categories, scored positive for at least one heterologous serotype and some scored positive for all serotypes tested. A comparative approach to identifying the strongest reaction amongst serotypes O, A and Asia 1 improved the accuracy of serotyping to 73–100% depending on the serotype and test system, but this method will be undermined where animals have been infected and/or vaccinated with multiple FMDV serotypes. Preliminary studies with stabilized recombinant capsid antigens of serotypes O and A that do not expose internal epitopes showed reduced cross-reactivity, supporting the hypothesis that capsid integrity can affect the serotype-specificity of the SP-ELISAs. The residual cross-reactivity associated with capsid surface epitopes was consistent with the evidence of cross-serotype virus neutralization.

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.

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.

RNA viruses have high mutation rates and exist within their hosts as large, complex and heterogeneous populations, comprising a spectrum of related but non-identical genome sequences. Next generation sequencing is revolutionising the study of viral populations by enabling the ultra deep sequencing of their genomes, and the subsequent identification of the full spectrum of variants within the population. Identification of low frequency variants is important for our understanding of mutational dynamics, disease progression, immune pressure, and for the detection of drug resistant or pathogenic mutations. However, the current challenge is to accurately model the errors in the sequence data and distinguish real viral variants, particularly those that exist at low frequency, from errors introduced during sequencing and sample processing, which can both be substantial. We have created a novel set of laboratory control samples that are derived from a plasmid containing a full-length viral genome with extremely limited diversity in the starting population. One sample was sequenced without PCR amplification whilst the other samples were subjected to increasing amounts of RT and PCR amplification prior to ultra-deep sequencing. This enabled the level of error introduced by the RT and PCR processes to be assessed and minimum frequency thresholds to be set for true viral variant identification. We developed a genome-scale computational model of the sample processing and NGS calling process to gain a detailed understanding of the errors at each step, which predicted that RT and PCR errors are more likely to occur at some genomic sites than others. The model can also be used to investigate whether the number of observed mutations at a given site of interest is greater than would be expected from processing errors alone in any NGS data set. After providing basic sample processing information and the site’s coverage and quality scores, the model utilises the fitted RT-PCR error distributions to simulate the number of mutations that would be observed from processing errors alone. These data sets and models provide an effective means of separating true viral mutations from those erroneously introduced during sample processing and sequencing.