The Immunogenicity of a Foot-and-Mouth Disease Virus Serotype O Vaccine in Commercial and Subsistence Cattle Herds in Zambia.

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.

Maternally Derived Antibodies to Foot-and-Mouth Disease Virus Modulate the Antigenic Specificity of Humoral Responses in Vaccinated Cattle.

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.

Comparison of vaccination schedules for foot-and-mouth disease among cattle and sheep in Mongolia.

Vaccines are a critical tool for the control strategy for foot-and-mouth disease (FMD) in Mongolia where sporadic outbreaks regularly occur. A two-dose primary vaccination course is recommended for most commercial vaccines though this can be logistically challenging to deliver among nomadic pastoralist systems which predominate in the country. Although there is evidence that very high potency vaccines can provide prolonged duration of immunity, this has not been demonstrated under field conditions using commercially available vaccines. This study compared neutralizing titres to a O/ME-SA/Panasia strain over a 6-month period following either a two-dose primary course or a single double-dose vaccination among Mongolian sheep and cattle using a 6.0 PD50 vaccine. Titers were not significantly different between groups except in sheep at six-months post vaccination when the single double-dose group had significantly lower titers. These results indicate the single double-dose regimen may be a cost-effective approach for vaccination campaigns supporting FMD control in Mongolia.

Evaluating serological tests for foot-and-mouth disease while accounting for different serotypes and uncertain vaccination status.

Controlling foot-and-mouth disease (FMD) by vaccination requires adequate population coverage and high vaccine efficacy under field conditions. To assure veterinary services that animals have acquired sufficient immunity, strategic post-vaccination surveys can be conducted to monitor the coverage and performance of the vaccine. Correct interpretation of these serological data and an ability to derive exact prevalence estimates of antibody responses requires an awareness of the performance of serological tests. Here, we used Bayesian latent class analysis to evaluate the diagnostic sensitivity and specificity of four tests. A non-structural protein (NSP) ELISA determines vaccine independent antibodies from environmental exposure to FMD virus (FMDV), and three assays measuring total antibodies derived from vaccine antigen or environmental exposure to two serotypes (A, O): the virus neutralisation test (VNT), a solid phase competitive ELISA (SPCE), and a liquid phase blocking ELISA (LPBE). Sera (n = 461) were collected by a strategic post-vaccination monitoring survey in two provinces of Southern Lao People's Democratic Republic (PDR) after a vaccination campaign in early 2017. Not all samples were tested by every assay and each serotype: VNT tested for serotype A and O, whereas SPCE and LPBE tested for serotype O, and only NSP-negative samples were tested by VNT, with 90 of them not tested (missing by study design). These data challenges required informed priors (based on expert opinion) for mitigating possible lack of model identifiability. The vaccination status of each animal, its environmental exposure to FMDV, and the indicator of successful vaccination were treated as latent (unobserved) variables. Posterior median for sensitivity and specificity of all tests were in the range of 92-99 %, except for the sensitivity of NSP (∼66%) and the specificity of LPBE (∼71 %). There was strong evidence that SPCE outperformed LPBE. In addition, the proportion of animals recorded as having been vaccinated that showed a serological immune response was estimated to be in the range of 67-86 %. The Bayesian latent class modelling framework can easily and appropriately impute missing data. It is important to use field study data as diagnostic tests are likely to perform differently on field survey samples compared to samples obtained under controlled conditions.

Predicting cross-protection against foot-and-mouth disease virus strains by serology after vaccination.

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.

Development of anti-Crimean-Congo hemorrhagic fever virus Gc and NP-specific ELISA for detection of antibodies in domestic animal sera.

Crimean-Congo hemorrhagic fever (CCHF) is a priority emerging disease. CCHF, caused by the CCHF virus (CCHFV), can lead to hemorrhagic fever in humans with severe cases often having fatal outcomes. CCHFV is maintained within a tick-vertebrate-tick cycle, which includes domestic animals. Domestic animals infected with CCHFV do not show clinical signs of the disease and the presence of antibodies in the serum can provide evidence of their exposure to the virus. Current serological tests are specific to either one CCHFV antigen or the whole virus antigen. Here, we present the development of two in-house ELISAs for the detection of serum IgG that is specific for two different CCHFV antigens: glycoprotein Gc (CCHFV Gc) and nucleoprotein (CCHFV NP). We demonstrate that these two assays were able to detect anti-CCHFV Gc-specific and anti-CCHFV NP-specific IgG in sheep from endemic CCHFV areas with high specificity, providing new insight into the heterogeneity of the immune response induced by natural infection with CCHFV in domestic animals.

Cross-Serotype Reactivity of ELISAs Used to Detect Antibodies to the Structural Proteins of Foot-and-Mouth Disease Virus.

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.

Avidity of Polyclonal Antibodies to Foot-and-Mouth Disease Virus in Bovine Serum Measured Using Bio-Layer Interferometry.

Foot-and-mouth disease (FMD) is a disease of cloven-hoofed livestock caused by FMD virus (FMDV). FMD can be controlled through the use of inactivated vaccines, and it is well established that the protection afforded by FMD vaccines correlates strongly with neutralising antibody titres. However, the overall strength of binding, referred to as avidity, is also an important parameter with respect to the ability of antibodies to neutralise virus infection, and there is evidence that avidity can affect the level of protection afforded by FMDV vaccines. Here, as an alternative to modified enzyme-linked immunosorbent assays (avidity ELISAs) incorporating a chaotropic wash step, we used bio-layer interferometry (BLI) to measure the avidity of bovine polyclonal antibodies against FMDV capsids. We conducted preliminary experiments using recombinant FMDV capsids, as well as peptides representing antigenic loops, to demonstrate that the binding of monoclonal antibodies targeting specific antigenic sites could be detected using BLI. Subsequent experiments using polyclonal sera derived from FMD vaccinated cattle provided evidence of a positive correlation between the neutralising titre of the serum and the avidity as measured by BLI. Furthermore, we observed an increase in BLI avidity, as well as in the titre, in vaccinated animals upon challenge with the live virus.

Toward the calibration of serological assays using sera collected from cattle and sheep following a single dose of foot-and-mouth disease vaccine.

Background and Aim: Serological assays are widely used to monitor the performance of foot-and-mouth disease (FMD) vaccines to estimate vaccination coverage and to ensure that vaccinated animals generate adequate immune responses. This study aimed to measure the FMD virus (FMDV)-specific responses in cattle and sheep after a single dose of a trivalent FMD vaccine containing serotypes A, O, and Asia-1, and to use these sera to calibrate virus neutralization tests (VNTs) and serotype-specific serological enzyme-linked immunoassays (ELISAs) that can measure post-vaccination responses. Materials and Methods: Sera were collected from cattle (n=10) and sheep (n=10) on 0, 21, and 56 days after immunization with an imported aqueous formulated FMD vaccine. These samples were tested by VNT using field FMDV isolates that are representative of the epidemiological risks in Central Asia (A/ASIA/Iran-05, A/ASIA/GVII, O/ME-SA/Ind-2001, O/SEA/Mya-98, O/ME-SA/PanAsia, and Asia-1 Shamir). Heterologous VNT antibody responses were compared to those measured using commercial FMDV-specific ELISAs for serotypes O, A, and Asia 1. Results: Administration of the FMD vaccine increased FMDV-specific antibody titers for both species in sera collected on day 21, but these elevated titers were short-lived and were decreased by day 56. Conclusion: These results highlight the short duration of immunity with a single dose of this aqueous vaccine and motivate further studies to assess immune responses in cattle and small ruminants after a two-dose course vaccination schedule. Further comparative data for VNT and serotype-specific ELISAs are needed to define cutoffs that can be used to monitor post-vaccination immune responses in low-containment laboratories where it is not possible to handle live FMDVs.

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses.

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.

Inter-laboratory comparison of 2 ELISA kits used for foot-and-mouth disease virus nonstructural protein serology.

Serologic assays used to detect antibodies to nonstructural proteins (NSPs) of foot-and-mouth disease virus (FMDV) are used for disease surveillance in endemic countries, and are essential to providing evidence for freedom of the disease with or without vaccination and to recover the free status of a country after an outbreak. In a 5-site inter-laboratory study, we compared the performance of 2 commercial NSP ELISA kits (ID Screen FMD NSP ELISA single day [short] and overnight protocols, ID.Vet; PrioCHECK FMDV NS antibody ELISA, Thermo Fisher Scientific). The overall concordance between the PrioCHECK and ID Screen test was 93.8% (95% CI: 92.0-95.2%) and 94.8% (95% CI: 93.1-96.1%) for the overnight and short ID Screen incubation protocols, respectively. Our results indicate that the assays (including the 2 different formats of the ID Screen test) can be used interchangeably in post-outbreak serosurveillance.

Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19.

Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.

Immunogenicity of imported foot-and-mouth vaccines in different species in Mongolia.

Foot-and-mouth disease (FMD) is a high impact viral disease of livestock for which vaccines are extensively used in control. Mongolia has regular incursions of FMD virus that are typically limited to the eastern region although large epidemics are occasionally reported in the normally disease-free western areas. Vaccines are imported and form an important component of the control strategy. In 2015, post-vaccination monitoring guidelines were published by the FAO-OIE recommending approaches for assessing the appropriateness of imported vaccines including small-scale immunogenicity studies. This study used these recommended approaches to guide the use of vaccine adjuvant type and the need for a one or two dose primary course in the national control programme considering cattle, sheep and Bactrian camels and also whether these vaccines were appropriate for the FMD virus lineages considered high risk to Mongolia (A/ASIA/Sea-97; O/SEA/Mya-98; O/ME-SA/PanAsia; O/ME-SA/Ind-2001). The results of these immunogenicity studies indicated that in cattle and sheep, oil-adjuvanted vaccines led to higher and more persistent neutralisation titres that were satisfactory against the target lineages if a two-dose primary course was utilised. In contrast, aqueous-adjuvanted vaccines were associated with lower titres that likely required a booster after 3 months. Levels of antibodies in Bactrian camels were significantly lower although it is unknown how these may correlate with protection under experimental or field exposure conditions. The results of this study have implications for vaccine policy in Mongolia and suggest further studies on the role of Bactrian camels in the epidemiology of FMD are necessary to indicate if further research on FMD vaccines are needed in this species.

Efficacy of a high-potency multivalent foot-and-mouth disease virus vaccine in cattle against heterologous challenge with a field virus from the emerging A/ASIA/G-VII lineage.

In 2015, outbreaks of foot-and-mouth disease (FMD) in the Middle East were discovered to be caused by a viral lineage (A/ASIA/G-VII), which has recently emerged from the Indian sub-continent. In vitro vaccine matching data generated by the World Reference Laboratory (WRLFMD) indicated that A/ASIA/G-VII field viruses were poorly matched with vaccines (A-SAU-95, A22 IRQ and A-IRN-05) that are already used in the region. In order to assess the likely performance of one of these commercially available FMD vaccines, sixteen cattle were vaccinated with a polyvalent vaccine which contained two serotype A components (A-SAU-95 and A-IRN-05) with a homologous potency of at least 6PD50, and two cattle were left unvaccinated as controls. Twenty-one days later, all 18 cattle were challenged by tongue inoculation with an FMDV field isolate A/IRN/22/2015 from the A/ASIA/G-VII lineage, in line with the European Pharmacopeia PPG test conditions. The two control animals developed generalised FMD, and 7/16 vaccinated animals developed at least one foot lesion, thus only 56.3% were defined as protected. For the vaccine components, there was a significant increase in the probability of protection with increasing serological titres for A-SAU-95 (p = 0.03), but not for A-IRN-05 (p = 0.42). Analysis of FMDV in blood and nasal swabs suggested that vaccination reduced shedding and potential onward spread of FMD virus even if the animal developed foot lesions. In summary, the results from this study suggest that whilst this vaccine would not be appropriate for use in an emergency situation (in previously FMD-free countries), it may be partially effective in the field in endemic countries where repeat prophylactic vaccination is practiced. For emergency reactive vaccination, the findings from this study support the idea that a new vaccine strain should be developed that is tailored to the A/ASIA/G-VII lineage.

Evaluation of two different swab transport systems in the detection of avian influenza virus excretion from infected Pekin ducks (Anas platyrhynchos).

The role of wild birds in the epidemiology and ecology of influenza A viruses has long been recognised (Alexander, 2007a). As a result of the emergence of a H5N1 highly pathogenic avian influenza (HPAI) virus and the apparent role of wild birds in its spread across Asia, Europe and Africa, avian influenza (AI) wild bird surveillance has been implemented in many countries including, since February 2006, a mandatory programme in the European Union (CEC, 2006a). In the present study the detection of virus excreted from Pekin ducks (Anas platyrhynchos) infected experimentally with A/mallard/England/2126/07 (H3N6) was investigated over a fourteen day period post-infection using cloacal and oropharyngeal swabs, with (wet) and without (dry) viral transport medium which were collected from each duck in alternating order. For influenza A virus matrix gene RNA detection, wet oropharyngeal swabs were significantly more sensitive than dry oropharyngeal on days 4-5 after infection. For cloacal samples, dry swabs were equivalent or superior to wet swabs throughout the study. Although differences in detection between dry and wet swabs were observed, the qualitative bird-level results were unaffected, meaning that the infection status of individual birds was correctly determined.