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.

A new blocking ELISA for detection of foot-and-mouth disease non-structural protein (NSP) antibodies in a broad host range.

Large-scale monitoring of foot-and-mouth disease (FMD) in livestock is imperative in an FMD control program. Detection of antibodies against non-structural proteins (NSP) of FMD virus (FMDV) is one of the best tools to estimate the prevalence of past infection; availability of such a well-validated test is therefore essential. Using a FMDV 3B protein-specific monoclonal antibody, we have developed a new NSP antibody blocking ELISA (10H9 bELISA) and validated it on large panels of sera from different susceptible species. The diagnostic sensitivity of the ELISA was 95% with a specificity of 98%, similar to the values found using a commercial kit (PrioCHECK FMD NS test). The 10H9 bELISA can be used in a broad range of FMD susceptible species making it a very useful tool in monitoring the foot-and-mouth disease control programs by detection of virus circulation in the vaccinated populations. KEY POINTS: • A new ELISA for detection of foot and mouth disease (FMD) antibodies. • Diagnostic sensitivity of 95% and specificity of 98%. • Tested with panels of validated sera from broad host range.

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.

Mapping of foot-and-mouth disease virus antigenic sites recognized by single-domain antibodies reveals different 146S particle specific sites and particle flexibility.

Vaccination with intact (146S) foot-and-mouth disease virus (FMDV) particles is used to control FMD. However, 146S particles easily dissociate into stable pentameric 12S particles which are less immunogenic. We earlier isolated several single-domain antibody fragments (VHHs) that specifically bind either 146S or 12S particles. These particle-specific VHHs are excellent tools for vaccine quality control. In this study we mapped the antigenic sites recognized by these VHHs by competition ELISAs, virus neutralization, and trypsin sensitivity of epitopes. We included two previously described monoclonal antibodies (mAbs) that are either 12S specific (mAb 13A6) or 146S specific (mAb 9). Although both are 12S specific, the VHH M3F and mAb 13A6 were found to bind independent antigenic sites. M3F recognized a non-neutralizing and trypsin insensitive site whereas mAb 13A6 recognized the trypsin sensitive VP2 N-terminus. The Asia1 146S-specific site was trypsin sensitive, neutralizing and also recognized by the VHH M8F, suggesting it involves the VP1 GH-loop. The type A 146S-specific VHHs recognized two independent antigenic sites that are both also neutralizing but trypsin insensitive. The major site was further mapped by cross-linking mass spectrometry (XL-MS) of two broadly strain reactive 146S-specific VHHs complexed to FMDV. The epitopes were located close to the 2-fold and 3-fold symmetry axes of the icosahedral virus 3D structure, mainly on VP2 and VP3, overlapping the earlier identified mAb 9 site. Since the epitopes were located on a single 12S pentamer, the 146S specificity cannot be explained by the epitope being split due to 12S pentamer dissociation. In an earlier study the cryo-EM structure of the 146S-specific VHH M170 complexed to type O FMDV was resolved. The 146S specificity was reported to be caused by an altered conformation of this epitope in 12S and 146S particles. This mechanism probably also explains the 146S-specific binding by the two type A VHHs mapped by XL-MS since their epitopes overlapped with the epitope recognized by M170. Surprisingly, residues internal in the 146S quaternary structure were also cross-linked to VHH. This probably reflects particle flexibility in solution. Molecular studies of virus-antibody interactions help to further optimize vaccines and improve their quality control.

Emergency FMD Serotype O Vaccines Protect Cattle against Heterologous Challenge with a Variant Foot-and-Mouth Disease Virus from the O/ME-SA/Ind2001 Lineage.

Vaccination is one of the best approaches to control and eradicate foot-and-mouth disease (FMD). To achieve this goal, vaccines with inactivated FMD virus antigen in suitable adjuvants are being used in addition to other control measures. However, only a limited number of vaccine strains are commercially available, which often have a restricted spectrum of activity against the different FMD virus strains in circulation. As a result, when new strains emerge, it is important to measure the efficacy of the current vaccine strains against these new variants. This is important for countries where FMD is endemic but also for countries that hold an FMD vaccine bank, to ensure they are prepared for emergency vaccination. The emergence and spread of the O/ME-SA/Ind-2001 lineage of viruses posed a serious threat to countries with OIE-endorsed FMD control plans who had not reported FMD for many years. In vitro vaccine-matching results showed a poor match (r1-value < 0.3) with the more widely used vaccine strain O1 Manisa and less protection in a challenge test. This paper describes the use of the O3039 vaccine strain as an alternative, either alone or in combination with the O1 Manisa vaccine strain with virulent challenge by a O/ME-SA/Ind-2001d sub-lineage virus from Algeria (O/ALG/3/2014). The experiment included challenge at 7 days post-vaccination (to study protection and emergency use) and 21 days post-vaccination (as in standard potency studies). The results indicated that the O3039 vaccine strain alone, as well as the combination with O1 Manisa, is effective against this strain of the O/ME-SA/Ind/2001d lineage, offering protection from clinical disease even after 7 days post-vaccination with a reduction in viraemia and virus excretion.

Novel Capsid-Specific Single-Domain Antibodies with Broad Foot-and-Mouth Disease Strain Recognition Reveal Differences in Antigenicity of Virions, Empty Capsids, and Virus-Like Particles.

Foot-and-mouth disease (FMD) vaccine efficacy is mainly determined by the content of intact virions (146S) and empty capsids (75S). Both particles may dissociate into 12S subunits upon vaccine manufacturing, formulation, and storage, reducing vaccine potency. We report the isolation of capsid-specific llama single-domain antibodies (VHHs) with broad strain recognition that can be used to quantify intact capsids in FMD vaccines by double antibody sandwich (DAS) ELISA. One capsid-specific VHH displayed remarkably broad strain reactivity, recognizing 14 strains representing the 13 most important lineages of serotype A, and two VHHs cross-reacted with other serotypes. We additionally show that the newly isolated VHHs, as well as previously characterized VHHs, can be used to identify antigenic differences between authentic 146S and 75S capsids, as well as corresponding genetically engineered virus-like particles (VLPs). Our work underscores that VHHs are excellent tools for monitoring the quantity and stability of intact capsids during vaccine manufacturing, formulation, and storage, and additionally shows that VHHs can be used to predict the native-like structure of VLPs.

Mathematical Quantification of Transmission in Experiments: FMDV Transmission in Pigs Can Be Blocked by Vaccination and Separation.

Quantitative understanding of transmission with and without control measures is important for the control of infectious diseases because it helps to determine which of these measures (or combinations thereof) will be effective to reduce transmission. In this paper, the statistical methods used to estimate transmission parameters are explained. To show how these methods can be used we reviewed literature for papers describing foot-and-mouth disease virus (FMDV) transmission in pigs and we used the data to estimate transmission parameters. The analysis showed that FMDV transmits very well when pigs have direct contact. Transmission, however, is reduced when a physical barrier separates infected and susceptible non-vaccinated pigs. Vaccination of pigs can prevent infection when virus is administered by a single intradermal virus injection in the bulb of the heel, but it cannot prevent infection when pigs are directly exposed to either non-vaccinated or vaccinated FMDV infected pigs. Physical separation combined with vaccination is observed to block transmission. Vaccination and separation can make a significant difference in the estimated number of new infections per day. Experimental transmission studies show that the combined effect of vaccination and physical separation can significantly reduce transmission (R < 1), which is a very relevant result for the control of between-farm transmission.

Quantifying the Transmission of Foot-and-Mouth Disease Virus in Cattle via a Contaminated Environment.

Indirect transmission via a contaminated environment can occur for a number of pathogens, even those typically thought of as being directly transmitted, such as influenza virus, norovirus, bovine tuberculosis, or foot-and-mouth disease virus (FMDV). Indirect transmission facilitates spread from multiple sources beyond the infectious host, complicating the epidemiology and control of these diseases. This study carried out a series of transmission experiments to determine the dose-response relationship between environmental contamination and transmission of FMDV in cattle from measurements of viral shedding and rates of environmental contamination and survival. Seven out of ten indirect exposures resulted in successful transmission. The basic reproduction number for environmental transmission of FMDV in this experimental setting was estimated at 1.65, indicating that environmental transmission alone could sustain an outbreak. Importantly, detection of virus in the environment prior to the appearance of clinical signs in infected cattle and successful transmission from these environments highlights there is a risk of environmental transmission even before foot-and-mouth disease (FMD) is clinically apparent in cattle. Estimated viral decay rates suggest that FMDV remained viable in this environment for up to 14 days, emphasizing the requirement for stringent biosecurity procedures following outbreaks of FMD and the design of control measures that reflect the biology of a pathogen.IMPORTANCE Effective control of a disease relies on comprehensive understanding of how transmission occurs, in order to design and apply effective control measures. Foot-and-mouth disease virus (FMDV) is primarily spread by direct contact between infected and naive individuals, although the high levels of virus shed by infected animals mean that virus can also be spread through contact with contaminated environments. Using a series of transmission experiments, we demonstrate that environmental transmission alone would be sufficient to sustain an outbreak. Key observations include that a risk of transmission exists before clinical signs of foot-and-mouth disease (FMD) are apparent in cattle and that survival of virus in the environment extends the transmission risk period. This study highlights the role a contaminated environment can play in the transmission of FMDV and presents approaches that can also be applied to study the transmission of other pathogens that are able to survive in the environment.

Emergency Foot-and-Mouth Disease Vaccines A Malaysia 97 and A22 Iraq 64 Offer Good Protection against Heterologous Challenge with A Variant Serotype A ASIA/G-IX/SEA-97 Lineage Virus.

The continuous emergence of foot-and-mouth disease virus (FMDV) serotype A variants in South East Asia is of concern for international FMDV antigen banks, especially when in vitro tests predict a low antigenic match. A vaccination-challenge study was performed by using two emergency FMDV vaccines with A22 Iraq 64 (A22 IRQ) and A Malaysia 97 (A MAY 97) strains, against challenge with a variant strain of FMDV A/Asia/G-IX/SEA-97 lineage at 7- and 21-day post-vaccination (dpv). At 7 dpv, three of five female calves vaccinated with A MAY 97 and four of five vaccinated with A22 IRQ did not show lesions on the feet and were considered protected, while at 21 dpv all five calves were protected with each vaccine, indicating equal efficacy of both vaccine strains. Calves were protected despite relatively low heterologous neutralizing antibody titers to the challenge virus at the time of challenge. All the calves developed antibodies to the non-structural proteins, most likely due to the direct intradermolingual (IDL) inoculation. Only one calf from the A MAY 97-7 group had infectious virus in the serum 1-3-day post-challenge (dpc), while no virus could be isolated from the serum of cattle challenged on 21 dpv. The virus could be isolated from the oral swabs of all calves, 1-7 dpc with viral RNA detected 1-10 dpc. Nasal swabs were positive for virus 1-6 dpc in a small number of calves. The time between vaccination and infection did not have an impact on the number of animals with persistent infection, with almost all the animals showing viral RNA in their oro-pharyngeal fluid (probang) samples up to 35 dpc. Despite the poor in vitro matching data and field reports of vaccine failures, this study suggests that these vaccine strains should be effective against this new A/Asia/G/SEA-97 variant, provided they are formulated with a high antigen dose.

Cross-Protection Induced by a A/MAY/97 Emergency Vaccine Against Intra-Serotype Heterologous Challenge with a Foot-and-Mouth Disease Virus from the A/ASIA/G-VII Lineage.

Since 2015, outbreaks of foot-and-mouth disease (FMD) in the Middle East have been caused by a new emerging viral lineage, A/ASIA/G-VII. Invitro vaccine matching data indicated that this virus poorly matched (low r1-value) with vaccines that were being used in the region as well as most other commercially available vaccines. The aim of this study was to assess the performance of two candidate vaccines against challenge with a representative field virus from the A/ASIA/G-VII lineage. The results from an initial full dose protection study provided encouraging data for the A/MAY/97 vaccine, while the A22/IRQ/64 vaccine only protected 2/7 vaccinated animals. In view of these promising results, this vaccine was tested in a potency test (PD50) experiment in which 5 cattle were vaccinated with a full dose, 5 cattle with a 1/3 dose and 5 cattle with a 1/9 dose of vaccine. At 21 days post vaccination these vaccinated cattle and 3 control cattle were challenged intradermolingually with a field isolate from the A/ASIA/G-VII lineage. The intra-serotype heterologous potency test resulted in an intra-serotype heterologous potency of 6.5 PD50/dose. These data support previous studies showing that a high potency emergency vaccine can protect against clinical disease when challenged with a heterologous strain of the same serotype, indicating that not only the r1-value of the vaccine, but also the homologous potency of a vaccine should be taken into account when advising vaccines to control an outbreak.

Replacement of foot-and-mouth disease virus cattle tongue titration by in vitro titration

Titration of foot-and-mouth disease cattle challenge virus in cattle tongue has been the standard for many years in many countries, although titration in animals has been replaced by in vitro methods for all other applications. The objective of the analysis was the replacement of in vivo titration of cattle challenge virus by in vitro titration. Using data from 32 in vivo titration experiments together with the in vitro titration results of the same samples obtained by plaque count on primary lamb or pig kidney cells, as well as data from the virus isolation control chart used in the laboratory, we show that the reproducibility of the in vitro titration is much higher than that of the in vivo titration. The titer on primary kidney cells was on average 1.4 log10 higher than the titer determined by titration in cattle tongue (PFU/ml compared to bovine ID50/ml), but the difference varied among different strains. The study also shows that the probability of infection in cattle tongue is high even when a lower challenge dose is used, which makes the variability between strains less important. Based on these results, we propose to change the standard dose for cattle challenge from 104 bovine ID50 to 105.4 PFU, and to replace the in vivo cattle tongue titration method with the in vitro titration method.

A foot-and-mouth disease SAT2 vaccine protects swine against experimental challenge with a homologous virus strain, irrespective of mild pathogenicity in this species.

FMDV serotype SAT2 is most frequently associated with outbreaks in ruminants. However, the risk of it spreading from cattle to pigs cannot be excluded. To assess the efficacy of an SAT2-type FMD inactivated vaccine against homologous challenge in pigs, a suitable challenge strain adapted to pigs was produced. After two passages in two pigs each, a FMDV stock of SAT2 challenge strain was produced. This material was used to infect two groups of five pigs. The first group being vaccinated 28 days before challenge and the other one left as an unvaccinated control. Clinical signs were recorded, virus shedding was assessed on mouth swabs, and neutralising antibody titres were determined. At least 80% of the vaccinated pigs were protected against clinical disease. Furthermore, no virus shedding was observed in any of the vaccinated pigs. This study shows that experimentally inoculated pigs can become infected with a SAT2 serotype. Furthermore, vaccination offers protection against generalisation and viral excretion, confirming the potential of vaccination as an important tool in the control of FMD in pigs.

Isolation of Single-Domain Antibody Fragments That Preferentially Detect Intact (146S) Particles of Foot-and-Mouth Disease Virus for Use in Vaccine Quality Control.

Intact (146S) foot-and-mouth disease virus (FMDVs) can dissociate into specific (12S) viral capsid degradation products. FMD vaccines normally consist of inactivated virions. Vaccine quality is dependent on 146S virus particles rather than 12S particles. We earlier isolated two llama single-domain antibody fragments (VHHs) that specifically recognize 146S particles of FMDV strain O1 Manisa and shown their potential use in quality control of FMD vaccines during manufacturing. These 146S-specific VHHs were specific for particular O serotype strains and did not bind strains from other FMDV serotypes. Here, we describe the isolation of 146S-specific VHHs against FMDV SAT2 and Asia 1 strains by phage display selection from llama immune libraries. VHHs that bind both 12S and 146S particles were readily isolated but VHHs that bind specifically to 146S particles could only be isolated by phage display selection using prior depletion for 12S particles. We obtained one 146S-specific VHH-M332F-that binds to strain Asia 1 Shamir and several VHHs that preferentially bind 146S particles of SAT2 strain SAU/2/00, from which we selected VHH M379F for further characterization. Both M332F and M379F did not bind FMDV strains from other serotypes. In a sandwich enzyme-linked immunosorbent assay (ELISA) employing unlabeled and biotinylated versions of the same VHH M332F showed high specificity for 146S particles but M379F showed lower 146S-specificity with some cross-reaction with 12S particles. These ELISAs could detect 146S particle concentrations as low as 2.3-4.6 µg/l. They can be used for FMD vaccine quality control and research and development, for example, to identify virion stabilizing excipients.

Efficacy of a high potency O1 Manisa foot-and-mouth disease vaccine in cattle against heterologous challenge with a field virus from the O/ME-SA/Ind-2001 lineage collected in North Africa.

Outbreaks of foot-and-mouth disease (FMD) in North Africa (2013) and the Gulf States (2013) of the Middle East have been caused by a FMD viral lineage (O/ME-SA/Ind-2001) that was before 2013 restricted to the Indian Sub-continent. This study was undertaken to assess the in vivo efficacy of a FMD virus emergency vaccine type O1 Manisa against heterologous challenge with a representative field virus (O/ALG/3/2014) from this emerging lineage. This widely available vaccine was selected since in vitro vaccine-matching results gave inconclusive results as to whether or not it would be protective. Three groups of five cattle were vaccinated with O1 Manisa (homologous potency ≥6PD50/dose) using study guidelines outlined in the European Pharmacopeia, and challenged at 21days post-vaccination by tongue inoculation. All animals that were vaccinated with the lowest dose (1/16) of vaccine developed generalised FMD, defined as vesicular lesions at the feet. One animal vaccinated with a 1/4 dose of the vaccine also developed generalised disease, as did two animals vaccinated with the full dose of vaccine. These results indicate that the heterologous potency of this high potency O1 Manisa vaccine was approximately 3.5 PD50/dose. These data support the use of the O1 Manisa vaccine for FMD control in areas where FMDV is endemic e.g. North Africa, and motivate further studies to evaluate other vaccine candidates (or multivalent combinations) that might be potentially used for emergency purposes in FMD-free settings.

The serological response against foot and mouth disease virus elicited by repeated vaccination of dairy cattle.

In Israel, cattle are annually vaccinated against foot and mouth disease (FMD). If infections with FMD virus occur in dairy farms it mainly involves heifers and calves, while older dairy cows seldom become infected. We hypothesized that this difference in susceptibility between adult cows and the young heifers and calves is due to stronger and more stable immune response elicited by multiple vaccinations. In order to test this hypothesis, 99 dairy cattle, divided into six groups according to number of prior vaccinations, were annually vaccinated with a trivalent vaccine (A, O and Asia-1) and followed during two consecutive years. In total 988 sera were sampled at 11 time points. Virus neutralization tests (VNT) were performed in order to determine the neutralizing antibody titers (NAT) against the vaccine homologous serotypes: O-4625, O-Manisa, Asia-1-Shamir and the heterologous serotype A-Turkey-20/2006. A similar NAT pattern was observed to all serotypes and therefore statistical analysis was restricted to O-4625 serotype. In the 'high vaccination' groups (cows that were vaccinated at least four times before the study), high NAT were found on the beginning of the trial and no or only a mild increase of NAT was observed following further vaccinations. Additionally, in the 'high vaccination' groups, the percentage of cows that had a NAT higher than 2.0 (log10) by the end of the 1st year was significantly higher than in the 'low vaccination' groups (cows vaccinated only three times or less before the study). We conclude that starting from the 5th vaccination, the NAT increase following vaccination is mild and NAT are persistent, suggesting reduction of the frequency of routine vaccination after multiple vaccinations is possible.

The long term effect of age and maternally derived antibodies against foot and mouth disease on the serological response following vaccination in young dairy calves.

In Israel, occurrence of foot and mouth disease (FMD) in dairy farms is rare. However, when FMD outbreaks occur, dairy calves are the most affected, despite routine vaccination. Contradictory findings exist regarding the effect of age and maternally derived antibodies (MDA) on the serological response following vaccinations against FMD in dairy calves. Furthermore, the long term effect of FMD vaccination regimen during early life was rarely assessed. This study was conducted in order to assess both the short and long term effects. In total 44 non-vaccinated calves were divided into four groups of different age. Calves were vaccinated up to four times and 484 serum samples were collected on 11 time points in a period of 70weeks. Virus neutralizing tests were performed in order to determine the neutralizing antibody titers (NAT) against the vaccine strains (homologous serotypes): O-4625, O-Manisa, ASIA-1-Shamir and the heterologous serotype A-Turkey-20/2006. A similar NAT pattern was observed to all serotypes and therefore statistical analysis was restricted to O-4625 serotype. The MDA titer was negatively associated with the age of the calves and the MDA half-life was 22days. We demonstrated that early vaccination of calves (younger than three months) resulted in low NAT, even after four repeated vaccinations, compared with vaccination of calves older than three months. The percentage of time in which these calves had a NAT above 2.0 (log10) between the age of six months and 1.5years was significantly lower compared to older calves (older than three months). Additionally, we found that by increasing the frequency of vaccination in calves older than three months, it is possible to reach high NAT by the age of one year. Adoption of such a vaccination regimen in Israel as well as other FMD endemic countries may allow better protection against FMD in dairy calves and reduction in FMD incidence.

Proper Timing of Foot-and-Mouth Disease Vaccination of Piglets with Maternally Derived Antibodies Will Maximize Expected Protection Levels.

We investigated to what extent maternally derived antibodies interfere with foot-and-mouth disease (FMD) vaccination in order to determine the factors that influence the correct vaccination for piglets. Groups of piglets with maternally derived antibodies were vaccinated at different time points following birth, and the antibody titers to FMD virus (FMDV) were measured using virus neutralization tests (VNT). We used 50 piglets from 5 sows that had been vaccinated 3 times intramuscularly in the neck during pregnancy with FMD vaccine containing strains of FMDV serotypes O, A, and Asia-1. Four groups of 10 piglets were vaccinated intramuscularly in the neck at 3, 5, 7, or 9 weeks of age using a monovalent Cedivac-FMD vaccine (serotype A TUR/14/98). One group of 10 piglets with maternally derived antibodies was not vaccinated, and another group of 10 piglets without maternally derived antibodies was vaccinated at 3 weeks of age and served as a control group. Sera samples were collected, and antibody titers were determined using VNT. In our study, the antibody responses of piglets with maternally derived antibodies vaccinated at 7 or 9 weeks of age were similar to the responses of piglets without maternally derived antibodies vaccinated at 3 weeks of age. The maternally derived antibody levels in piglets depended very strongly on the antibody titer in the sow, so the optimal time for vaccination of piglets will depend on the vaccination scheme and quality of vaccine used in the sows and should, therefore, be monitored and reviewed on regular basis in countries that use FMD prophylactic vaccination.

Full protection of swine against foot-and-mouth disease by a bivalent B-cell epitope dendrimer peptide.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. We have reported (Cubillos et al., 2008) that a synthetic dendrimeric peptide consisting of four copies of a B-cell epitope [VP1(136-154)] linked through thioether bonds to a T-cell epitope [3A(21-35)] of FMDV [B4T(thi)] elicits potent B- and T-cell specific responses and confers solid protection in pigs to type C FMDV challenge. Herein we show that downsized versions of this peptide bearing two copies of a B-cell epitope from a type O isolate and using thioether [B2T(thi)] or maleimide [B2T(mal)] conjugation chemistries for their synthesis elicited in swine similar or higher B and T-cell specific responses than tetravalent B4T(thi). Moreover, while partial protection was observed in animals immunized with B4T(thi) (60%) and B2T(thi) (80%), B2T(mal) conferred full (100%) protection against FMDV challenge, associated to high levels of circulating IgG2 and mucosal IgGA, and entirely prevented virus shedding. Interestingly, B2T(mal) is also the most advantageous option in terms of synthetic practicality. Taken together, the results reported here point out to B2T(mal) as a highly valuable, cost-effective FMDV candidate vaccine.

Quantification of transmission of foot-and-mouth disease virus caused by an environment contaminated with secretions and excretions from infected calves.

Foot-and-mouth disease virus (FMDV) infected animals can contaminate the environment with their secretions and excretions. To quantify the contribution of a contaminated environment to the transmission of FMDV, this study used calves that were not vaccinated and calves that were vaccinated 1 week prior to inoculation with the virus in direct and indirect contact experiments. In direct contact experiments, contact calves were exposed to inoculated calves in the same room. In indirect contact experiments, contact calves were housed in rooms that previously had held inoculated calves for three days (either from 0 to 3 or from 3 to 6 days post inoculation). Secretions and excretions from all calves were tested for the presence of FMDV by virus isolation; the results were used to quantify FMDV transmission. This was done using a generalized linear model based on a 2 route (2R, i.e. direct contact and environment) SIR model that included information on FMDV survival in the environment. The study shows that roughly 44% of transmission occurs via the environment, as indicated by the reproduction ratio R0(2R)environment that equalled 2.0, whereas the sum of R0(2R)contact and R0(2R)environment equalled 4.6. Because vaccination 1 week prior to inoculation of the calves conferred protective immunity against FMDV infection, no transmission rate parameters could be estimated from the experiments with vaccinated calves. We conclude that a contaminated environment contributes considerably to the transmission of FMDV therefore that hygiene measures can play a crucial role in FMD control.

No significant differences in the breadth of the foot-and-mouth disease serotype A vaccine induced antibody responses in cattle, using different adjuvants, mixed antigens and different routes of administration.

Inactivated whole virus foot-and-mouth disease (FMD) vaccines are used worldwide for protection against FMD, but not all vaccines induce protection against all genetic variants of the same FMD virus serotype. The aim of this study is to investigate whether the "breadth" of the antibody response against different strains of the same FMD virus serotype in cattle could be improved by using a different adjuvant, a mix of antigens and/or different routes of administration. To this end, six groups of five cattle were vaccinated with different FMD virus serotype A strain vaccines formulated with Montanide ISA 206 VG adjuvant. Antibody responses for homologous and heterologous cross-reactivity against a panel of 10 different FMD virus serotype A strains were tested by a liquid-phase blocking ELISA. Results of cattle vaccinated with ISA 206 VG adjuvanted vaccine were compared with results obtained in a previous study using aluminium hydroxide-saponin adjuvant. No significant effect of adjuvant on the breadth of the antibody response was observed, neither for mixing of antigens nor for the route of administration (subcutaneous vs. intradermal). Comparison of antigen payload, however, increased both homologous and heterologous titres; a 10-fold higher antigen dose resulted in approximately four times higher titres against all tested strains. Our study shows that breadth of the antibody response depends mainly on the vaccine strain; we therefore propose that, for vaccine preparation, only FMD virus strains are selected that, among other important characteristics, will induce a wide antibody response to different field strains.