P.L. Eblé

Passive immunization of pigs with bispecific llama single-domain antibody fragments against foot-and-mouth disease and porcine immunoglobulin

Foot-and-mouth disease (FMD) is a contagious viral disease of cloven-hoofed animals that occasionally causes outbreaks in Europe. We aim to develop an immunotherapy that confers rapid protection against FMD in outbreak situations. For this purpose, we previously isolated llama single-domain antibody fragments (VHHs) binding to FMDV or porcine immunoglobulin (pIg). The pIg binding VHHs can be genetically fused to other VHHs, resulting in so-called VHH2s. As compared to non-pIg binding VHHs such VHH2s have a 100-fold increased serum half-life which is essential for effective immunotherapy. We have now produced three bispecific VHH2s by fusion of three FMDV binding VHHs (clones M3, M8 and M23) to a pIg binding VHH (VI-4). The resulting yeast-produced VHH2s bound FMDV and pIg with high affinity (K(D) about 1nM) and neutralized FMDV in vitro as efficiently as their monovalent counterparts. To evaluate their therapeutic potential all three VHH2s were intramuscularly injected into pigs that were challenge infected with FMDV 24h later. Administration of one of these VHH2s (M23ggsVI-4) reduced the viremia significantly (P=0.0034) and reduced viral shedding almost significantly (P=0.11). However, it did not prevent development of clinical signs or transmission of FMDV. These results suggest that immunotherapy using bispecific VHH2s binding to FMDV and pIg is possible in principle, but should be improved by increasing VHH2 dosage or using more potent VHH2s.

No between-pen transmission of foot-and-mouth disease virus in vaccinated pigs.

Many studies have shown transmission of foot-and-mouth disease virus (FMDV) within groups of pigs, even when vaccinated, but only limited information is available on transmission between pens. Three new experiments were carried out in two replicates, which consisted of infectious pigs housed in a central pen surrounded by four separate pens. First, all pigs were non-vaccinated and pens were separated by a walkway of 40-70 cm. Second, all pigs were non-vaccinated again but pens were adjacent. Third, this was repeated with all pigs vaccinated. From the experiments it is concluded that a single pen wall of solid wood between adjacent pens reduces the FMDV transmission 10- to 20-fold compared to within-pen transmission, for both non-vaccinated and for vaccinated pigs. Vaccination of pigs reduces the pen-to-adjacent pen R to values significantly below 1, whereas previous studies showed that it does not reduce the within-pen R(0) to values below 1.

Passive immunization with llama single-domain antibody fragments reduces foot-and-mouth disease transmission between pigs

We aim to develop a method that confers rapid protection against foot-and-mouth disease (FMD) by passive immunization with recombinant llama single-domain antibody fragments (VHHs). Previously constructed genetic fusions of two VHHs (VHH2s) that either neutralizes FMDV or binds to porcine immunoglobulin to increase the serum half-life, conferred only limited protection to pigs. We therefore now generated VHH3s containing an additional FMDV binding VHH. Two VHH3s neutralized FMDV more potently than single VHHs and were highly produced by yeast cells. Injection of a mixture of these two VHH3s 24h before FMD challenge infection of pigs reduced and delayed the development of clinical disease, viraemia and viral shedding. Furthermore, it significantly (P=0.023) delayed FMD transmission. Thus, we have shown a proof of concept of passive FMD immunoprophylaxis using VHHs.

Intradermal vaccination of pigs against FMD with 1/10 dose results in comparable vaccine efficacy as intramuscular vaccination with a full dose.

The aim of this study was to investigate whether intradermal (ID) vaccination against foot-and-mouth disease (FMD) is suitable as an alternative for the usually used intramuscular (IM) route. We compared vaccine efficacy in groups of pigs in which vaccine administration differed with respect to antigen payload of the vaccine, administrated volume and administration route. When compared with pigs that were IM vaccinated with a full dose vaccine with a standard antigen payload, pigs vaccinated ID with 1/10 dose of the same vaccine were equally protected against clinical disease and subclinical virus shedding. The ID vaccinated pigs were protected against virus shedding at a significant lower VN-titre as compared to IM vaccinated pigs, suggesting that immune responses other than neutralising antibodies also contributed to protection. We conclude that the ID route might be a good alternative for IM application, as ID application might induce a very efficient immunological response against FMD and, moreover, because the dose required by the ID route is lower compared to the IM route, ID application may reduce the production costs per dose of FMD vaccine markedly.

Estimation of the transmission of foot-and-mouth disease virus from infected sheep to cattle

The quantitative role of sheep in the transmission of foot-and-mouth disease virus (FMDV) is not well known. To estimate the role of sheep in the transmission of FMDV, a direct contact transmission experiment with 10 groups of animals each consisting of 2 infected lambs and 1 contact calf was performed. Secretions and excretions (oral swabs, blood, urine, faeces and probang samples) from all animals were tested for the presence of FMDV by virus isolation (VI) and/or RT-PCR. Serum was tested for the presence of antibodies against FMDV. To estimate FMDV transmission, the VI, RT-PCR and serology results were used. The partial reproduction ratio R0p i.e. the average number of new infections caused by one infected sheep introduced into a population of susceptible cattle, was estimated using either data of the whole infection chain of the experimental epidemics (the transient state method) or the final sizes of the experimental epidemics (the final size method). Using the transient state method, R0p was estimated as 1.0 (95% CI 0.2 - 6.0) using virus isolation results and 1.4 (95% CI 0.3 - 8.0) using RT-PCR results. Using the final size method, R0p was estimated as 0.9 (95% CI 0.2 - 3.0). Finally, R0p was compared to the R0’s obtained in previous transmission studies with sheep or cattle only. This comparison showed that the infectivity of sheep is lower than that of cattle and that sheep and cattle are similarly susceptible to FMD. These results indicate that in a mixed population of sheep and cattle, sheep play a more limited role in the transmission of FMDV than cattle.

Efficacy of a high potency O 1 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.

Intratypic heterologous vaccination of calves can induce an antibody response in presence of maternal antibodies against foot-and-mouth disease virus

BackgroundMaternal antibodies can interfere with foot-and-mouth disease vaccination. In this study we determined whether intratypic heterologous vaccination could help to improve herd immunity.ResultsIn unvaccinated calves, a half-life of maternal antibodies of 21 days was determined. At two weeks of age, calves without maternal antibodies showed a good antibody response against both vaccines used in the trial, while in calves with maternal antibodies no antibody response to homologous vaccination (A Turkey 14/98) but a limited antibody response to intratypic heterologous vaccination (A22 Iraq) was observed.ConclusionTwo weeks old calves without maternal antibodies respond well to vaccination, but when emergency vaccination is carried out in a region that uses prophylactic vaccination, using an intratypic heterologous vaccine strain may improve the immunity in calves with maternal antibodies.

Vaccination of cattle only is sufficient to stop FMDV transmission in mixed populations of sheep and cattle.

We quantified the transmission of foot-and-mouth disease virus in mixed cattle-sheep populations and the effect of different vaccination strategies. The (partial) reproduction ratios (R) in groups of non-vaccinated and vaccinated cattle and/or sheep were estimated from (published) transmission experiments. A 4 × 4 next-generation matrix (NGM) was constructed using these estimates. The dominant eigenvalue of the NGM, the R for a mixed population, was determined for populations with different proportions of cattle and sheep and for three different vaccination strategies. The higher the proportion of cattle in a mixed cattle-sheep population, the higher the R for the mixed population. Therefore the impact of vaccination of the cattle is higher. After vaccination of all animals R = 0·1 independent of population composition. In mixed cattle-sheep populations with at least 14% of cattle, vaccination of cattle only is sufficient to reduce R to < 1.

Identification of factors associated with increased excretion of foot-and-mouth disease virus

We investigated which variables possibly influence the amount of foot-and-mouth disease virus (FMDV) shed in secretions and excretions by FMDV infected animals, as it is likely that the amount of FMDV shed is related to transmission risk. First, in a separate analysis of laboratory data, we showed that the total amount of FMDV in secretions and excretions from infected animals is highly correlated with maximum titres of FMDV. Next, we collected data from 32 published scientific articles in which FMDV infection experiments were described. The maximum titres of FMDV reported in different secretions and excretions (the response variable) and the experimental conditions in which they occurred (the explanatory variables), were recorded in a database and analyzed using multivariate regression models with and without random effects. In both types of models, maximum titres of FMDV were significantly (p<0.05) associated with types of secretions and excretions, animal species, stage of the disease and days post infection. These results can be used to prioritize biosecurity measures in contingency plans.

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.