Paul V. Barnett

The Cell Attachment Site on Foot-and-Mouth Disease Virus Includes the Amino Acid Sequence RGD (Arginine-Glycine-Aspartic Acid)

The amino acid sequence RGD (arginine-glycine-aspartic acid) is highly conserved in the VP1 protein of foot-and-mouth disease virus (FMDV), despite being situated in the immunodominant hypervariable region between amino acids 135 and 160. RGD-containing proteins are known to be important in promoting cell attachment in several different systems, and we report here that synthetic peptides containing this sequence are able to inhibit attachment of the virus to baby hamster kidney (BHK) cells. Inhibition was dose-dependent and could be reversed on removal of the peptide. A synthetic peptide corresponding to a portion of the same hypervariable region but not containing the RGD sequence did not inhibit virus attachment under the same conditions. Antibody against the RGD region of VP1 blocked attachment of the virus to BHK cells, and neutralizing monoclonal antibodies, which neutralize virus by preventing cell attachment, were blocked by RGD-containing peptides from binding virus in an ELISA test. Cleavage of the C-terminal region of virus VP1 in situ with proteolytic enzymes reduced cell attachment, and antiserum against a peptide corresponding to this region was also able to inhibit attachment of virus to BHK cells. These results indicate that the amino acid sequence RGD at positions 145 to 147 and amino acids from the C-terminal region of VP1 (positions 203 to 213) contribute to the cell attachment site on FMDV for BHK cells.

Relationship Between Clinical Signs and Transmission of an Infectious Disease and the Implications for Control

Livestock experiments provide precise parameters for incubation and infectious periods for foot-and-mouth disease virus. Control of many infectious diseases relies on the detection of clinical cases and the isolation, removal, or treatment of cases and their contacts. The success of such “reactive” strategies is influenced by the fraction of transmission occurring before signs appear. We performed experimental studies of foot-and-mouth disease transmission in cattle and estimated this fraction at less than half the value expected from detecting virus in body fluids, the standard proxy measure of infectiousness. This is because the infectious period is shorter (mean 1.7 days) than currently realized, and animals are not infectious until, on average, 0.5 days after clinical signs appear. These results imply that controversial preemptive control measures may be unnecessary; instead, efforts should be directed at early detection of infection and rapid intervention.

Neutralizing Epitopes of Type O Foot-and-Mouth Disease Virus. II. Mapping Three Conformational Sites with Synthetic Peptide Reagents

Four neutralizing monoclonal antibodies (MAbs), recognizing three functionally independent, conformational sites on type O foot-and-mouth disease virus (FMDV) failed to react with immobilized structural proteins or synthetic peptides but bound to the isolated capsid protein VP1 and peptides in solution. Inhibition ELISA techniques were, therefore, applied using peptide antigens and anti-peptide sera to block MAb binding to virus particles, permitting the identification of those portions of the VP1 protein contributing to the epitopes. The binding site of one MAb, which neutralized a range of type O FMDV isolates, was shown to have components within regions 146 to 150 and 200 to 213 of VP1 with a critical involvement of the amino acids at positions 146 and 206 or 207. The determinants recognized by two other MAbs which were directed at similar, but not identical, epitopes from a second site included components from the 200 to 213 and 143 to 146 regions with amino acids 143 and 144, respectively, appearing critical for the inhibition of the virus binding of the two antibodies. These results demonstrate that the two previously identified immunogenic tracts of VP1 are brought into proximity in the quaternary structure of the virion to form an antigenic domain containing several conformational epitopes, some of which are functionally independent. A fourth, strain-specific MAb was effectively blocked from reacting with virus by peptides corresponding to residues 161 to 180 and 200 to 213.

Longevity of antibody and cytokine responses following vaccination with high potency emergency FMD vaccines.

The ability of high potency emergency foot-and-mouth disease (FMD) vaccines to promote sustainable immune responses in sheep and pigs following a single application was examined. All vaccine formulations induced a rapid seroconversion in both species, as expected, which was maintained at near peak titres for up to 6 months in sheep and 7 months in pigs. The Montanide ISA 206 formulation gave the best results in sheep. Vaccinated pigs challenged with homologous FMDV were protected from disease at 7 months post vaccination. Systemic levels of cytokines IL-6, IL-8, and in some pigs IL-12, increased following vaccination and were often maintained at an increased level for the duration of the trials. These initial results suggest that high potency vaccines may promote longer lasting immunity than the conventional lower potency vaccines in ruminants and a comparable response in pigs. Results indicate that in an outbreak situation, should emergency vaccination be done with these high potency vaccines, protection should be conferred for a long enough period for the outbreak to be brought under control without the need to revaccinate. Given the increased interval for re-vaccination the use of high potency vaccines for routine prophylactic campaigns could provide a more cost-effective and efficient means of maintaining herd immunity and is an area thus worthy of further examination.

Further studies on the early protective responses of pigs following immunisation with high potency foot and mouth disease vaccine

The ability of an emergency oil adjuvanted foot-and-mouth disease (FMD) vaccine to elicit early protective immunity in pigs against direct contact homologous challenge was examined. All vaccinates showed reduced viraemia and shedding of FMDV, and certain animals were protected, showing no clinical signs. IL-6, IL-8 and IL-12 were consistently detected in challenged animals that had been vaccinated. Other cytokines--IL-1, IL-2, TNF, TGF and interferons--were not detected. This demonstrates that the vaccine did not induce a systemic inflammatory response, nor a systemic elevation of T lymphocyte activity. Although the IL-6 and IL-8 did not relate to protection, IL-12 production was highest in the protected vaccinated pigs. Thus, the induction of monocytic cell activity, demonstrable by the production of IL-6, IL-8 and IL-12, appears to play a critical role in FMDV emergency vaccine induction of the innate immune defences which relate to early protection against FMD. The possible modes of defence in which such cytokine activity would be involved are discussed.

Neutralizing epitopes of type O foot-and-mouth disease virus. I: Identification and characterization of three functionally independent, conformational sites

Eleven neutralizing monoclonal antibodies (MAbs) were produced to the O1BFS 1860/67 strain of foot-and-mouth disease virus (FMDV), and were characterized for their ability to bind viral and subviral antigens in different ELISA tests and to neutralize heterologous type O isolates. Neutralization escape variants of the homologous virus, isolated under pressure from five of these MAbs, were used in cross-neutralization tests with all of the 11 antibodies. These studies identified three functionally independent, conformational, neutralizing sites. The most conformationally dependent site bound antibody which neutralized a range of type O virus isolates. A second site was less dependent on conformation and was recognized by antibody that was strain-specific. The least conformational site bound MAbs which showed limited cross-neutralization of other type O strains. This latter site appeared to be immunodominant and contained several overlapping epitopes which showed some differences in their specificities. Isoelectrofocusing and sequencing studies of the variants strongly suggested that polypeptide VP2 contributes to the immunodominant site.

Foot-and-mouth disease vaccine potency testing: determination and statistical validation of a model using a serological approach

European foot-and-mouth disease vaccine manufacturers are required to quantify the efficacy of their product in accordance with the European Pharmacopoeia (EP). The method used most often to establish the potency of foot-and-mouth disease vaccines requires viral challenge of vaccinated cattle. Alternative approaches, such as challenge-free serological assessments have many advantages over existing methods and could be used if robust statistical models could be developed that related antibody titres to protection from challenge. Logistic regression analysis of data from two independent research laboratories, representing six of the seven main serotypes of FMD, permitted the parameterisation of these models and indicated that a significant relationship existed between antibody titre and probability of protection. Furthermore, no significant differences were observed in the parameters of logistic models fitted to different strains within the serotypes A, O, and SAT-3, or when strains from serotypes A, O, and Asia-1, or SAT-1 and SAT-3, were combined. However, significant differences in the model parameters did exist between different laboratories. Using these models a bootstrap analysis suggested that for vaccines that induced consistently high titres, as few as six to eight individual animals could be used to establish with confidence the minimum protective doses that would protect 50% of vaccinated animals. We conclude that a serologically evaluated truncated test that eliminates the need to virus challenge cattle is a credible alternative for quantifying vaccine potency.

Experimental evaluation of foot-and-mouth disease vaccines for emergency use in ruminants and pigs: a review

Changes to foot-and-mouth disease (FMD) control policies since 2001 mean that emergency vaccination must be considered more readily as a control measure in the future. Since field application of vaccine for emergency use has only rarely been applied, the effectiveness of single dose administration, as a control measure in an outbreak situation, is poorly understood. In this review we consider all the available experimental data from studies utilizing either experimental or readily available, commercially produced vaccines, in order to assess their likely effectiveness as an additional means of controlling FMD transmission and spread in an emergency. Overall it is concluded that such vaccines offer an additional and valuable means of FMD control for both ruminants and pigs. They are able to reduce clinical disease, sub-clinical infection and excretion and onward transmission of virus. However, to be most effective, vaccination should be rapidly applied to give maximum opportunity for immunity to develop. We also identify areas for future research and emphasize the importance of vaccine efficacy studies in providing data for models that can help to predict the efficacy of differing FMD control strategies.

Interspecies Major Histocompatibility Complex-Restricted Th Cell Epitope on Foot-and-Mouth Disease Virus Capsid Protein VP4

T-cell epitopes within viral polypeptide VP4 of the capsid protein of foot-and-mouth disease virus were analyzed using 15-mer peptides and peripheral blood mononuclear cells (PBMC) from vaccinated outbred pigs. An immunodominant region between VP4 residues 16 and 35 was identified, with peptide residues 20 to 34 (VP4-0) and 21 to 35 (VP4-5) particularly immunostimulatory for PBMC from all of the vaccinated pigs. CD25 upregulation on peptide-stimulated CD4(+) CD8(+) cells-dominated by Th memory cells in the pig-and inhibition using anti-major histocompatibility complex class II monoclonal antibodies indicated recognition by Th lymphocytes. VP4-0 immunogenicity was retained in a tandem peptide with the VP1 residue 137 to 156 sequential B-cell epitope. This B-cell site also retained immunogenicity, but evidence is presented that specific antibody induction in vitro required both this and the T-cell site. Heterotypic recognition of the residue 20 to 35 region was also noted. Consequently, the VP4 residue 20 to 35 region is a promiscuous, immunodominant and heterotypic T-cell antigenic site for pigs that is capable of providing help for a B-cell epitope when in tandem, thus extending the possible immunogenic repertoire of peptide vaccines.

Chimeric foot-and-mouth disease viruses: Evaluation of their efficacy as potential marker vaccines in cattle

Previous work in pigs, has demonstrated that full protection against foot-and-mouth disease (FMD) can be achieved following vaccination with chimeric foot-and-mouth disease virus (FMDV) vaccines, in which the VP1 G-H loop had been substituted with that from another serotype. If proven to be effective in other economically important species such as cattle, such vaccine constructs could be trialed as potential marker vaccines. Here, we determine if G-H loop chimera FMDV vaccines can: (i) protect cattle from virus challenge and (ii) induce an antibody response that would enable the identification of infection, regardless of vaccination status. Inactivated, oil adjuvanated, chimeric vaccine constructs, based on the backbone sequence of the A(12)119 serotype virus, fully protected cattle from challenge 21 days post-vaccination. Differentiation assays developed for use in this study were able to identify sub-clinical infection, which in one vaccinated animal, persisted beyond day 32 post-challenge. This paper emphasises the importance of epitopes outside of the VP1 G-H loop for protective immunity in cattle, and demonstrates that chimeric FMDV vaccines could prove to be useful marker vaccines for the future.