Natural and vaccine induced immunity to FMD.

A brief overview of the foot-and-mouth disease (FMD) literature over the last 100 years will give the impression that a great deal is known about the immune response of livestock to infection and vaccination. At the practical level, this is indeed the case and our knowledge is more than adequate in relation to the production and supply of potent vaccines for the control of the disease. The deficiencies in our understanding of the immune response are at the fundamental level and, arguably, stand in the way of its rational manipulation to achieve goals such as life-long immunity conferred by vaccination. Most of the research activity to date has focused on T cell dependency of the immune response of livestock and important B (and probably T) cell epitopes and has been used by researchers to design highly sophisticated novel vaccines and delivery systems. None of these, to the author's knowledge, exceeds the potency obtained with a good commercial vaccine. Although it is not yet possible to see a clear direction for the development of improved formulations, it is important to reflect on our current knowledge of natural and vaccine-induced immunity and some of the issues surrounding modern inactivated FMD vaccines. This process will perhaps help to discriminate the fact from the fiction and serve to focus on precisely what is needed or desirable for improved products.

Heterotypic recognition of recombinant FMDV proteins by bovine T-cells: the polymerase (P3Dpol) as an immunodominant T-cell immunogen.

In this study we have examined the recognition of VP0, VP1, VP2, VP3 and P3Dpol by PBMC and CD4+ T-cells from infected, vaccinated-challenged, and multiply-vaccinated (O1, A24, C1 or ASIA1) cattle using recombinant proteins of an O1 serotype virus. The structural protein VP1 was recognised in an homotypic context whereas VP2, VP3, VP4 and P3Dpol were also recognised by T-cells from animals exposed to heterotypic viruses. Only the non-structural protein P3Dpol was consistently recognised by T-cells from the majority of animals examined and heterotypic recognition correlated with the presence of serologically detectable P3Dpol in purified virus. Thus, P3Dpol is a major cross-reactive immunodeterminant of FMDV, eliciting heterotypic T-cell responses and, therefore, with possible potential for inclusion in a subunit vaccine.

The expression of the proteins of equine herpesvirus 1 which share homology with herpes simplex virus 1 glycoproteins H and L.

Several expression systems were used in studies aimed at characterizing the equine herpesvirus 1 (EHV-1) glycoprotein H and L homologues of HSV-1 (EHV-1 gH and gL) and the products were compared to the authentic proteins synthesized in virus infected cells. Using an in vitro transcription/translation system two gH species were detected (an unprocessed 89 kDa and a processed 116 kDa product). Three low molecular weight proteins were found in the case of gL (21.8 kDa, 22.9 kDa and 26.9 kDa) and these showed a slight reduction in mobility on the addition of microsomal membranes to the reactions. A gL fusion protein was produced in pGEX-2T, expression being confirmed by Western blotting using a gL-specific antiserum raised against a peptide incorporating the 13 carboxyl terminal amino acids of the protein. A gH specific peptide antiserum precipitated both gH and two smaller proteins from EHV-1 infected cells thought to be two forms of gL. Insect cells infected with gH or gL baculovirus recombinants were used to vaccinate C3H (H-2k) mice. Some protection against EHV-1 infection was conferred to the gH inoculated mice. The results will enable further studies on the importance of the gH and gL interaction in the pathogenesis of EHV-1 to be evaluated and their potential in contributing to a subunit vaccine to be assessed.

Isolation of capsid proteins of foot-and-mouth disease virus by chromatofocusing.

A method for the isolation of foot-and-mouth disease virus (FMDV) capsid proteins was developed. The FMDV capsid proteins VP1, VP2, VP3 and VP0 were isolated from sucrose gradient purified virus by chromatofocusing in a pH 7.4-4.0 gradient on Polybuffer exchanger PBE 94. Under the conditions used the proteins eluted in the sequence VP1, VP2, VP0 (when present) and VP3. Capsid protein VP4 did not elute and could not be isolated by this method. Protein concentration in the eluate was monitored by the use of a radiolabelled marker and recoveries of approximately 50% of the input marker could be achieved when using up to 15 mg of virus and a 30-ml column. The high capacity and relative simplicity of chromatofocusing make it a useful alternative to other methods of purifying proteins.

Maturation of functional antibody affinity in animals immunised with synthetic foot-and-mouth disease virus.

A good correlation exists between specific neutralising antibody titre and protection against challenge with foot-and-mouth disease virus (FMDV) in infected or virus-vaccinated cattle, but not in the case of animals immunised with synthetic FMDV peptides. Therefore, mechanisms other than simple neutralisation are likely to be important in vivo. Antibody affinity may influence the protective capacity of sera from immunised animals and experiments were carried out to measure the functional affinity for synthetic FMDV peptide of sera from guinea pigs and cattle given various synthetic vaccines. In guinea pigs given a single dose of synthetic vaccine, antibody affinity increased with time after immunisation. In cattle, however, administration of a second dose of peptide 21 days after the first markedly retarded the process of affinity maturation. For guinea pig sera of equivalent neutralising activity, those of higher functional affinity had higher protective indices than those of lower functional affinity. Knowledge of the importance of antibody affinity in protection against FMD is important for an improved understanding of the mechanisms of protection and for the design of novel vaccines.

Natural and vaccine-induced immunity to foot and mouth disease: the prospects for improved vaccines.

The review considers both the immune responses of livestock to foot and mouth disease (FMD) virus after infection or vaccination, and the characteristics and properties of FMD viruses and vaccines which are relevant to protection. Particular attention is given to possible approaches which could be used to improve conventional vaccines or produce novel vaccines against this most important disease.

Foot-and-mouth disease: the risk for Great Britain after 1992.

Mass annual vaccination against foot-and-mouth disease, previously applied by eight member states in the European Community (EC), was progressively phased out during 1990-91. The other four member states (the United Kingdom, Denmark, the Republic of Ireland and Greece) either never have vaccinated or ceased to do so several years ago. The EC should increase its international competitiveness if it maintains its present foot-and-mouth disease-free, non-vaccinating status. Freedom from disease and a harmonised disease control policy will also permit unrestricted movement of livestock and animal products throughout the EC when the single market is completed in 1992. Vaccination against foot-and-mouth disease on continental Europe has greatly reduced the number of outbreaks during the last 30 years and this reduction has been of indirect benefit to Great Britain. However, the cessation of vaccination will result in a higher proportion of fully susceptible cattle and in the event of outbreaks will increase the likelihood of the rapid dissemination of virus and increase the risk that the infection will enter Great Britain. The main risks of entry are likely to be associated with live animals in which the disease can be mild or inapparent, ie, sheep and goats, and with airborne virus originating from pigs on the nearby continent especially in Brittany and the Benelux countries where they are present in very high densities.

Prospects for improved foot and mouth disease vaccines.

Improved foot and mouth disease (FMD) vaccines which have increased efficiency and stability are highly desirable. Work which aims to increase the stability of FMD virus particles for vaccine use is described and the use of these particles and of antigenic fragments of the virus for controlled release vaccine products is considered.

Optimisation of the immune response to foot-and-mouth disease vaccines.

Considering the many variables influencing the immune response of the host to vaccination against foot-and-mouth disease (FMD), the properties and characteristics of the vaccine and recommendations concerning its mode of application offer the best opportunity for the manufacturer to provide safe, effective protection of livestock. There are a number of critical elements in the production of FMD vaccines, such as the selection of appropriate strain(s), which have a direct bearing on the quality of the immune response. Equally, development of effective immunity depends on proper and timely application of good quality vaccine. In contrast, several of the potential variables in vaccination against FMD such as the use of oil adjuvants for cattle are probably less critical than is sometimes perceived. This presentation considers some of the many variables involved in the production and use of FMD vaccines.

Thermal stability of foot-and-mouth disease virus.

The thermal stabilities of 146S component of seven strains of foot-and-mouth disease virus were found to differ considerably. Inactivation of infectivity with acetylethyleneimine (AEI) reduced the thermal stabilities of all but one of the viruses. Treatment of AEI inactivated and control virus preparations with glutaraldehyde stabilized 146S particles to a considerable extent, whereas treatment with dimethyl suberimidate was less effective. In similar experiments with 75S, natural empty particles, the thermal stabilities were lower than those of the corresponding 146 S particles. Treatment of 75S particles with AEI appeared to have no direct effect on the protein-protein interactions involved in 75S capsid integrity. As with 146S particles, glutaraldehyde stabilized 75S particles.

Observations and implications of proteolysis in preparations of foot-and-mouth disease virus.

The integrity of the VP1 protein of foot-and-mouth disease virus was assessed by polyacrylamide gel electrophoresis (following storage at 4 degrees C of conventional tissue culture preparations and concentrated preparations of the virus. There was little evidence of VP1 degradation in tissue culture filtrates whereas considerable degradation was observed throughout a range of different concentrates. The use of the proteolytic enzyme inhibitor 'Trasylol' appeared to inhibit VP1 degradation in some virus preparations. Vaccination experiments with guinea pigs indicate that cleavage of O BFS 1860 virus by a range of proteolytic enzymes was always associated with a lowered stimulation of neutralising antibody and total antibody. Experiments with six other strains treated with trypsin gave similar results to those obtained with O BFS 1860.

An international collaborative study on foot and mouth disease virus assay methods. 2. Quantification of 146S particles.

Workers in 11 laboratories in Europe and one in North America participated in a collaborative study to assess the variability of a sucrose gradient procedure used for the quantification of foot and mouth disease virus (FMDV). To this end, a range of standards was distributed from one of the participating laboratories. A series of adenine preparations were used to assess the various spectrophotometers/UV monitors and it showed most to be accurate and linear in their responses. The FMDV and MS2 ribophage standards were used to assess the sucrose gradient procedure and indicated low levels of within laboratory variation whereas between laboratory variation was greater. Recalculation of results from the unprocessed data in the coordinating laboratory permitted the identification and reduction of one source of between laboratory variation. Despite the observed variations, the results indicated that meaningful comparisons could be made between the data of different laboratories provided that a procedure similar to the one described in this report is used.

The detection and inhibition of proteolytic enzyme activity in concentrated preparations of inactivated foot-and-mouth disease virus.

Proteolytic enzyme activity was detected in a large number of concentrated preparations of inactivated foot-and-mouth disease virus. Several lines of evidence indicated that at least some of this activity could be attributed to BHK cells, although low levels of microbial contamination in many of our preparations could not be discounted and would certainly enhance the cellular proteolytic activity. From an experiment with different concentrations of trypsin, it was concluded that the proteolytic activities of virus concentrates were sufficient to cause significant degradation of VP1. It was also shown that Trasylol and ox serum were effective inhibitors of proteolytic enzymes in concentrated virus preparations stored at 4 degrees C for 8 months.

Tryptic peptide analysis of the structural proteins of vesicular stomatitis virus.

The individual structural polypeptides of vesicular stomatitis virus have been examined by tryptic peptide analysis of 35S-methionine preparations labelled in vivo and 125I-preparations labelled in vitro. Isolates of the two classical serotypes of the virus (Indiana and New Jersey) and of a sub-type of the Indiana serotype, Brazil virus, were compared. The study showed that the major internal proteins of all three viruses gave similar maps, whereas the surface glycoproteins gave distinct maps that had very few spots in common. The map of the glycoprotein of Brazil virus, which has been shown previously to be more closely related serologically to Indiana virus than to New Jersey virus, did not show any greater similarity to the Indiana virus than to the New Jersey virus glycoprotein. On the other hand, peptide maps of the nucleoprotein and matrix protein showed Indiana and Brazil viruses to be more closely related to each other than to New Jersey virus.

Synthetic peptide vaccines against foot-and-mouth disease. I. Duration of the immune response and priming in guinea-pigs, rabbits and mice.

The immunogenicity of two aphthovirus-specific synthetic peptides was investigated. One peptide copied the sequence of amino acids 141 to 160 from the capsid VP1 of the aphthovirus strains O1 BFS 1860 and O1 Kaufbeuren (O peptide), the other copied the equivalent sequence from aphthovirus strain A24 Cruzeiro (A peptide). Peptide coupled to keyhole limpet haemocyanin (KLH) stimulated a long-lasting immune response in guinea-pigs and rabbits. Significant levels of antibody were detectable at least one year after vaccination, although the reactivity of the antibody depended on the species and the peptide used. In some circumstances the peptides were able to prime the immune system such that a subsequent dose of peptide boosted antibody production. This effect, also, was dependent on the species of experimental animal and on the peptide used, an observation which has important implications for the use of such peptides as vaccines.

Synthetic peptide vaccines against foot-and-mouth disease. II. Comparison of the response of guinea-pigs, rabbits and mice to various formulations.

The immune response of guinea-pigs to vaccination with either of two aphthovirus-specific synthetic peptides was investigated. One peptide copied the sequence of amino acids 141 to 160 from the VP1 of aphthovirus strains O1BFS 1860 and O1 Kaufbeuren (O peptide), the other copied the equivalent sequence from aphthovirus strain A24 Cruzeiro (A peptide). The immune response was enhanced when the peptides were conjugated to a carried protein, but the choice of carrier protein and cross-linking agent was not critical in obtaining enhancement. The response was greatest when the peptide or peptide-carrier conjugate was adjuvanted in Freund's complete adjuvant (FCA). The O peptide was poorly immunogenic and did not confer protection against challenge with infectious virus, whereas the A peptide had good immunogenicity and did confer protection. This reflected the relative immunogenicity of the parent viruses. Rabbits, three strains of guinea-pigs and seven strains of mice were vaccinated with the O peptide conjugated to keyhole limpet haemocyanin (KLH). Considerable variation was observed between the responses of each strain and it is proposed that this relates to their repertoire of immune response genes.

International bank for foot-and-mouth disease vaccine: stability studies with virus concentrates and vaccines prepared from them.

An international bank foot-and-mouth disease (FMD) vaccine has been established at the Pirbright Laboratory of the AFRC Institute for Animal Health. The bank is based on concentrated virus preparations stored in the gaseous phase of liquid nitrogen and is capable of producing up to 0.5 million cattle doses of each of five common strains of the virus (FMDV) for the member nations of the bank. This paper describes the initial and subsequent testing of the virus concentrates and vaccines prepared from them. There was no evidence of deterioration of the virus concentrates during liquid nitrogen storage. High levels of protection of cattle and guinea-pig were achieved when vaccines were used immediately following preparation from the recently thawed virus concentrates. In contrast, storage of vaccines at 4 degrees C for one or more months resulted in loss of potency in guinea-pigs and was attributed, in part to proteolytic enzymes in the virus concentrates.

Heterotypic recognition of foot-and-mouth disease virus by cattle lymphocytes.

Lymphoproliferation against foot-and-mouth disease (FMD) virus was examined using peripheral blood mononuclear cells from vaccinated cattle. Ten weeks after revaccination the optimum conditions for proliferation were obtained with 1 microgram/ml of purified virus after 5 to 6 days in culture. This contrasted with the response at 20 months post-revaccination, when the response required less antigen and showed a peak response after 3 to 4 days in culture. Proliferation was specific for FMD virus, but was cross-reactive between serotypically distinct strains of the virus. The proliferative response to isolated virus proteins (VP) involved all three major capsid proteins (VP1, -2 and -3), although the proliferation of lymphocytes from heterotypically vaccinated cattle was due to VP3. Furthermore, the response induced by purified virus, chemically fixed virus and subunit virus particles was indistinguishable and thus it is likely that processing was required for the induction of proliferation. Together these data strongly suggest that FMD virus-induced lymphoproliferation is T cell-mediated and that VP3 may contain dominant, cross-reactive sequences.

The stability and potency of vaccines prepared from inactivated foot-and-mouth disease virus concentrates.

The stability of 146S particles in concentrates of foot-and-mouth disease virus stored at 4 degrees C was similar to that of 146S particles in a conventional virus preparation. Proteolytic degradation of VPl was not observed in the stored conventional virus preparation or inhibitor-supplemented concentrate but was observed in a supplement-free concentrate. The potencies of vaccines made from the conventional and concentrated preparations and stored in parallel at 4 degrees C appeared to decrease after 16 weeks. The vaccines made from the supplement-free concentrate and the Trasylol supplemented concentrate appeared to be at least as potent as the conventional vaccine and were clearly superior to vaccine made from ox serum supplemented concentrate.