Innate immune defenses induced by CpG do not promote vaccine-induced protection against foot-and-mouth disease virus in pigs.

Emergency vaccination as part of the control strategies against foot-and-mouth disease virus (FMDV) has the potential to limit virus spread and reduce large-scale culling. To reduce the time between vaccination and the onset of immunity, immunostimulatory CpG was tested for its capacity to promote early protection against FMDV challenge in pigs. To this end, CpG 2142, an efficient inducer of alpha interferon, was injected intramuscularly. Increased transcription of Mx1, OAS, and IRF-7 was identified as a sensitive measurement of CpG-induced innate immunity, with increased levels detectable to at least 4 days after injection of CpG formulated with Emulsigen. Despite this, CpG combined with an FMD vaccine did not promote protection. Pigs vaccinated 2 days before challenge had disease development, which was at least as acute as that of unvaccinated controls. All pigs vaccinated 7 days before challenge were protected without a noticeable effect of CpG. In summary, our results demonstrate the caution required when translating findings from mouse models to natural hosts of FMDV.

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.

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.

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.

International bank for foot-and-mouth disease vaccine: assessment of Montanide ISA 25 and ISA 206, two commercially available oil adjuvants.

The International Vaccine Bank at Pirbright has recently installed large-scale vaccine formulation equipment for the preparation of oil-adjuvanted vaccines. Such vaccines are claimed to offer a number of advantages over Al(OH)3, particularly their ability to raise better immunity in pigs. This paper reports on the potency in pigs, cattle and guinea-pigs of foot-and-mouth disease vaccines prepared with two novel oil adjuvants, Montanide ISA 25 and 206 (Seppic, Paris). The results indicate that vaccines adjuvanted with these oils retain potency for a longer period than our conventional aqueous formulation, following storage at +4 degrees C, and elicit good antibody responses in both pigs and cattle regardless of injection route. In addition they gave no evidence of toxicity or prolonged pyrexia following their administration. Local reactions at the site of inoculation were not observed in cattle vaccinated intramuscularly, even following a booster dose. Pigs vaccinated intramuscularly only showed local reactions if the volume administered exceeded the 2.0 ml dose or the animals received a second vaccination. These observations on the efficacy of such oil formulated vaccines suggest that they have potential as an alternative to the current aqueous formulation.

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.

A protective anti-peptide antibody against the immunodominant site of the A24 Cruzeiro strain of foot-and-mouth disease virus and its reactivity with other subtype viruses containing the same minimum binding sequence.

A synthetic peptide vaccine of the general sequence Cys-Cys-(200-213)-Pro-Pro-Ser-(l41-158)-Pro-Cys-Gly(peptide A40), where the numbered residues refer to the VP1 sequence of foot-and-mouth disease virus (FMDV) strain A24 Cruzeiro, has previously been shown to elicit neutralizing and protective antibodies in guinea-pigs and cattle. To examine this immunogenic tract in more detail monoclonal antibodies (MAbs) were raised to this peptide. One such MAb C1.1, which recognized the homologous peptide, bound to native virus, neutralized infectivity in vitro and passively protected mice from challenge. Using overlapping dodecameric peptides the minimum binding 'footprint' of this MAb incorporated residues 149-154 which were respectively Gly-Ser-Leu-Ala-Ala-Arg. Since this 'footprint' occurs in several other A subtype strains of FMDV, the extent to which MAb C1.1 could cross-react was also examined. Using a liquid-phase competition ELISA, only viruses with a sequence that encompassed the same minimum binding 'footprint', namely A27 Cundinamarca Colombia/76, A Argentina/79, and A Venceslau Brazil/76 reacted with similar affinity against MAb C1.1. However, further serological examination of C1.1 with these viruses by indirect ELISA, in vitro neutralization and passive protection showed clear functional disparity. In contrast to the liquid-phase ELISA, the ability of C1.1 to react with electrostatically bound virus varied significantly depending on the subtype examined. Moreover, the capacity of this MAb to neutralize these subtypes showed wide divergence which was mirrored by the protection data.

Recognition of foot-and-mouth disease virus and its capsid protein VP1 by bovine peripheral T lymphocytes.

The role of T cells in immunity to foot-and-mouth disease virus is still poorly defined compared to that of the humoral response. In this paper we describe a systematic, longitudinal study on the cellular recognition of FMDV and its subunit protein VP1 by bovine peripheral blood T lymphocytes. Multiple vaccination with a single virus serotype induced a serotype cross-reactive proliferative T cell repertoire that varied in magnitude between individual animals and with the serotype of the vaccine used. Primary proliferative T cell responses of vaccinated and acutely infected cattle were weak relative to the magnitude of responses determined for the same animals after boosting. In contrast, the level of circulating antibody produced after both primary and secondary exposure to virus was good. Phenotypic analysis of lymphocytes from vaccinated or infected cattle showed a small increase in CD8+ T cells after infection compared to vaccination. However, in general the profiles of circulating lymphocytes elicited were similar. Thus, we were not able to use proliferative or phenotypic analyses to distinguish between vaccinated and convalescent cattle. T cell recognition of VP1 by multiply-vaccinated cattle was serotype-specific implying that the cross-reactive responses observed with whole virus may be attributed to proteins other than VP1. In contrast to other studies, immunization with recombinant VP1 induced only low levels of neutralizing antibody and failed to elicit profound proliferative responses or protection ever after two immunizations.

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.

Emergency vaccination against foot-and-mouth disease: rate of development of immunity and its implications for the carrier state.

Emergency foot-and-mouth disease (FMD) vaccines prepared from antigens held in the International Vaccine Bank at Pirbright were administered to cattle and pigs and the levels of protection were assessed following challenge by contact with infected pigs. Both Al(OH)3/saponin and oil-based cattle vaccines proved to be extremely effective and protected soon after vaccination (4 days postvaccination), whereas the pigs were seldom protected before 21 and 28 days postvaccination, probably due to lower levels of antibody and overwhelming challenge conditions. Early production of cattle occurred in the absence of significant levels of circulating antibody as measured by enzyme-linked immunosorbent assay, a neutralization assay and a passive protection test. A large number of the cattle vaccinated with the O1 Lausanne strain of FMD and subsequently challenged with this virus became persistently infected and there appeared to be a correlation with the time interval between vaccination and challenge. When the same cattle were vaccinated approximately 4 months later with a different strain of FMD, C1 Oberbayern, and challenged with this strain, the number of persistently infected animals was considerably lower. The results are discussed in the context of the use of emergency vaccines to prevent the dissemination of FMD from disease foci.

Detection of foot-and-mouth disease viral sequences in clinical specimens and ethyleneimine-inactivated preparations by the polymerase chain reaction.

The polymerase chain reaction method (PCR) has been applied to the diagnosis of foot-and-mouth disease viral RNA in tissues and, particularly, oesophageal-pharyngeal fluid (probang) samples from cattle. Using primer sets which corresponded to conserved regions of the VP1 sequence of the viral genome, it was possible to amplify sequences regardless of the serotype/strain of the virus. In comparison with infectivity assays, the PCR was generally more sensitive although there were a number of examples where only infectivity was detectable. In experiments with uninfected probang samples deliberately seeded with a dilution series of virus, the PCR proved to be approximately 10(4) times more sensitive than infectivity assays. This greater sensitivity was attributed, in part, to the ability of the PCR to amplify specifically from non-infectious RNA preparations. This enabled the identification, by sequencing, of viral RNA from chemically inactivated virus concentrates typical of those used for commercial vaccine production. Amplification of specific PCR products was also achieved with virus eluted from commercial vaccine, including preparations which had been stored for more than 10 years at 4 degrees C. The PCR technique is of considerable value, therefore, both as a complement to infectivity assays and as a powerful tool in vaccine-associated studies.

Caracterización detallada de algunos anticuerpos monoclonales que reconocen sitios antigénicos en el VP1 del virus de la fiebre aftosa

La mayoría de los anticuerpos monoclonales (AcMs) desarrollados contra las cepas O1 Campos, A24 Cruzeiro y C Indaial de la fiebre aftosa y que previamente mostraron reacción con el VP1 en "Western Blots", reconocieron péptidos sintéticos correspondientes a la secuencia 141-158 de esta proteína. Una número significativo de los AcMs que reconocieron péptidos sintéticos, lo hicieron de forma heteróloga u homóloga. Así muchos de los AcMs A24 Cruzeiro reconocieron un péptido O1 Kaufbeuren y atribuyeron su reacción cruzadas a la región 141-158 del VP1. Un AcMc3 Indaial reconoció péptidos correspondientes a tres serotipos y la actividad cruzada fue atribuida a la secuencia 200-213 del VP1. Generalmente, la reacción cruzada de un AcM dado se observó con el virus y con el péptido, pero no con los dos antígenos.

Most monoclonal antibodies (MAbs) raised against the O1 Campos, A24 Cruzeiro and C3 Indaial strains of foot-and-mouth disease virus (FMD), and which were previously shown to react with VP, in Western Blots, recognized synthetic peptides corresponding to the 141-158 sequence of this protein. A significant number of the MAbs which recognized synthetic peptides did so in a heterologous as well as homologous manner. Thus, many of the A24 Cruzeiro MAbs recognized an O 1 Kaufbeuren peptide and the cross-reactivity was ascribed to thE 141-158 region of the VP1. One C3 Indaial MAb recognized peptides corresponding to three serotypes and the cross-reactivity was ascribed to the 200-213 sequence of the VP1. Usually, the cross-reactivity of a given MAb was observed with either the virus or the peptide but not with both antigens.

La importancia de PANAFTOSA en los programas de investigación y de desarrollo para el control y erradicación de la fiebre aftosa en las Américas

La contribución del Centro Panamericano de Fiebre Aftosa para el conocimiento de las enfermedades vesiculares de los animales, especialmente la fiebre aftosa, ha sido de gran trascendencia para los programas de control iniciados durante las últimas cuatro décadas. En esta revisión se examina la importancia de la investigación en el pasado y los enfoques futuros para la investigación y el desarrollo sobre el control y erradicación de la enfermedad de las Américas.

The contribution made by the Pan American Foot-and Mouth Disease Center to our knowledge of vesicular diseases of animals and, in particular, foot-and-mouth disease, has assisted greatly the control programs initiated during the last four decades. This review examines the relevance of past research and explores future directions for research and development in relation to the control and eradication of the disease from the Americas.

La importancia de PANAFTOSA en los programasa de investigación y de desarrollo para el control y erradicación de la fiebre aftosa en las Américas / The relevance of the research and development programs of PANAFTOSA to the control and eradication of foot and mouth disease in the Americas

La contribución del Centro Panamericano de Fiebre Aftosa para el conocimiento de las enfermedades vesiculares de los animales, especialmente la fiebre aftosa, ha sido de gran transcedencia para los programas de control iniciados durante las últimas cuatro décadas. En esta revisión se examina la importancia de la ingestigación en el pasado y los enfoques futuros para la investigacion y el desarrollo sobre el control y erradicación de la enfermedad de las Américas

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.

Cross-reactive and serotype-specific antibodies against foot-and-mouth disease virus generated by different regions of the same synthetic peptide.

Synthetic peptides based on the VP1 proteins of two serotypes of foot-and-mouth disease virus (FMDV) and having the general formula C-C-(200-213)-P-P-S-(141-158)-P-C-G induce heterologous as well as homologous protection against challenge. Substitution of the sequence consisting of residues 200 to 213 (200-213 sequence) with a second copy of the homologous 141-158 sequence (i.e., homodimers) resulted in failure of either serotype peptide to protect heterologously. The antiviral and antipeptide titers of sera from guinea pigs immunized with the homodimeric 141-158 peptides showed serotype specificity and, with the data from the heterodimeric peptide vaccines, suggested that the C-terminal 141-158 sequence was more effectively recognized by the immune system than the N-terminal sequence. Whereas heterologous antiviral titers as measured by enzyme-linked immunosorbent assay and virus neutralization tests have not been observed with sera from cross-protected animals, epitope-mapping studies established that there was heterologous recognition of an octapeptide within the 200-213 sequence. That the 200-213 sequence was required for the induction of heterologous protection was also confirmed with a number of peptides, including hybrids based on the 200-213 sequence of one virus and the 141-158 sequence of a second virus. Thus, peptides of the general formula given above induce serotype-specific and serotype-cross-reactive protective antibodies and are unique in their induction of significant levels of heterologous protection, a property which has never been reported for whole FMDV vaccines.

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.

MHC class II restricted recognition of FMDV peptides by bovine T cells.

A putative synthetic vaccine for foot-and-mouth disease (FMDV15) has proved less successful in a host species, cattle, than predicted by results in a small-animal model. Possible reasons for this include non-recognition by T cells influenced by major histocompatibility complex (MHC)-linked immune response gene control. It is now possible to type for human leucocyte antigen (HLA) DR-like bovine MHC (BoLA) class II polymorphisms with a one-dimensional isoelectric focusing (IEF) technique. Using this method 14 unrelated cattle were selected with eight different BoLA class II IEF types. After immunization with FMDV15, 13 cattle generated a T-cell response to FMDV15. However, the fine specificity and magnitude of the response was related to BoLA class II type. The non-response by one animal and low response by two other animals were associated with two of the BoLA class II types. Response to the region 149-158 was immunodominant and animals which did not respond to this region had low responses to the whole peptide. Using FMDV-specific T-cell lines five BoLA class II types associated with responder animals were able to present FMDV15 in an MHC class II-restricted fashion, indicating that this peptide is capable of binding to different MHC class II molecules and may account for the broad response observed. The restriction patterns of the lines indicated that the IEF method does not distinguish all functional polymorphisms. At least two of the IEF-defined types could each be split into two distinct specificities and revealed that the three sets of animals with identical IEF types in fact expressed distinct restriction elements.

Studies on glycoprotein 13 (gp13) of equid herpesvirus 1 using affinity-purified gp13, glycoprotein-specific monoclonal antibodies and synthetic peptides in a hamster model.

Hamsters were immunized with either an affinity-purified preparation of equid herpesvirus 1 (EHV-1) glycoprotein 13 (gp13) or synthetic peptides representing three sequences within the homologous glycoprotein of EHV-4, resulting in the production of anti-peptide (in the case of peptide-immunized animals) or antivirus antibodies. The sera from gp13-immunized hamsters contained antibodies which showed virus-neutralizing activity and complement-mediated antibody lysis of EHV-1-infected target cells. These hamsters were protected from EHV-1 challenge. The characteristics of a panel of anti-gp13 monoclonal antibodies (P28, P17, 14H7, 16E4 and 16H9) were assessed both in vivo and in vitro. 16E4 and P28 showed high levels of complement-mediated neutralization of virus, complement-mediated lysis of virus-infected target cells and passive protection of hamsters. Furthermore, epitope mapping studies demonstrated that this glycoprotein contains a neutralizing epitope recognized by EHV-1-immune horse serum. The data imply that gp13 has potential as a candidate antigen for a molecular vaccine.