Very fast (and safe) inactivation of foot-and-mouth disease virus and enteroviruses by a combination of binary ethyleneimine and formaldehyde.

For FMD vaccine production, inactivation of the FMD virus is the most critical step. Formerly, from 1940 onwards, the virus was inactivated with formaldehyde. This inactivation was relatively slow, about 0.2 - 0.3 log 10 per hour. Because formaldehyde not only reacts with the virus produced but with many other components in the medium, such as proteins and amino acids, its concentration can become rate-limiting and inactivation plots may show tailing-off, resulting in residual infectivity. Many of the bad stories of post-vaccination outbreaks date back to the use of formaldehyde-inactivated vaccines (e.g. the outbreaks in France in 1981 and in Eastern Germany causing the Danish outbreak in 1982). Much faster and safer inactivation was obtained with aziridines and in the 1980s binary ethyleneimine (BEI) was introduced in practically all vaccine production laboratories. If inactivation plots are made of every production batch, as is now required by the European Pharmacopoeia, and these plots show proper inactivation rates, vaccines can considered to be completely safe. Under optimal conditions, inactivation rates are in the range of 0.5 - 1.0 log 10 per hour. In general, the inactivation takes 40-48 hours,which will guarantee complete inactivation of all virus particles in a batch. Since formaldehyde (FA), the 'classical' inactivating agent, inactivates at a rate of 0.3 logs per hour only, a significant contribution of FA to the inactivation of BEI can hardly be expected. However, here it is shown that FA added during the BEI-inactivation process strongly augments inactivation rates with a hundred to thousand-times (to 2.5-3.5 logs per hour). This will enable inactivation during a working day or just overnight with even higher safety levels of the vaccines. Also, it is known that formaldehyde cross-links viral proteins which will stabilise the antigen. The short inactivation times will limit proteolytic destruction of 146 S antigen and increase antigen yields. It is expected that by the cross-linking activity of FA the stability of the antigen (and of vaccines) and the endurance of the immune response will be favourably influenced.

Development and performance of inactivated vaccines against foot and mouth disease.

The historical background of foot and mouth disease (FMD) vaccine production is briefly described. Improvements achieved through the use of monolayer and suspension cultures are outlined. Elements that are crucial in the production of modern vaccines are discussed, such as inactivation of viral antigen, successive concentration and purification of the antigen and the final formulation of the vaccine. Storage of concentrated antigen at ultra-low temperatures creates greater flexibility for the producer and has also enabled national and international organisations to establish vaccine banks. The purification of FMD viral antigens, including the removal of non-structural proteins (NSPs), enables the immune responses of vaccinated animals to be distinguished from the responses of animals infected with live FMD virus. Consequently, the combined use of purified vaccine and tests for the detection of antibodies against NSPs essentially provides a marker system to distinguish between vaccinated animals that subsequently become infected and those that have not. Bearing in mind the good record of modern vaccines in the control of outbreaks and the possibility of screening vaccinated herds for carriers, the author proposes that the OIE reconsider the differences between the requirements for regaining export status following the use of stamping-out as opposed to vaccination in outbreak situations.

Developments in foot-and-mouth disease vaccines.

The current status of foot-and-mouth disease (FMD) vaccine production is reviewed. The production of antigen in bovine tongue epithelium (Frenkel culture) is described and improvements in monolayer and suspension cultures of cell lines are outlined. Inactivation of viral antigen and safety tests are discussed. A 'minimum safety level' is recommended: at the end of the inactivation process, antigen batches of any size should contain less than one virus particle. After inactivation the antigen can be formulated into a vaccine or purified and concentrated for storage at ultra-low temperatures in a vaccine bank. Vaccines prepared with the adjuvants Al(OH)3 and saponin are compared with (double) oil emulsion vaccines. Because oil vaccines can protect both cattle and pigs and induce long-term protection, they are most suitable for use in ring vaccinations. A new generation of vaccines, based on constructed modified-live viruses or (bio-) synthetic peptides, is briefly reviewed.

Protection of guinea-pigs against foot-and-mouth disease virus by immunization with a PhoE-FMDV hybrid protein.

A hybrid protein was constructed containing two antigenic determinants of the structural protein VP1 of foot-and-mouth disease virus, inserted in a cell surface-exposed region of Escherichia coli outer membrane protein PhoE. Immunization of guinea-pigs with partially purified protein resulted in high levels of neutralizing antibodies and complete protection against challenge with the virus.

Expression of foreign epitopes in P-fimbriae of Escherichia coli.

Hypervariable regions (HRs) of the major subunit of F11 fimbriae were exploited for insertion of foreign epitopes. Two insertion vectors were created that contain a unique cloning site in HR1 or HR4 respectively. Several oligonucleotides, coding for antigenic determinants derived from different pathogens, were cloned in both insertion vectors. Hybrid fimbrial subunits were generally shown to be assembled in fimbriae when the length of the inserted peptide did not exceed 14 amino acids. The inserted peptides appeared to be exposed in the fimbrial filament. One hybrid fimbrial protein induced detectable levels of antibodies against the inserted epitope if injected into mice.

Development and properties of a cell culture produced vaccine for hog cholera based on the Chinese strain.

The Chinese strain of hog cholera virus (HCV) was adapted to suspension cultures of the established swine kidney cell line SK6. The strain designated "Cedipest", is produced on the basis of a seedlot system. The masterseed virus was identified in vitro and in vivo, and was found free from extraneous pig pathogenic viruses by repeated animal inoculation followed by appropriate serological tests. A distinct and reproducible relationship was ascertained between infectivity in vitro and protection. Pigs inoculated with 400-600 TCID50 of the Cedipest strain proved fully protected against challenge with greater than 100 pig LD50 of a virulent strain of HCV at 7 days and at 6 month post vaccination.

Antigenic sites on foot-and-mouth disease virus type A10.

A set of monoclonal antibodies was used to isolate nonneutralizable foot-and-mouth disease virus variants, and the RNAs of the variants were sequenced. Cross-neutralization studies and mapping of the amino acid changes indicated two major antigenic sites. The first site was trypsin sensitive and included the VP1 140 to 160 sequence. The second site was trypsin insensitive and included mainly VP3 residues. Two minor sites were located near VP1 169 and on the C terminus of VP1. Comparison with poliovirus type 1 and human rhinovirus 14 showed a similarity in the immunogenicity of comparable sites on the viruses.

Evidence for more than one important, neutralizing site on foot-and-mouth disease virus. Brief report.

Using polyclonal sera raised against foot-and-mouth disease virus in susceptible animals, evidence was obtained for the existence of at least one further important antigenic site in addition to the neutralizing site on VP1 140-160.

Multiple variants in foot-and-mouse disease virus (FMDV) populations: the Achilles heel for peptide and rec. DNA vaccines?

Variants of type A10 FMDV were isolated by passage of virus in BHK-cells in the presence of a neutralizing anti-peptide serum or monoclonal antibodies. These variants which were no longer neutralized by the particular anti-peptide serum or monoclonal antibody were easily obtained from (crude) virus populations ("cattle" virus and BHK-adapted virus). The rapidity of isolation (in two or three passages) suggested that these variants are already present in normal virus populations. All (plaque purified) variants isolated so far seem to be different: A panel of 20 monoclonal antibodies and an anti-peptide serum showed different neutralization patterns for all isolates and parent virus. Electrofocusing patterns of many variants were found to be different showing changed charges for VP2 as well as for VP1. Thus in FMDV both VP1 and VP2 are probably involved in antigenic sites. Normally the variants escape our attention because in neutralization assays only a limited quantity of infective units are used, representing only the "top of the iceberg". In classical inactivated virus vaccines many of these variants will be represented and therefore can be expected to be "primed" immunogenetically. This will not be the case with peptide vaccines or in case of recombinant DNA products, where only one virus clone is represented. In addition, and probably more important, inactivated virus vaccines will raise antibodies against completely independent epitopes that each have a limited chance to be changed in the variants present in the challenge virus population. Thus if peptide vaccines can be composed in such a way that antibodies are raised against completely different antigenic sites the chance of break through of variants will be strongly limited and it is expected that the efficient protection of inactivated virus vaccines can be approached.

Challenging settled opinions in classic foot-and-mouth disease vaccine preparation.

In a previous study we challenged the generally accepted opinion that inactivation of FMDV by formaldehyde (FA) is an (unsafe) non-linear process. Our data showed that under proper conditions inactivation will be linear without "tailing-off". For more than forty years two other fixed beliefs existed with respect to FMD vaccine preparation: Virus must first be adsorbed to Al(OH)3-gel before being inactivated. Concentrations of formaldehyde are critical and must be within a very narrow range. Until recently the prescription of adsorption of the virus prior to inactivation made proper control of inactivation kinetics impossible. However, by eluting the virus from the gel by caesium chloride density centrifugation, the kinetics of inactivation can be studied. For adsorbed and non-adsorbed virus, identical inactivation curves were found. Antigenicity was found to be of identical "quality" for adsorbed and non-adsorbed virus. The second dogma was challenged by inactivating non-adsorbed virus with FA-concentrations of up to 6 times the one originally prescribed. In parallel inactivation was performed with acetyl-ethylene-immine (AEI). The antigen was purified after inactivation. Antigen yields after purification were systematically lower at high FA-concentrations, however, antigenicity seemed only slightly changed by the treatments: In an immunosorbent (ELISA) assay using a panel of 22 monoclonal antibodies (McAb) only few McAb's reacted differently with FA-treated antigen if compared with AEI-treated or fresh (A10) FMDV. The FA-treatment induced decreases, as well as increases in reactivities. The AEI-treated virus reacted almost identically to non-treated 146S particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of fusion proteins containing antigenic determinants of foot-and-mouth disease virus.

Part of the genome of foot-and-mouth disease virus (FMDV) type 01,BFS, including the sequence encoding the capsid polypeptide VP1, was cloned in Escherichia coli following a new cloning strategy. The clone containing the VP1 sequence was used for the construction of two expression plasmids encoding VP1 fusion proteins. Subsequently, substantial amounts of the two VP1-beta-galactosidase fusion proteins, containing either one (amino acid region 140-160) or two (amino acid regions 140-160 and 200-213) antigenic determinants of the virus, were synthesized by E. coli bacteria. The protein containing the amino acid region 140-160 of VP1 fused to beta-galactosidase efficiently induced antibodies in rabbits specifically reacting with FMDV type 01,BFS. The same protein was also capable of eliciting neutralizing antibodies. The fusion protein containing both antigenic determinants did not efficiently induce antibodies reacting with FMDV.

Epitope mapping of the outer structural protein VP1 of three different serotypes of foot-and-mouth disease virus.

All overlapping hexapeptides of the outer structural protein VP1 of type O1, type A10, and type C1 were reacted with the appropriate anti-virus, anti-viral subunit and anti-VP1 sera. The results suggest that anti-virus sera may contain activities against viral subunit and VP1 as well as against virus. Furthermore the antigenic peptides associated with the intact virion of all three serotypes are found at similar locations on their respective VP1s, and produced neutralizing activities when used for vaccination. The results further offer an explanation for the often observed cross-reactions between serotypes, especially at the level of the viral subunit and VP1. The reliability of predictions of useful peptides from hydrophilicity profiles and secondary structure predictions is questioned. Predictions based on variation profiles appear to be more useful.

An epitope located at the C terminus of isolated VP1 of foot-and-mouth disease virus type O induces neutralizing activity but poor protection.

Both whole virus particles and isolated VP1 of foot-and-mouth disease virus type O1 induce neutralizing antibodies. Results obtained with pigs vaccinated with either isolated VP1 or intact particles and subsequently challenged show that neutralizing activity induced by intact virus correlates well with protection in pigs, whereas neutralizing activity induced by isolated VP1 confers little or no protection. Further evidence suggests that the epitope responsible for the induction of neutralizing antibodies by VP1 is located at the C-terminal end of the protein between residues 200 and 210.

Small peptides induce antibodies with a sequence and structural requirement for binding antigen comparable to antibodies raised against the native protein.

Antisera were raised against the chemically synthesized peptide corresponding to each epitope of three foot-and-mouth disease virus strains. Peptide synthesis was further used to determine which amino acid residues in each epitope are important for the specificity of antisera raised against the whole virus. The specificity of the antibody paratope for its epitope was shown to depend on structure as well as sequence. Anti-virus sera demonstrated a greater specificity for the homologous peptide than did the anti-peptide sera. Two of the three peptides were able to induce neutralizing antibodies against the homologous virus. The specificities of the antibodies present in the anti-peptide sera were also inferred from the reactions of each with related sets of peptides. The cross-reactions observed for the anti-peptide sera were readily explained in terms of the antibody specificities determined to be present. The findings also suggest that the diversity of antibodies raised against small peptides is limited and is determined by the immune system. A similar limited response to the native protein was observed, which may account for the high frequency with which anti-peptide sera react with the native homologous protein.

Conditions for proper formaldehyde inactivation of foot and mouse disease alhydrogel vaccines.

The inactivation of FMDV by formaldehyde (FA) was studied under different conditions, both as free virus and (as in routine vaccine production) after adsorption of the virus to aluminium hydroxide gel (alhydrogel). In the latter case infectivity was monitored after elution of the virus from the gel by isopycnic ultracentrifugation of the virus-alhydrogel mixture in CsCl. By this method good virus recoveries were obtained. Adsorption of the virus to alhydrogel (without formaldehyde) did not reduce infectivity significantly. Both adsorbed and non-absorbed virus lost infectivity at a rate of about one log10 per day (at pH 8.5, 25 degrees C - no formaldehyde). Kinetics of FA inactivation of adsorbed and non-adsorbed virus were also identical, with a fast reduction in the initial phase. After this initial phase inactivation became linear and rather slow. No 'tailing-off' was observed. Some additives (f.i. LAH and especially Tris) reduced the inactivation rate. These findings might explain some data of others who observed 'tailing-off'.

Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid.

A procedure is described for rapid concurrent synthesis on solid supports of hundreds of peptides, of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner an immunogenic epitope of the immunologically important coat protein of foot-and-mouth disease virus (type O1) is located with a resolution of seven amino acids, corresponding to amino acids 146-152 of that protein. Then, a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope was synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined. It was found that the leucine residues at positions 148 and 151 were essential for reaction with antisera raised against intact virus. A lesser contribution was derived from the glutamine and alanine residues at positions 149 and 152, respectively. Aside from the practical significance for locating and examining epitopes at high resolution, these findings may lead to better understanding of the basis of antigen-antibody interaction and antibody specificity.

Formaldehyde inactivation of foot-and-mouth disease virus. Conditions for the preparation of safe vaccine.

The inactivation of foot-and-mouth disease virus by formaldehyde was studied under different conditions, both as free virus and (as in routine vaccine production) after adsorption of the virus to aluminium hydroxide gel (alhydrogel). In the latter case infectivity was monitored after elution of the virus from the gel by isopycnic ultracentrifugation of the virus-alhydrogel mixture in CsCl. By this method good virus recoveries were obtained. Adsorption of the virus to alhydrogel (without formaldehyde) did not reduce infectivity significantly. Both adsorbed and non-absorbed virus lost infectivity at a rate of about one log10 per day (at pH 8.5, 25 degrees C--no formaldehyde). Kinetics of formaldehyde inactivation of adsorbed and non-adsorbed virus were also identical, with a fast reduction in the initial phase (in case of O1 and A10-virus approximately one log10/hour). After this initial phase inactivation became linear and rather slow (for O1 and A10-virus 0.2 log10/hour). No "tailing-off" was observed. Under standard conditions (0.04 per cent formaldehyde, pH 8.5, 25 degrees C) CD-virus was inactivated approximately 1.5 times faster than O1 and A10-virus. At 4 degrees C the inactivation of the three strains continued at about one log10/day. Increased lactalbumin hydrolysate concentrations reduced the inactivation rate, especially at the formaldehyde concentration of 0.02 per cent, which was originally applied. Quaternary amines like Tris strongly inhibited formaldehyde activity. These findings might explain some data of others who observed "tailing off". Analysis of formaldehyde inactivated antigen by SDS-PAGE and electrofocusing showed that extensive cross-linking occurs especially of VP1, probably with other virus proteins but also with non-virus proteins from the medium. VP2 and VP3 are less affected. Cross-linking was enhanced when the virus had been adsorbed to alhydrogel during inactivation. Progressive cross-linking was observed during storage of the vaccine at 4 degrees C, which also indicated that inactivation continued at this temperature. These data show that formaldehyde inactivated adsorbate vaccines can be safe.

Innocuity testing of foot-and-mouth disease vaccines. I. Formaldehyde-inactivated alhydrogel vaccines.

Conditions that contribute to efficient innocuity testing of formaldehyde (FA)-inactivated alhydrogel vaccines were investigated. Under our conditions good yields of 146S antigen were obtained if the antigen was eluted by potassium phosphate buffer (pH 7.4) and concentrated by ultrafiltration. Non-inactivated virus added to the vaccine and adsorbed overnight could be recovered if residual FA was removed from the vaccine by washing the gel thoroughly with Frenkel culture medium before the addition of the virus. It was shown that the presence of high concentrations of inactivated virus in the concentrated eluate could prevent the detection of small amounts infectious virus in intradermolingual tests in cattle. This interference phenomenon was not found if (more susceptible) monolayers of foetal calf thyroid cells were used for the detection of virus. Intensive pre-washing of the gel with Frenkel culture medium, elution with potassium phosphate, concentration by ultrafiltration and the use of thyroid cells for the final detection of surviving virus is therefore advised for safety testing.