WO1991003255A1

WO1991003255A1 - Polypeptide vaccines against foot-and-mouth disease virus

Info

Publication number: WO1991003255A1
Application number: PCT/GB1990/001329
Authority: WO
Inventors: Nigel Richard Parry; Elizabeth Jane Ouldridge; David John Rowlands
Original assignee: The Wellcome Foundation Limited
Priority date: 1989-09-08
Filing date: 1990-08-28
Publication date: 1991-03-21
Also published as: AU6358190A; GB8920357D0

Abstract

A polypeptide, suitable for use as a vaccine against foot-and-mouth disease virus (FMDV) serotype O or A, presents an epitope comprising the amino acid sequence from residue 142 to residue 158 of the VP1 capsid protein of foot-and-mouth disease virus (FMDV) O1Kaufbeuren, or the corresponding sequence of another FMDV strain of serotype O, with the proviso that the amino acid residue 148 is serine; the said polypeptide being (i) no more than 50 amino acid residues long or (ii) a chimaeric protein comprising the sequence of a carrier protein and a foreign sequence of no more than 50 amino acid residues which comprises the sequence of a said epitope.

Description

POLYPEPTIDE VACCINES AGAINST FOOT-AND-MOUTH DISEASE VIRUS The present invention relates to peptides suitable for use as vaccines against foot-and-mouth disease.

Foot-and-mouth disease (FMD) is the most

economically important affliction of domestic livestock.

One of the greatest problems in its control with

conventional vaccines prepared from inactivated virus particles is the occurrence of the virus as seven distinct serotypes within which there is considerable antigenic variation. By definition, there is no demonstrable

cross-protection between these serotypes, so that an animal which has recovered from infection with a virus of one serotype is as susceptible to infection with virus of another serotype as an animal which has not previously been infected. Moreover, antigenic variation within a serotype can affect the success of vaccination programmes.

According to the present invention, there is provided a polypeptide suitable for use as a vaccine against foot-and-mouth disease virus (FMDV) serotypes O and A, which polypeptide presents an epitope comprising the amino acid sequence from residue 142 to residue 158 of the VP1 capsid protein of FMDV O₁ Kaufbeuren, or the corresponding sequence of another FMDV strain of serotype 0, with the proviso that the amino acid residue 148 is serine; the said polypeptide being

(i) no more than 50 amino acid residues long, or (ii) a chimaeric protein comprising the sequence of a carrier protein and a foreign sequence of not more than 50 amino acid residues which comprises the sequence of a said epitope.

The polypeptides of the invention comprise a defined antigenically effective sequence. This sequence is composed of VP1 residues 142 to 158 of FMDV O₁ Kaufbeuren (Kurz et al. Nuc. Acids Res. 9, 1919-1931, 1981) or the corresponding residues of another strain of FMDV serotype O. However, the residue at VP1 position 148 is serine rather than the natural leucine. In this way, the polypeptides can raise neutralising antibody not only against FMDV serotype O but also against FMDV serotype A.

Based on FMDV O₁ Kaufbeuren, the epitope of interest comprises residues PNLRGDSQVLAQKVART. Amino acid residues are shown using the one letter code (Eur. J.

Biochem. 138. 9-37, 1984). The corresponding sequence for another strain of FMDV of serotype O can be readily

determined by lining up the VP1 sequence of O₁ Kaufbeuren with the VP1 sequence of the other strain and substituting a serine (S) residue at position 148 for the natural residue.

A first type of polypeptide according to the invention is composed of up to 50 amino acid residues, for example up to 40 or up to 30 or up to 20 amino acid

residues, which presents an epitope as defined above.

Further amino acids may therefore be added to one or both ends of the epitope. One, two, three, four or five

additional residues may be provided at the N-terminus or C-terminus or at both terminii of the defined epitope.

Where additional residues are provided at either or both ends of the epitope, preferably these are the natural residues. These can be deduced from the sequence of the VP1 protein. Preferred flanking sequences for the epitope are therefore the flanking sequences which naturally occur either side of the amino acid sequence for the epitope in the overall sequence of the VP1 protein. Also, a cysteine residue may be provided at the N-terminus or C-terminus. In particular, a cysteine residue may be added to the

C-terminus alone. This is in order to facilitate carrier coupling and/or to enhance the immunogenicity of the

polypeptide.

A preferred polypeptide starts with a VP1 residue numbered from 138 to 142 and ends with a VP1 residue

numbered from 158 to 162. The polypeptide may therefore correspond to, for example, VP1 residues 138-162, 138-160, 140-162, 140-160, 141-162 or 141-160. A preferred

polypeptide, based on FMDV O₁ Kaufbeuren and including a non-natural C-terminal cysteine residue, has the sequence VPNLRGDSQVLAQKVARTLPC.

The polypeptide may have a free C-terminal carboxy group. Alternatively, it may be in the form of a C-terminal amide. Pharmaceutically acceptable salts of the polypeptide may be employed. The polypeptide may be coupled to a carrier in order to creature an immunogen which is

antigenically active. Any appropriate physiologically acceptable carrier may be employed. A conjugate between the polypeptide and the carrier may be formed. The carrier may be for example bovine serum albumin, thyroglobulin,

ovalbumin, keyhole limpet hemocyanin (KLH) or hepatitis B core antigen.

A second type of polypeptide according to the invention is a chimaeric protein which presents the defined epitope. The chimaeric protein is typically a carrier protein which has been modified so that its amino acid sequence comprises a foreign sequence of up to 50 amino acids which includes the sequence of the desired epitope. Some amino acids of a protein may be replaced by the foreign amino acid sequence. Alternatively, the foreign amino acid sequence is fused to a protein. An intervening linker of up to 10 amino acids, for example of up to 5 amino acids, may be provided between the epitope and the carrier. The foreign amino acid sequence may vary in length as described for the first type of polypeptide according to the

invention.

The epitope is exposed on the surface of the chimaeric protein so that it is presented to the immune system. The chimaeric protein may take the form of a particle or form part of a particulate aggregation. Such an aggregation may comprise plurality of chimaeric proteins and/or may be a viral particle. A protein to which a foreign amino acid sequence comprising the epitope may be fused may be a particle-forming protein such as hepatitis B surface antigen (HBsAg, EP-A-0175261) or hepatitis B core antigen (HBcAg, JP-A-63196299). The foreign sequence may be inserted into the sequence of a viral protein exposed on the surface of the virus (GB-A-2125065). The viral protein may be a capsid protein of a virus.

The foreign sequence may therefore be provided at one of the antigenic sites of a picornavirus such as

poliovirus (EP-A-0302801). The epitope may be presented at one of the antigenic sites, for example site 1, 2 or 3, on a capsid protein of an attenuated strain of type 1 poliovirus, or at an antigenic site of type 2 or 3 poliovirus. Other picornaviruses, suitably modified, may be used, e.g. Bovine enterovirus.

The amino acid sequence of an antigenic site of a picornavirus may be replaced completely or partly by the foreign amino acid sequence. Preferably the foreign amino acid sequence is provided in place of some or all of

antigenic site 1 of an attenuated strain of type 1

poliovirus. The attenuated strain is typically the Sabin 1 vaccine strain. Antigenic site 1 of a type 1 poliovirus is composed of amino acid residues 91 to 102 of the VP1 capside protein.

The polypeptides of the invention are synthetic polypeptides. They may be prepared by chemical synthesis, in particular the first type of polypeptide of up to 50 amino acid residues long. Solid-phase or solution methods of peptide synthesis may be employed. A polypeptide can be built up therefore by a process comprising condensing single amino acids and/or preformed peptides or two or more amino acids in the order in which amino acids occur in a

polypeptide of the invention. The polypeptide may be synthesised so as to possess a free C-terminal carboxy group or a C-terminal amide group. If desired, the polypeptide may be converted into a pharmaceutically acceptable salt.

In solid-phase synthesis, the amino acid sequence of the desired polypeptide is built up sequentially from the C-terminal amino acid which is bound to an insoluble resin. When the desired polypeptide has been produced, it is cleaved from the resin. When solution-phase synthesis is employed, the polypeptide may again be built up from the C-terminal amino acid. The carboxy group of this acid remains blocked throughout by a suitable protecting group, which is removed at the end of the synthesis.

Whichever technique, solid phase or solution-phase, is employed each amino acid added to the reaction system typically has a protected α-amino group and an activated carboxy group. An amino group may be protected by the fluoren-9-ylmethoxycarbonyl (Fmoc) or t-butoxycarbonyl (Boc) group. A carboxy group may be activated as a

pentafluorophenyl or 1-oxo-2-hydroxy- dihydrobenzotriazine ester. Each condensation step may be effected in the

presence of dicyclohexylcarbodiimide or

1-hydroxybenzotriazole.

Side chain functional groups are typically protected too, for example the side chain amino group of a lysine, the side chain hydroxy group of a threonine or the side chain sulphydryl group of a cysteine. After each step in the synthesis, the α-amino protecting group is removed.

However, any side-chain protecting groups are generally only removed at the end of the synthesis although they may be retained if desired.

The polypeptides may be prepared with a C-terminal carboxy or amide group as desired. In solid phase peptide synthesis, this may be determined by how the C-terminal amino acid is linked to the resin support and/or how the final peptide is cleaved from the resin. Typically the resin is a styrene and/or divinylbenzene polymer. The

C-terminal amino acid may be linked to the resin via an ester linkage which can be cleaved by a strong acid such as HBr in trifluoroacetic acid or HF to give the peptide with a C-terminal carboxy group. Ammonolysis can give the

corresponding amide instead.

An alternative method of obtaining a polypeptide amide by solid phase synthesis is to arrange for the

C-terminal amino acid of the polypeptide to be linked to the resin via a peptide aminobenzhydryl bond. This can be formed by coupling with dicyclohexylcarbodiimide and can be cleaved with HF, typically in the cold. For solution phase synthesis, whether a C-terminal carboxy or amide group is present may depend upon how the carboxy group of the

C-terminal amino acid is blocked and, at the end of the synthesis, unblocked. A polypeptide with a C-terminal carboxy group can be converted into one with a C-terminal amide group and vice versa.

Both types of polypeptide according to the invention may be prepared by recombinant DNA methodologies. Thus a DNA sequence encoding the desired polypeptide is provided. An expression vector is prepared which

incorporates the DNA sequence and which is capable of expressing the polypeptide when provided in a suitable host. The DNA sequence is located between translation start and stop signals in the vector. Appropriate transcriptional and translational control elements are also provided, in particular a promoter for the DNA sequence and a

transcriptional termination site. The DNA sequence is provided in the correct frame such as to enable expression of the polypeptide to occur in a host compatible with the vector.

In the case of a chimaeric protein, a DNA fragment encoding the foreign amino acid sequence is inserted into a vector at a location which enables the epitope of interest to be expressed, as part of the chimaeric protein, exposed on the surface of the protein. The chimaeric protein is then expressed. Cells harbouring the vector are cultured so as to enable expression to occur. Depending on the type of chimaeric protein, the protein may self-assemble into particles.

Any appropriate host-vector system may be employed. The vector may be plasmid. In that event, a bacterial or yeast host may be used for example E.coli or S. cerevisiae. Alternatively, the vector may be a viral vector. This may be used to transfect cells of a mammalian cell line, such as CHO cells, in order to cause polypeptide expression.

An epitope according to the invention may be linked to one or more helper T-cell (Th-cell) epitopes. A Th-cell epitope is a site capable of eliciting help for antibody production. A Th-cell epitope is capable of binding class II major histocompatibility (MHC) molecules on the surface of host antigen-presenting cells and B-cells subsequently interacting with the T-cell receptor in the form of a trimolecular complex in order to induce B-cells to differentiate and proliferate.

A Th-cell epitope may be linked to the first type of polypeptide of the invention in a variety of ways.

Glutaraldehyde polymerisation may be used, in which the polypeptide of the invention is copolymerised with a

polypeptide which presents a Th-cell epitope via their amino groups. The polypeptide of the invention and the

polypeptide presenting the Th-cell epitope may be conjugated together via a heterobifunctional cross-linking agent such as m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS).

The polypeptide of the invention may alternatively by linked at its C-terminus or N-terminus to a polypeptide presenting a Th-cell epitope via a peptide bond. This may be achieved by co-linear synthesis of the polypeptide of the invention and the polypeptide presenting the Th-cell epitope or by use of recombinant DNA technology as above to express a fusion protein in which the two polypeptides are fused together. In any of the methods, any suitable Th-cell epitope may be used.

A preferred polypeptide presenting Th-cell epitopes is hepatitis B core antigen (HBcAg). A first type of polypeptide of the invention may be chemically coupled to HBcAg. Recombinant DNA technology can be used to produce a fusion protein according to the second type of polypeptide of the invention, comprising HBcAg to the amino terminus of which is linked the sequence of a polypeptide of the

invention. The epitope of interest may be fused directly to the amino terminus of HBcAg. Alternatively, the sequence may be fused to the HBcAg via an intervening linker. Such a linker may be composed of one or more, for example up to ten, amino acid residues.

The polypeptides of the invention are useful as vaccines against FMD, in particular FMDV serotypes O and A. An effective amount of the polypeptide is administered to a host animal such as a cow or pig. The polypeptide may be administered orally or parenterally, for example

subcutaneously or intramuscularly.

Typically, a polypeptide is administered orally or parenterally in an amount of 50 to 1000 μg per dose, more preferably from 50 to 250 μg per dose. A single dose may be given or several of doses may be administered over a period of time .

For vaccination purposes a polypeptide of the invention, however presented, is typically formulated with a veterinarily acceptable carrier or diluent. Conventional formulations, carrier, adjuvants and diluents may be

employed. Aluminium hydroxide or any other acceptable adjuvant may be used. A polypeptide could be linked to an immunostimulating complex (iscom: Morein et al, Nature 308, 457-460, 1984) or incorporated into liposomes.

The following Examples illustrate the present invention.

EXAMPLE 1: Preparation of peptides

The peptides shown below were synthesised using an adaptation of the Merrifield method (Merrifield, JACS, 85, 2149-2154, 1963) described by Houghten (Houghten, PNAS, 82, 5131-5135, 1985):

peptide 148 = L : VPNLRGDLQVLAQKVARTLPC

peptide 148 = R : VPNLRGDRQVLAQKVARTLPC

peptide 148 = S : VPNLRGDSQVLAQKVARTLPC

peptide 148 = T : VPNLRGDTQVLAQKVARTLPC

peptide 148 = I : VPNLRGDIQVLAQKVARTLPC

All these peptides are based on the sequence of FMDV O₁ Kaufbeuren. Peptide 148 = L is the natural sequence without any substitution at position 148. Peptide 148 = S is a peptide according to the invention. Each peptide was synthesised on a p-methylbenzhydrylamine divinylbenzene resin. The α-amino

protecting group on each amino acid was t-butoxycarbonyl (Boc). Each coupling cycle was as follows:

1.Wash resin with dichloromethane - 10 minutes 2.Wash with 5% diisopropylethylamine in

dichloromethane - 2 minutes × 3

3.Dichloromethane wash - 1 minute × 2

4. Couple t-butoxycarbonyl amino acid in

dichloromethane, 0.3M diisopropylcarbodiimide - 60 minutes. For N and Q coupling was effected in dimethylformamide, 0.3M diisopropylcarbodiimide and 0.125M hydroxybenzotriazole.

5.As 3

6.Deprotect with 50% trifluoroacetic acid in dichloromethane - 20 minutes

7.Dichloromethane wash - 1 minute × 6

8.Return to 2.

When coupling cycles were completed the peptide was cleaved off the resin using hydrogen fluoride for 1 hour with an anisole scavenger 10%. The peptide was thus

obtained with a carboxy-terminal amide group. It was then ether washed, dried, dissolved in 15% acetic acid and lyophilized.

Example 2: Test of peptides 1. Materials and Methods

Viruses. The O₁BFS 1860 and C₃ Indaial viruses used in this study are FMDV vaccine strains from Coopers Animal Health Ltd, Pirbright, Surrey. The low and high passage O₁Kaufbeuren strains are the 7th and 64th passage isolates described by Strohmaier et al. (J. gen. Virol. 59, 295-306), 1982). The B and C variants of A₁₂ 119 virus are those identified by Rowlands et al. (Nature, London 306, 694-697, 1983). 0 Colombia 9834 virus was obtained from the VECOL Laboratories, Colombia. The remaining type O viruses are primary field isolates obtained from the FMDV World Reference Laboratory, Institute for Animal Health,

Pirbright, Surrey and subsequently passaged three times in baby hamster kidney (BHK)21 cells. One of these viruses, 0 Thailand 1/80, was found to be a mixed antigenic population and two plaque-picked clones (clones 2 & 10), respectively susceptible and resistant to neutralization by O^BFS 1860 antisera, were isolated (Ouldridge et al., in "Foot and Mouth Disease" Proceedings of the 17th Conference, Paris, October 1986, 223-228, Office International des Epizooties). All virus stocks were passaged in BHK21 cells in the presence of Eagle's medium, supplemented with 10% tryptose phosphate broth and stored at -20°C as clarified tissue culture harvests plus an equal volume of glycerol.

Antibody preparations. Antisera were raised in Dunkin-Hartley guinea-pigs. Anti-virus serum was obtained by two inoculations 28 days apart of 20 μg of

acetylethyleneimine-inactivated virus particles purified by the method of Brown & Cartwright (Nature, London 199. 1168-1170, 1963). Antisera were raised to two inoculations of 500 μg of peptide 35 days apart, blood being collected 28 days after the second inoculation. All antigens were emulsified with an equal volume of incomplete Freund's adjuvant just prior to intramuscular injection. Equal volume pools of five guinea-pig antisera to each antigen were used for specificity testing.

Neutralization assay. This was performed as a two-dimensional micro-cpe test using BHK21 cells as the indicator of residual virus infectivity as described by Rweyemamu et al. (J. Hygiene, Cambridge 81, 107-123, 1978). Antibody titres are expressed as log₁₀ of the reciprocal serum dilution giving 50% neutralization of 100 tissue culture infective doses of virus (log₁₀ SN₅₀/100 TC1D₅₀). The antigencic relationship of viruses based on their neutralization by each serum is given by the ratio:

Neutralization titre against the heterologous virus r =

Neutralization titre against the homologous virus The significance of differences in the values of r obtained was evaluated according to the criteria of

Rweyemamu & Hingley (J. Biol. Standardization 12, 295-303, 1984). 2. Effect of amino acid 148 on the specificity of peptide antisera

Pooled antisera to the five individual peptides differing only in the amino acid at position 148, and an antiserum to an equimolar mixture of three of these peptides totalling 500 μg per dose, were compared for neutralizing specificity. Their activity was assessed against two O₁ strains (BFS and Kaufbeuren) that showed complete homology of the 141-160 sequence, three type O viruses readily distinguishable by polyclonal anti-virus serum (O₆ V₁, O Thailand 1/80 Clone 10 and 0 Philippines), two variants (B anc C) of A.-119 virus and C₃ Indaial. The mean serum titres from triplicate independent experiments are shown in Table 1, and the virus relationships (r values) derived from them are shown in Table 2. All the antisera with the single exception of that to the peptide containing the L - S substitution, neutralized the two O₁ viruses to a similar extent. As expected, antiserum to the native sequence peptide neutralized the selected heterologus O strains no better than anti-virus serum, but showed some neutralization of the C₃ virus, which also has a leucine residue at

position 148. There was, however, no detectable recognition in these assays of the B variant of A₁₂119 virus which also has leucine at 148.

The specificity of antisera to peptides containing substitutions at position 148 with amino acids occurring at the same position in other O viruses was not as anticipated. Antisera to peptides with L - T and L - I changes, which occur in the O₆V1 and 0 Philippines strains respectively, continued to give the highest titres against the OiBFS and O₁ Kaufbeuren viruses, but there was no improved relative neutralization of the strains possessing the specific amino acid at position 148. Surprisingly, however, these

anti-peptide antisera showed some neutralization of A₁₂119C virus. Antiserum to the L - R peptide also did not

neutralize the O virus with the homologous amino acid at 148 (O Tai 1/80, Clone 10) any better than the native sequence, but showed much improved neutralization of O₆V1 (148=T) and some activity against A₁₂-119B (148=L). This result was confirmed by testing the five individual sera comprising the antiserum pool to the L - R peptide which, although varying in titre, all produced the same profile of reactions with the eight strains (data not shown). The most significant result was the finding that antiserum to the peptide with the A₁₂119C specific substitution of L - S, although of low titre, neutralized O₁BFS, O₁ Kaufbeuren and _A12119C equally. This was corroborated by titrating the serum in the more sensitive mouse index test (Skinner, in Proceedings of the XVth International Veterinary Congress, Stockholm, part II, 208-210, 1953), in which the infectivity titres of O₁BFS and A₁₂119C were reduced by 3.5 and 3.9 log₁₀ respectively compared to 5.2 and <0.2 log₁₀ for the native anti-peptide antiserum. 3. Cross-protection of guinea-pigs with a single peptide

The relationship of the observed heterotypic cross-neutralization to immunity was assessed in a

guinea-pig protection test. Eight animals were inoculated twice with the L - S peptide as described, except that the first inoculum was emulsified with complete Freund's

adjuvant, and test bled 27 days after the second injection. At 28 days post boost three animals were challenged with 1000 guinea-pig infectious doses of O₁BFS and four with the same dose of A₁₂119C, one animal having died of unknown causes during the response period. Four unimmunized

guinea-pigs were also challenged with each virus. All eight of the non-immune animals developed lesions, whereas only one guinea-pig, that with the lowest serum titre, from each of the immunized groups developed lesions (Table 3).

Table 1 Neutralization (log₁0 SN₅₀/100 TCID₅₀) of FMDV strains by antisera to peptides corresponding to the

OBFS 1860 141- 160 VP1 sequence with selected amino acid substitutions at position 148

Antiserum to peptides substituted at position 148 Anti-virus

Virus strain 148=L 148=R 148=S 148=T 148=1 148-Mix particle

L, R, S (OBFS 1860)

Natural

a. a. 148

O₁BFS 1860 L 2.74 2.68 1.30 2.26 2.46 2.51 2.75

O₁ Kaufbeuren L 2.67 2.61 1.30 2.26 2.33 2.59 2.78

O₆V1 T 1.88 2.40 <0.95 1.44 1.99 1.61 1.22

O Tai 1/80 C1. 10 R 1.80 1.98 <0.95 1.33 1.55 1.92 1.79

O Phil I 1.21 1.37 <0.6 1.34 1.17 ND 1.09

A₁₂ 119C S <0.95 <0.95 1.43 2.13 1.96 1.56 <1.05

A₁₂ 119B L <0.95 1.63 <0.6 <1.03 <0.9 ND <1.05

C_» Indaial L 1.72 <0.60 <0.6 <0.83 <0.75 ND <1.05

Claims

1. A polypeptide which presents an epitope comprising the amino acid sequence from residue 142 to residue 158 of the VP1 capsid protein of foot-and-mouth disease virus (FMDV) O₁Kaufbeuren, or the corresponding sequence of another FMDV strain of serotype 0, with the proviso that the amino acid residue 148 is serine; the said polypeptide being

(i) no more than 50 amino acid residues long or

(ii) a chimaeric protein comprising the sequence of a carrier protein and a foreign sequence of no more than 50 amino acid residues which comprises the

sequence of a said epitope.

2. A polypetide according to claim 1, which is no more than 30 amino acid residues long or which is a said chimaeric protein wherein the foreign sequence is no more than 30 amino acid residues long.

3. A polypeptide according to claim 2, which is composed of the amino acid sequence of a said epitope having up to 5 additional amino acid residues provided at the N-terminus thereof and/or up to 5 additional amino acid residues provided at the C-terminus thereof.

4. A polypeptide according to claim 1, which is no more than 50 amino acid residues long and is provided with a cysteine residue at the N-terminus or C-terminus.

5. A polypeptide according to claim 1 which is a said chimaeric protein composed of hepataiis B core antigen having the foreign sequence linked to the N-terminus thereof.

6. A process for the preparation of a polypeptide as claimed in claim 1 which process comprises condensing single amino acids and/or preformed peptides of two or more amino acids in the order in which amino acids occur in the said polypeptide.

7. A process for the preparation of a polypeptide as claimed in claim 1, which process comprises: (i) preparing an expression vector which incorporates a DNA sequence encoding the said polypeptide and which is capable of expressing the said polypeptide when provided in a suitable host, and

(ii) providing the said expression vector in the said host such as to enable expression of the polypeptide to occur.

8. A conjugate comprising a polypeptide as defined in claim 1 linked to a physiologically acceptable carrier.

9. A vaccine suitable for use against FMDV serotype 0 or A, which vaccine comprises a polypeptide as defined in claim 1 formulated with a veterinarily acceptable carrier or diluent.

10. A method of vaccinating an animal against FMDV serotype 0 or A, which method comprises administering to the animal an effective amount of a polypeptide as defined in claim 1.