CA1287447C - Peptides useful in vaccination against enteroviruses - Google Patents

Abstract

ABSTRACT
PEPTIDES USEFUL IN VACCINATION AGAINST ENTEROVIRUSES

A synthetic peptide, suitable for use in vaccination against or diagnosis of a disease caused by an enterovirus and especially by a poliovirus, is the peptide coded for by codons 286-290 in the RNA sequence coding for the structural capsid protein VP1 of poliovirus type 3 Sabin strain or by equivalent codons of another enterovirus or is an antigenic equivalent thereof, the numbers of the codons being counted from the 5'-terminus of the nucleotide sequence coding for the VP1 capsid protein.

Description

~2~3~7~'~7 "PEPTIDES USEFUL IN VACCINATION AGAINST ENTEROVIR~SES"
This invention relates to peptides having biological activity, particularly for use in vaccines for diseases caused by enteroviruses and in particular polioviruses.
Polioviruses are divisible into three serotypes on the basis of their neutralization reactions with specific immune sera. However, they have similar virological properties and clinical effects and the nucleic acid and amino-acid sequences of all three serotypes are strikingly similar (Stanway et al Nucleic Acids Research _ , 5629 - 5643, 1983; Stanway et al Proc. Natl. Acad. Sci.
USA 81, 1539 - 1543, 1984).
We have previously identified the location oE a single major antigenic site involved in the neutralization of poliovirus type 3 (Minor et al, Nature 301, 674 - 679, 1983; Evans et al, Nature 304, 459 - 462, 1983). In GB-A-2 128 621 we have described and claimed a synthetic polypeptide, suitable for use in vaccination against or diagnosis of a disease caused by an enterovirus, which comprises an antigenically effective hexapeptide coded for by codons 93 to 98 in the RNA sequence coding for the structural capsid protein VPl of a poliovirus type 3 Sabin strain or by equivalent codons of another enterovirus. The codon numbers herein are counted from the 5'-terminus of the nucleotide sequence coding for the VPl capsid protein.

~2~7~a~7 In contrast to this, it has been suggested that neutralization of poliovirus type`l involves multiple independent antigenic sites (Emini et al J. Virol. 46, 466-474, 1983). If correct, this di~ference between closely related viruses would make the satisfactory prediction of viral peptide sequences for use as vaccines more difficult than hitherto supposed.
We have now identified a second antigenically significant peptide coded for by an RNA sequence within the genome region coding for the structural capsid protein VPl of an enterovirus.
Accordingly the present invention provides a synthetic peptide, suitable for use in vaccination against or diagnosis of a disease caused by an enterovirus, which is the peptide coded for by codons 286-290 in the RNA sequence coding for the structural capsid protein VP1 of poliovirus type 3 Sabin strain or by equivalent codons of another enterovirus or is an antigenic equivalent of the said peptide, the antigenic equivalent ~eing:
(i) a said peptide modified by the inclusion therein of one or more changes to the amino acid sequence;
(ii) a first longer peptide which incorporates the sequence of a said peptide or a said modified peptide and which has up to four extra amino acid residues attached to the C-terminal end of the said sequence or up to four extra amino acid residues attached to the N-terminal e~d of said seguence or up to four extra amino acid residues attached to the C-terminal end and up to ~our extra amino acid residues attached to the N-terminal end of said sequence;
(iii) a second longer peptide which incorporates the sequence of a said peptide, a said modified peptide or a said first longer peptide, to which 12l~7~7 - 2a -sequence is linked directly, by means of a further amino acid residue or by means of a disulphide bridge between Cys residues attached to each sequence, a sequence of up to eighteen amino acid residues which comprises a hexapeptide sequence coded for by codons 93 to 98 in said RNA
sequence or by equivalent codons of another enterovirus, or (iv) a third longer peptide which incorporates the a sequence of a said peptide, a said modified peptide or a said first longer peptide, to which sequence is linked directly, by means of a further amino acid residue or by means of a disulphide bridge between Cys residues attached ta each sequence, a sequence comprising residues 58 to 59 of the VP3 capsid protein of an enterovirus of the same type but starting with a residue numbered no lower than 53 and ending with a resi~ue numbered no higher than 68; each said antigenic equivalent being capable of raising antibodies capable of neutralizing the same strain and type of enterovirus as said peptide to ~hich said antigenic equivalent corresponds and the numbers of the codons being counted from the ~'-terminus of the nucleotide saquence for the VP1 capsid protein.
The peptides of the invention therefore comprise an antigenically effective peptide unit coded for as indicated. ~hey are not naturally-occurring peptides, such as the VPl capsid protein i.tself, which have been recovered in a suitably pure form after disrupting an enterovirus.
Rather, the hand of man has been involved in the making of the peptides of the invention. In particular, the peptides ~ -",Y `

12~ 7 of the invention can be prepared by chemical synthesis rom single amino acids or s~aller preformed peptides; or by employing the methods o~ genetic engineering to produce an organism which makes the peptides in recoverable ~orm.
By 'lequivalent codons" is meant a sequence of codons in the RNA sequence coding for the structural capsid protein VPl of another enterovirus, corresponding to the codon sequence 286-290 in the RNA sequence coding for the structural capsid protein VPl of the poliovirus type 3 Sabin strain. The "equivalent codons"
are therefore the counterpart codons in the RNA sequence coding for ~he capsid protein VPl of another enterovirus to codons 286-290 for the poliovirus type 3 Sabin strain.
The counterpart codons can readily be determined by lining up the base sequence coding for the VPl protein in the RNA sequence of another enterovirus with the corresponding base sequence of the poliovirus type 3 Sabin strain. While it is possible that the equivalent codons in the other enterovirus may also be 286-290, this is not necessarily the case. In the poliovirus type 3 Leon strain, which is the virulent progenitor of the attenuated Sabin strain, the equivalent codons are 286-290. The equivalent codons are 2a6-290 too or the poliovirus type 2 Sabin strain. However, in the poliovirus type 1 Sabin strain, there are si~ equivalent codons, ~287~ ~7 287-292. Thus, when the base sequences for the Sabin strains of each of the three types of poliovirus are lined up (Toyoda et al, ~. Mol. Biol. 174, 561-585, l9a4), the result is as follows:

type 3 Sabin:codon 286 287 288 289 290 AGG AAC AAC UUG GAC
type 2 Sabin:codon 286 287 288289 290 AAA GAU GGGCUC ACC
type l Sabin:codon 287 288 289290 291 292 AAG GAU GGU ACG CUU ACA

An "antigenic equivalent" of any particular "natural" peptide sequence coded for by an existing enterovirus twhether wild-type or mutant) is a peptide which, if not itself immunogenic, when linked to material which renders it immunogenic is capable of inducing the same or a very similar antibody response as the "natural" peptide, i.e.
the antibody produced, though possibly not precisely identical, would neutralize the same strain and type of enterovirus and hence antigenicity is effectively equivalent.
An antigenic equivalent of a "natural" peptide sequence may be a peptide of the same length which, however, is not coded for by a wild-type or known mutant enterovirus but includes one or more changes to the amino acids in the sequence which does not affect the antigenicity. Thus, one or more amino acids of a "natural" peptide sequence may be ~Z~74~7 replaced by, respectively, one or more other amino acids which preserve the physico-chemical character of the original, i.e. in terms of charge density, hydrophilicity/hydrophobicity, size and configuration, and 5 hence preserve the immunological structure. For example, Thr may be replaced by Ser and vice versa, Asp may be replaced by Glu and vice versa and Asn may be replaced by Gln and vice versa.
An antigenic equivalent may also be a longer peptide which comprises a "natural" peptide sequence but still has equivalent antigenicity. The "natural" peptide sequence will thus be exposed in the longer peptide so as to be available to induce the appropriate immune response and not "buried" in the interior of the longer peptide and consequently unable, itself, to provoke an immune response.
Yet ~urther antigenic equivalents may be formed by modifying reactive groups within a "natural" sequence or modifying the N-terminal amino and/or C-terminal carboxyl group. Such equivalents can include salts formed with acids and/or bases, particular physiologically acceptable inorganic and organic acids and bases. Other equivalents may include modified carboxyl groups to produce esters or amides or may include typical amino acid protecting groups such as N-t-butoxycarbonyl. Preferred modifications of this type are those which enable the production of a more stable, active peptide which will be less prone to enzymic degradation in vivo.

lZ~7~7 A combination of two or more of the types of variations of a "natural" sequence described above may be used to arrive at an antigenic equivalent peptide of the invention. For example, a peptide sequence which has been derived from a "naturall' sequence by changing one or m~re of the amino acids in the "natural" sequence may be incorporated in a longer peptide.
The present invention will now be described with particular reference to polioviruses, though it will be appreciated that the concept of the invention is considered - to apply equally well to other enteroviruses, i.e. viruses which are found in the intestine, e.g. ECHO (Enteric Cytopathic Human Orphan) and Coxsackie B viruses. In accordance with convention, the bases referred to herein are as follows:
A = Adenine G = Guanine C = Cytosine U = Uracil.
Similarly, in accordance with convention, the following abbreviations are used for the amino acid radicals:
Alanine = Ala Arginine = Arg Asparagine = Asn 25 Aspartic acid = Asp Cysteine = Cys Glutamine = Gln Glutamic Acid = Glu ~Z~7~47 Glycine = Gly Histidine = His Isoleucine = Ile Leucine = Leu Lysine = Lys Methionine = Met Phenylalanine = Phe Proline = Pro Serine = Ser Threonine = Thr Tryptophan = Trp Tyrosine = Tyr Valine = Val ~herever these amino acids are mentioned, they lS cover both the D- and L-configurations. However, it is preferred in accordance with the invention that the amino acids should take the natural, i.e. the L-, configuration.
The Figure of the accompanying drawing shows the RNA sequence for the VPl capsid protein in the poliovirus type 3 Sabin strain. Within this sequence, codons 93-98 and 286-290 are underlined.
It has not yet been unequivocably established whether the nucleotide sequence coding for the VPl capsid protein of the poliovirus type 3 Sabin strain actually commences with the codons GGU AUU . . . as shown in the Figure or with the codon GGC . . . which is the twelfth codon ~L287~4~

in the Figure. Nevertheless, herein the codons for the nucleotide sequence of the VPl capsid protein of poliovirus type 3 Sabin strain are counted from the first codon in the Figure, GGU.
In accordance with this notation, the appropriate RNA
sequence coded for by codons 286-290 for Sabin type 3 poliovirus and the corresponding tripeptide are as follows:

AGG AAC AAC W G GAC
Arg-Asn-Asn-Leu-Asp Table 1 below sets out the codons and amino acid residues of the Leon strain and a mutant strain of poliovirus type 3, of Sabin strain poliovirus type 2 and of the Sabin and Mahoney strains of poliovirus type 1 in 15 comparison to codons 286-290 for the Sabin strain of poliovirus type 3.
A preferred peptide according to the invention, suitable for use in vaccination against or diagnosis of a disease caused by type 3 poliovirus, comprises the sequence (I):
Ao-Al-A2-Leu-A3 (I) in which (i) Ao is Arg, each of Al and A2 is independently Asn or Gln and A3 is Asp or Glu or (ii), with the others of Ao to A3 being as defined under (i), Ao is Lys or A~ or A2 is Asp or Glu. Preferably, A~ ir Arg, both A1 and A2 are Asn and A3 is Asp.
A preferred peptide, suitable for use in vaccination against or diagnosis of a disease caused by type ~Z874 ~7 g

2 poliovirus, comprises the sequence (II):
Lys-Al-Gly-Leu-A3 (II) in which Al is Asp or Glu and A3 is Thr or Ser. More preferably, Al is Asp and A3 is Thr.
A preferred peptide, suitable for use in vaccination against or diagnosis of a disease caused by type 1 poliovirus, comprises the sequence (III):
~ ys-A4-Gly-As-Leu-A6 (II) in which A4 is Asp or Glu, and each of A5 and A6 is inde~endently Ser or Thr. More preferably, A4 is Asp and A5 and A6 are both Thr.
The present invention also includes peptides longer than the basic pentapeptide, in the case of polioviruses types 2 and 3, or hexapeptide, in the case of poliovirus type 1.
Further amino acids and/or peptides can be linked to one or both ends of the basic peptide chain. 1, 2, 3 or 4 extra amino acid residues may be attached to the C-terminal of the basic peptide and/or 1, 2, 3 or 4 extra amino acid residues can be attached at the N-terminal of the hasic peptide. Preferably, these additional amino acids correspond to those in a "natural" sequence. Thus, a longer peptide according to the invention may correspond to codons 282 to 292 in the RNA sequence coding for the VPl capsid protein of poliovirus type 3 Sabin strain or equivalent codons of another poliovirus such as poliovirus type 1 or 2 Sabin strain. Such peptides ~Z~ 7 in relation to the Sabin strains are:
(type 1) Gly-Val-Asp-Tyr-Lys-Asp-Gly-~hr-Leu-Thr-Pro-Leu (type 2) Gly-Val-Asp-Tyr-Lys-Asp-Gly-~eu-Thr-Pro-Leu (type 3) Gly-Val-Asp-Tyr-Arg-Asn-Asn-Leu-Asp-Pro-Leu The longer polypeptides may terminate in a Cys residue at one or both ends. Alternatively, the basic peptide chain itself or longer peptides containing this chain may be linked at one or both ends to a protein and/or some other carrier.
When, for example, the basic amino acid sequence o a "natural" peptide or of an antigenic equivalent thereof is included in a longer peptide, the additional amino acids attached to the basic peptide preferably correspond to the amino acids linked to the "natural" peptide in the corresponding natural VPl capsid protein. In type 3 Sabin poliovirus, the first N-terminal amino acid which may be added to the basic pentapeptide is Tyr (coded for by U~U as can be seen from the Figure of the accompanying drawing).
The first C-terminus amino acid which may be added in this instance is Pro (coded for by CCC). Appropriate further amino acids in this case can be determined from the Figure.
La~ger compounds are such that the basic peptide sequence is positioned so as to be readily available to lZ~7~7 induce the appropriate immune response and, in particular, so that it is not "buried" in the interior of the molecule.
Thus, for example, repeats of basic peptide may be linked together either by non-covalent or, preferably, covalent bonds. Where no appropriate amino acid is contained in the peptide sequence of the present invention, additional acids can be attached at either terminus for this purpose, in particular Cys which will enable covalent bonding through formation of a disulphide linkage.
Alternatively, a longer peptide may be formed into a loop by including groups which can link together at each terminus of the chain. A loop can of course be created by formation of an amide link between the N-terminus and C-terminus which can occur irrespective oE the amino acids at those termini. Also, a longer peptide may comprise sequences for different types of poliovirus. Such a peptide would be useful as a vaccine for or in the diagnosis o~ two or all three types of poliovirus.
A peptide of the invention may comprise the basic peptide sequence linked to the sequence o a polypeptide according to GB-A-2 128 621. The polypeptides of GB-A-2 128 621 are hexapeptides coded for by codons 93-9a in the RNA
sequence coding for the structural capsid protein VPl of poliovirus type 3 Sabin strain or by equivalent codons of another enterovirus or are antigenic equivalents of such a hexapeptide. Preferably, the basic peptide sequence acco}ding to the present invention and the polypeptide according to GB-A-2 128 621 which are linked together are in 12~7~47 respect of the same type, more preferably the same strain, of enterovirus.
A preferred hexapeptide according to GB-A-2 128 621, suitable for use as a vaccine for or in the diagnosis of type 3 poliovirus, has the formula (IV):

11 12 13 14 15 (IV-) in which (A) Alo is Glu, All is Gln, A12 is Pro, A13 is Thr, A14 is Thr and A15 is Arg, or (B) with the remainder of Alo to A15 being as defined under (A), (a) Alo is Gly or (b) A13 is ~le, Ser, Ala or Asn or (c) A14 is Asn, Ser or Ile or (d) A15 is Gln, Trp or Gly or (e) A13 is Ile and A14 is Asn or Ala This hexapeptide can therefore be linked to the preferred type 3 poliovirus tripeptide of formula (I) above.
A preferred hexapeptide according to Ga-A-2 128 621, suitable for use as a vaccine for or in the diagnosis of type 2 poliovirus, has the formula (IV) in which Alo is Asp, All is Ala, A12 is Pro, A13 is Thr, A14 is Lys and A15 is Arg. This hexapeptide may therefore be linked to the preferred type 2 poliovirus tripeptide of forMula (II)above.
A preferred hexapeptide according to GB-A 2 128 621, suitable for use as a vaccine for or in the diagnosis of type 1 poliovirus, has the formula (IV) in which All is Ala, A12 is Ser, A13 is Thr, A15 is Asn and either (A) Alo is Ser and A14 is Lys or (B) Alo is Pro and A14 is Thr. This hexapeptide may therefore be linked to the preferred type 1 poliovirus tripeptide of formula (III)above-~8~

The hexapeptides of GB-A-2 128 621 can be built up into longer polypeptides, and these longer polypeptides may too be linked to peptides according to the present invention.
For example a preferred type 3 poliovirus octapeptide according to GB-A-2 128 621 has the formula (V):

Alo-All-A12-A13-A14-A15-A16-A17 tV) in which Alo is Glu, Al~ is Thr or Ser and A15 is Arg. More preferably, (A) Alo is Glu, All is Gln, A12 is Pror A13 is Thr, A14 is Thr, A15 is Arg, A16 is Ala and A17 is Gln, or (B) with the others of Alo to A17 being as defined in (A) (a) Alo is Gly, or (b) A13 is Ile, Ala or Asn, or (c) A14 is Asn, Ser or Ile, or (d) A15 is Gln or Trp, or (e) A16 is Thr or Val, or (f) A17 is Leu, Pro, Arg or His; or further (g) A13 is Ser, Ile or Asn and A16 is Thr, or (h) A13 is Ile, A14 is Asn or Ala and A16 is Thr.
A preferred type 2 poliovirus octopeptide has the formula (V) in which: Alo is ~sP~ All iS Ala~ A12 is Pro~
A13 is Thr, A14 is ~ys, A15 is Arg, A16 is Ala and A17 is Ser.
A preferred type 1 poliovirus octapeptide has the 12~4'~7 formula (V) in which: All is Ala, A12 is Ser, A13 is Thr, A15 is Asn, A16 is Lys, A17 is Asp and either tA) Alo is Ser and A14 is Lys or (B) Alo is Pro and A14 is Thr.
Further amino acids and/or peptides can be linked to one or both ends of these eight amino acid polypeptide chains according to GB-A-2 128 621. For example, a dodecapeptide or an octadecapeptide may be formed respectively by bonding:
(poliovirus type 3) Glu-Val-Asp-Asn-, (poliovirus type 2) Glu-Val-Asp-Asn-, (poliovirus type 1) Thr-Val-Asp-Asn, to residue Alo of formula (IV), or (poliovirus type 3) Ala-Ile-Ile-Glu-Val-Asp-Asn and -Lys-Leu-Phe, (poliovirus type 2) Ala-Ile-Ile-Glu-Val-Asp-Asn- and -Arg-Leu-Phe, or (poliovirus type 1) Ala-Ile-Ile-Thr-Val-Asp-Asn- (when A1o is Ser and A14 is Lys) or Thr-Thr-Met-Thr-Val-Asp-Asn- (when Alo is Pro and A14 is Thr) and -Lys-Leu-Phe, to residues Alo and A17 of formula (IV).
The basic peptide sequence according to the present invention may be linked to a polypeptide sequence according to GB-A-2 128 621 directly or by one or more amino acid residues or by a disulphide bridge between Cys residues attached to each sequence. Not only may peptides of the same poliovirus type be lin~ed ~2~37~'~7 together but also peptides of different types may be linked so as to form a single peptide useful as a vaccine for or in the diagnosis of two or all three types of poliovirus.
A peptide of the invention may also comprise the basic peptide sequence linked to another peptide comprising residues 58 and 59, for example residues 58 to 61, of the VP3 capsid protein of the same type, preferably of the same strain, of enterovirus, in particular poliovirus, as that to which the basic sequence corresponds. ~or poliovirus type 3, the other peptide may also comprise VP3 residues 77 and 79.
It is believed that for poliovirus type 3 the pentapeptide coded for by codons 286-290 in the RN~ sequence coding for VPl and VP3 residues 58,59,77 and 79, which are thought to constitute a subsidiary antiqenic site, form a single operationally distinct antigenic site~
Thus, the invention provides a peptide suitable for use in vaccination against or diagnosis of a disease caused by type 3 poliovirus, which com?rises a type 3 pentapeptide of formula (I) above linked to a VP3 peptide of formula (~I) ortVIII):

A7-A~-Ag~LYS (VI) 7 A8 Ag Lys Alo~ All (VIII) in which ~i) each of A7 and Alo is independently Glu or Asp, each of A8, Ag and All is independently Thr or Ser and Alo and All are linked to the Lys residue and Alo respectively either dir~ctly or through intervenin~ amino acid residues or 7~

(ii), with the others of A7 to All being as defined under (i), A7 is Asn or Gln or A8 is Arg, Asn or Gln. The peptide of formula (~TII)may therefore correspond to VP3 amino acid residues 58 through to 79 of, for example, Sabin or Leon strain type 3 poliovirus. Preferably, A7 is Glu, A8 and A
are both Ser, Ag is Thr and Alo is Asp.
The invention also provides a peptide suitable for use in vaccination against or diagnosis of a disease oaused by type 2 poliovirus, which comprises a type 2 pentapeptide of formula (I) above linked to a VP3 peptide of formula (V) or (VI):

A12-A13-A14-Arg ( ) A12 A13~A14~Ar9...His , A15 (VI) in which each of A12 and A13 is independently Thr or Ser, A14 is Gln or Asn, A15 is Asp or Glu and the His residue and A15 are linked to the Arg residue and the His residue respectively either directly or through intervening amino acid residues. The peptide of formula (VI) may therefore correspond to VP3 amino acid residues 58 through to 79 of, for example, Sabin strain type 2 poliovirus. Preferably, A12 is Thr, A13 is Ser, A14 is Gln and A15 is Asp.
The invention further provides a peptide suitable for use in vaccination against or diagnosis of a disease caused by type 1 poliovirus, which comprise a type 1 hexapeptide of formula (II) above linked to a VP3 peptide of formula (VII) or (VIII):

3~2~

A -Ala-Lys-Lys (VII) Al6-Ala-Lys-~ys.~His~Al7 (VIII) in which A16 is Thr or Ser, A17 is Asp or Glu and the His residue and A17 are linked to the Lys residue shown adjacent to the His residue in formula (VIII) and the His residue respectively either directly or through intervening amino acid residues. The peptide of formula (VIII) may therefore correspond to VP3 amino acid residues 58 through to 79 of, for example, Sabin or Mahoney strain type 1 poliovirus.
Preferably, A16 is Ser and A17 is Asp. The hexapeptide of formula (II) may be linked to the peptide of formula (VII) or (VIII), and the type 3 and 2 pentapeptides of formula (I~ may be linked to the peptide of formula (III) or (IV) and the peptide of formula (V) or (VI) respectively in the same manner as the basic peptide sequence according to the invention may be linked to a polypeptide according to GB-A-2 128 621.
A peptide of the present invention, if not itself immunogenically active, may be linked to a carrier in order to create a conjugate which will be immunogenically active.
The carrier in that case may be a protein such as bovine serum albumin, thyroglobulin, ovalbumin or keyhole limpet hemocyanin, or palmitic acid. For immunization oE humans, the carrier must be a physiologically acceptable carier acceptable to humans and safe. Preferably however, the peptide is linked to tetanus toxoid and/or diptheria toxoid thus providing both an immunogen and a multivalent vaccine at ~2~7~4~7 the same time. Alternatively, the peptide may be chemically bonded to inert carriers where they can be used to assay and/or isolate by affinity chromatography antibodies to the appropriate virus. Examples of such inert carriers are dextrans e.g. sepharose.
The present invention also provides a process for the preparation of a peptide of the invention, which process comprises identifying either (a) the codons in the RNA
sequence coding for the structural capsid protein VPl oE an enterovirus which are or which are equivalent to codons 286 to 290 for a poliovirus type 3 Sabin strain or (b) the corresponding codons in a D~A sequence corresponding to said RNA sequence; and producing a synthetic peptide comprising the peptide sequence corresponding to the codons thus identified .
A peptide of the invention may be produced by chemical synthesis, for example by one of the generally known methods. In these methods, the peptide is usually built up either from the N-terminus or, more usually, the C-terminus using either single amino acids or preformed peptides containing two or mo~e amino acid residues. Particular techniques for synthesising peptides include the classical methods where the peptides of increasing size are usually isolated before each amino acid or preformed peptide addition. Alternatively, solid phase peptide synthesis may be employed where the peptide is built up attached usually to ' ' ~2~ 7 a resin e.g. ~ Merrifield resin. In these syntheses, groups on the amino acids will generally be in protected form using standard protecting groups such as t-butoxycarbonyl. If necessary, these protecting groups are conveniently cleaved once the synthesis is complete, though they may be retained where they do not affect the ability of the compound including the peptide to provoke an appropriate immune response. Other modifications of the peptide may either be introduced during ~he synthesis or at the end of it.
A still further possible method for producing the peptides of the invention is by employing the techniques of genetic engineering whereby a DNA sequence coding for the peptide is introduced into a plasmid which itself is introduced into an oeganism e.g. a bacterium, which can be lS induced to make the peptide in recoverable ~orm. The present invention thus not only covers the peptide, but also a DNA or RNA sequence coding for the peptide which can be used in such a synthesis. However, in view of the small number of amino acids in the pepetide chain of the invention, the most appropriate methods of production are the synthetic methods for building up the chains described above~
The peptides of the present invention have a particular application in vaccinating patients against diseases caused by enteroviruses, in particular polioviruses.
Vaccination is achieved by administering to a patient an effective amount of a peptide of the invention, either as ~2~ 7 such or linked to a carrier. Typically, from 100 ug to 1 mg of the peptide is administered intramuscularly to a human.
When used for this purpose, the material must be such, particularly of such a size, that it produces an immune reaction. The peptide is usually therefore coupled to an immunogenically active carrier such as the proteins mentioned hereinbefore or be in the form of a longer peptide including the peptide sequence, which may be achievea by linking the peptide to a synthetic polypeptide such as poly-lys.
The vaccines may include not just one peptide in accordance with the present invention, but two or more. By including several different peptides, for example one for each of the three different types of poliovirus, a patient may be vaccinated against all three types of poliovirus and the vaccine can also take account o variations in the peptide between different viruses of the same type.
Further, a peptide according to the present invention may therefore be administered with a polypeptide according to GB-A-2 128 621 and/or a VP3 peptide as described above. The peptides may be mixed together or administered separately over a period of time in any order. Preferably, peptides in respect oE the same type, more preEerably the same strain, of enterovirus are administered. Preferred polypeptides according to GB-A-2 128 621 and preferred VP3 peptides which may be administered simultaneously with or separately from a peptide according to the present invention are those mentioned above. Alternatively, a polypeptide according to GB-A-2 128 621 and/or a VP3 peptide may be linked to the same carrier as a polypeptide o~ the present invention.
It is also preferred to formulate vaccine compositions as physical mixtures which include other antigens particularly those commonly used in infant vaccines, such as tetanus, diphtheria and whooping cough. However, as indicated before, such antigens may, if desired, be linked chemically to the peptide of the invention in order to render it immunogenic.
Although the peptides of the present invention, when in immunogenic form, can act as vaccines to protect a patient by inducing the production of the appropriate antibodies, it is possible that, in addition, the peptide may have a chemotherapeutic effect. Thus it is believed that the same peptide sequence which can evoke the production of antibodies may be the se~uence in the viral capsid protein which enables the virus to attach itself to a cell within a patient and thereby cause the infection. Thus the peptide of the present invention may have a competitive effect and, by occupying the appropriate cell receptor sites, prevent the virus itself from infecting the patient. Generally, immunogens comprising the peptides of the present invention will be administered by injection which will usually be intramuscular but can be by routes, such as intraperitoneally or subcutaneously.

1~87~7 The peptides of the present invention can also be used to prime the immune system of a patient to exhibit an enhanced response to vaccination against diseases caused by enteroviruses. An effective amount, typically 100 ug to 1 mg, of a peptide of the invention can be administered to a patient and, after a suitable amount of time has elapsed, the patient can be vaccinated against a disease caused by a corresponding enterovirus in the conventional manner. Less material, both of the peptide of the invention and of that required for the conventional vaccination, may be needed and fewer challenges may be required to achieve ef~ective vaccination.
The present invention also provides a pharmaceutical composition useful as a vaccine against a disease caused by an enterovirus which composition comprises a pharmaceutically acceptable carrier or diluent and, as active ingredient, a peptide of the present invention. The actual form of the peptide in this composition, i.e.
whether it is lin~ed to another compound or not, will depend upon the use to which the composition is to be put.
The composition may, for example, comprise an effective amount of the peptide in a suitable diluent such as Freunds Complete Adjuvant (FCA) or physiologically acceptable saline.
An alternative use for the peptides of the present invention is in the diagnosis of infection by enteroviruses. This diagnosis may be carried out by the detection of the 12~3~7~7 presence or absence of antibody to the approriate virus in the patient. For this purpose, the peptides are usually bonded to inert carriers as mentioned hereinbefore and, in such form, they can also be used as an affinity chromatography medium in the isolation of antibodies to the virus. The peptide of the invention may therefore form a component of a test kit, suitable for use in determining antibody against an enterovirus, which kit also includes means for determining antibody bound to the peptide. Any suitable immunoassay system, for example radioimmunoassay system, may be used to determine the antibody.
The following Examples illustrate the invention.
Example 1: Identification of the antigenic site coded for by codons 286-290 in the RNA sequence coding for the VPl capsid protein, and a subsidiacy antigenic site coded for by codons including 58,59,77 and 79 in the RNA
se~uence coding for the VP3 capsid protein, of poliovirus type 3 Sabin strain.
1 Identification of an antigenic site coded for .
by VPl codons 286-288.
Mutants which were resistant to neutr~lization by specific monoclonal antibodies were isolated by plaque formation by antibody-treated virus under an agar overlay containin~ antibody (Minor et al 1983). They were isolated from the type 3 poliovirus strain P3 Leon/USA/1937 (Minor et al 1983, Evans et al 1983) and its attenuated derivative the - ~287~'~7 Sabin vaccine strain. The mutants were characterized by their susceptibility to neutralization by a panel of twelve monoclonal antibodies as shown in Table 2 below. Mutants obtained from a total of 213 plaques derived from P3/Leon/37 virus could be classified into 16 distinct groups on the baiss of the pattern of their neutralization. Similarly fifteen distinct groups of mutants were selected from the Sabin vaccine strain from a total of 129 plaques.
Evidence for a second, independent antigenic site was obtained using a highly strain-specific monoclonal antibody, 138, which neutralizes the Sabin type 3 vaccine virus or most strains derived from it, but not P3/Leon/37 or other strains (Ferguson _ al 1982). All mutants of the Sabin strain which were resistant to the twelve antibodies which selected mutants with substitutions in the first site tsee GB-A-2 128 621), were found to be still sensitive to antibody 138, while all mutants selected for resistance to antibody 138 were fully sensitive to these site 1 antibodies.
Antibody 138 was therefore thought to be directed against an independently mutable site, distinct from the first site.
This second site was identified as follows.
Nucleotide sequencing studies (Stanway et al 1983, 1984) indicate that there are only two predicted amino acid differences between Leon 12alb tthe Sabin strain) and P3/Leon USA 1937 in the structural portion of the genome, one in the region coding for protein VP3, the other at codon 286 from 12l~7~47 the 5' end of the region coding for VPl r which is a lysine in P3/Leon/37 and an arginine in the Sabin vaccine strain. A
recombinant plasmid was prepared from whole cloned cDNA
copies of the genomes of Sabin type 3 vaccine virus and P3/Leon/1937 such that the recombinant genome contained the VP3 region of the Sabin strain and the VPl of Leon (G
Westrop, unpublished). Virus was recovered by transfection of cells with this plasmid (Racaniello and Baltimore 1981) and characterized by partial sequencing of the genome in the regions coding for VPl and VP3. This recombinant virus failed to react with antibody 138, suggesting that the specific site did not involve VP3 of the Sabin strain, but included the amino acid at position 286 from the N terminus of VPl.
Mutants resistant to 138 isolated from Sabin virus proved to have a base substitution in the adjacent codon (287) leading to the substitution of an aspartate residue for asparagine.
Additionally, a series of isolates was obtained from a hypogammaglobulinaemic vaccinee (222f) who excreted a vaccine derived type 3 poliovirus for a prolonged period after administration of a monovalent type 3 Sabin vaccine (MacCallum 1971)~ Six of these strains proved to be different from each other on the basis of Tl oliogonucleotide maps of their R~A, but all failed to react with antibody 138 (P Minor unpublished). Sequencing studies revealed that the ~2~i79~ :~7 amino acid at codon 288 from the N-terminus of VPl was aspartate for all six excreted strains and asparagine for the parental Sabin vaccine virus.
The findings with recombinant virus, mutant virus and excreted strains strongly im~ly that antibody 138 recognises a strain specific antigenic site encompassing codons 286-288 from the N-terminus of VPl.
2. Identification of the antigenic site coded for by VPl codons 286-290 and of the subsidiary antigenic site Next, monoclonal antibodies were prepared by the fusion of splenocytes from immunised Balb/C mice with myeloma cells as described (Ferguson et al 1984). The mice were immunised with antigenically abnormal virus, either an antigenically drifted strain (P3/23127/Finland/84) implicated in an outbreak of poliomyelitis in Finland ~Lenikki et al 1985) or poliovirus type 3 Sabin strain which had been treated with trypsin as described (Fricks et al 1985).
Hybridoma supernatants were screened using a modified single radical diffusion assay (antigen blocking test) and monoclonal antibodies were generally used as ascites prepared in syngeneic mice (Ferguson et al 1984). Immunisation schedules and other protocols were similar in all ~usions~
Monoclonal antibodies ~enerated from animals immunised with both types of virus were able to neutralise both untreated virus and all mutants having substitution within the principal antigenic site, the region in the VPl :~2~ 7 capsid protein from amino acids 89 to 100. Mutants were selected with four of these antibodies as follows. Antigenic variants were selected by plaque formation on He~2c cells by virus treated with antibody under an agar overlay containing the antibody as described (Minor et al 1983). Putative mutants were subject to two cycles of selection, and small working pools grown up from the secondary plaque plugs.
The antigenic patterns of reaction of the mutants picked are shown in Table 3, together with two mutants which have been previously described. Mutant 1 had an amino acid substitution in VPl at position 98, where a glycine residue was found instead of an arginine. Mutant 2 had a mutation in VPl at position 287, where an aspartate residue was found in place of an asparagine. The thirteen mutants ell into three distinct non overlapping groups, implying the existence of three independent antigenic sites.
The genomic RNA of mutants 3 to 13 was sequenced by primer extension, through five regions corresponding to areas containing antigenically significant mutations in type 1 or type 3 poliovirus. These included regions coding for residues 89 to 100, 220 to 222 and 286 to 290 of VPl, residues 50 to 80 of VP3 and residues 160 to 180 of VP2. The eesul~s are presented in Table 4. Mutants 2 to 8 had single base s~bstitutions resulting in predicted amino acid changes in VPl at residLes 287 and 290, and in VP3 at residues ~8, 59, 77 and 79. It was notable that the reactions of ~2~7~

antibodies 1023, 840, 251, 557, 1084 and 1007 were affected both by mutations within VP3 at residues 58 and 59 and by mutations within VPl at residues 287 and 290. It is believed that the second antigenic site comprising residues 286 to 290 of VPl and the subsidiary antigenic site comprising residues 58,59,77 and 79 form a single operationally distinct antigenic site.
Example 2: Synthesis of Cys-Glu-Val-Asp-Asn-Glu-Gln-Pro-Thr-Thr-Arg-Ala-Gln-Lys-Leu-Phe-Ala-Met-Gly-Val-Asp-Tyr-Arg-Asn-Asn-~eu-Asp-Pro-Leu-Cys (Peptide 1) and Cys-Gly-Val-Asp-Tyr-Arg-Asn-Asn-Leu-Asp-Pro-Leu-Cys tPeptide 2) The required peptides were synthesised by the Fmoc-polyamide mode of solid phase peptide synthesis (Brown et al 1983 and references cited therein). The general protocol was as follows:
Polydimethylacrylamide gel resin (a copolymer of dimethylacrylamide-ethylenebisacrylamide-acryloylsarcosine methyl ester) containing 0.3 milliquivalents of sarcosine per gram resin, was treated with ethylenediamine overnight.
After thorough washing, the acid labile linkage agent,

4-hydroxymethylphenoxyacetic acid, was added as its symmetrical anhydride. After thorough washing this afforded the low loading acid labile resin that was used to prepare the peptides under discussion.
Fmoc-amino acids were coupled tin a twelve fold i2~4~7 excess) as their preformed symmetrical anhydrides: the Fmoc-amino acid (2 equiv) was dissolved in dichloromethane with a few drops of N,N-dimethylformamide (DMF) if required to aid dissolution. N,N-Dichlorohexylcarbodiimide tDCC) (1 equiv) was added and the mixture stirred at room temperature for 10 minutes. The precipitated ~,N-dicyclohexylurea (DCU) was filtered off, the filtrate evaporated to dryness and the residue dissolved in DMF. This solution was added to the deprotected and washed resin and the coupling reaction allowed to proceed.
Asparagine and glutamine residues were added as follows: l-hydroxybenzotriazole (1 equiv) and DCC (1 e~uiv) were dissolved in DMF at 0C. After stirring for ten minutes at 0C a solution of Fmoc-asparagine (or glutamine) (1 equiv) lS in DMF was added. This mixture was stirred for a further ten minutes at 0C and then the entire mixture added to the resin and coupling allowed to proceed.
A typical synthetic cycle was as follows:
Reagent Duration Operation 20 DMF 5 x 1 min Wash 20% Piperidine/DMF 1 x 3 + 1 x 7 min Deprotection DM~ 10 x 1 min Wash Preformed symmetrical anhydride or active ester 60 - 120 min Coupling DMF 5 x 1 min Wash 8~7~ ~

The completeness of coupling at each stage was monitored using ninhydrin and trimethylbenzenesulphonic acid test reagents.
The coupling of the first residue to the S derivatised resin was carried out in the presence of N,N-dimethylaminopyridine (DMAP) (0.1 equiv).
As the C-terminal dodecapeptide sequence ~as common to both peptides, the synthesis was carried out on twice the scale, half being used to continue the first peptide and cysteine being added to the other half to give the second peptide.
The quantities used were as follows:
Acid Labile Resin (0.1 g; 0.3 mequiv g 1); for each cycle, Fmoc-amino acid ~3.6 mmol) and DCC (0.38 g; 1.8 lS mmol); for the first cycle, DMAP ~0.022 g; 0.18mmol); and, for asparagine and glutamine residues HOBT (0~24 g; 1.8 mmol). The cycles were carried out on the following basis:

Fmoc-amino acid-OHQuantities Coupling Time Fmoc-Cys(Trt)-OH2.10 g; 3.6 mmol1 hour 20 Fmoc-Leu-OH 1.28 g; 3.6 mmol 1 hour Fmoc-Pro-OH 1.22 g 3.6 mmol 1 ho~r Fmoc-Asp(0But)-OH1.48 g; 3.6 mmol1 hour Fmoc-~eu-OH 1.28 g; 3.6 mmol 1 hour Fmoc-Asn-OH 0.64 g; 1.8 mmol 1 hour 25 Fmoc-Asn-OH 0.64 g; 1.8 mmol 3 hour Fmoc-Arg(Mtr)-OH2.20 g; 3.6 mmol1 hour :~2~37'1~'7 Fmoc-Tyr(Bu )-OH 1.64 g; 3.6 mmol 1 hour Fmoc-Asp(OBu )-OH 1.48 g; 3.6 mmol 1 hour Fmoc-Val-OH 1.22 g; 3.6 mmol 1 hour Fmoc-Gly-OH 1.08 g; 3.6 mmol 1 hour Resin split in half after deprotection, to half was added:
Boc-Cys(Trt)-OH 0.83 g; 1.8 mmol 1 hour giving the second peptide.
To the other half of the resin was added:
10 Fmoc-Met-OH 0.67 g; 1.8 mmol 1 hour Fmoc-Ala-OH 0.56 g; 1.8 mmol 1 hour Fmoc-Phe-OH 0.74 g; 1.8 mmol 1 hour Fmoc-Leu-OH 0.64 g; 1.8 mmol 1 hour Fmoc-Lys(Boc)-OH 0.84 g; 1.8 mmol 1 hour Fmoc-Gln-OH 0.33 g; 0.9 mmol 1 hour ~ 2 hour~
Fmoc-Ala-OH 0.56 g; 1.8 mmol 1 hour Fmoc-Arg(Mtr)-OH 1.10 g; 1.8 mmol 1 hour Fmoc-Thr(Bu )-OH 0.72 g: 1.8 mmol 1 hour 20 Fmoc-Thr(Bu )-OH 0.72 g; 1.8 mmol 1 hour Fmoc-Pro-OH 0.61 g; 1.8 mmol 1 hour Fmoc-Gln-OH 0.33 g; 0.9 mmol 5 hoùrs Fmoc-Glu(OBut)-OH 0.77 g; 1.8 mmol 1 hour Fmoc-Asn-OH 0.32 g; 0.9 mmol 1 1/2 hours Fmoc-Asp(OBut)_OH 0.74 g; 1.8 mmol 1 hour ~287~ ~7 Fmoc-Val-OH 0.61 g, 1.8 mmol l hour Fmoc-Glu(OBu )-OH 0.77 g; 1.8 mmol l hour Boc-Cys(Trt)-OH 0.83 9; 1.8 mmol l hour After washing both peptide resins were shrunk by washing with dichloromethane and diethyl ether.
Peptide l The peptide was cleaved from the resin and the side chain protecting groups were removed by treating the peptide resin with 95~ trifluoroacetic acid (TFA)/5% ethane dithion (EDT) (3 x 1 hour). After filtration, evaporation of each fragment afforded a residue which on trituration with diethyl ether afforded three white solids (104 m~, 80 mg and 41 mg). Hplc (uBondpak C18; linear gradient 5 - 95%
0.1% TFA/CH3 CN - 0.1~ TFA/H20 over 20 minutes) showed the product to consist of four major compounds, i.e. Mtr protecting groups still present. The Mtr (~-methoxy-2,3,6-trimethylbenzenesulphonyl) group used for the peotection of the arginine side chain is cleaved considerably more slowly than the t-butyl based side chain protecting groups used for other functional residues and, therefore, extended treatment with trifluoroacetic acid was necessary to facilitate its removal. The three Eractions were therefore combined and re-treated with TFA for a further five hours. ~Iplc showed one major peak and several smaller ones. The combined materials were dissolved in 10%
acetic acid and then subjected to exclusion chromatography on Sephadex G-25 Superfine eluted with 10~ acetic acid.
The eluate was monitored at 254 nm and fractions ~2t37~'~'7 corresponding to the required compound were combined and lyophilised af~ording the product as a white fluffy solid (130 mg). This compound did not give a molecular ion when subjected to fast atom bombardment mass spectrometry.
Peptide 2 The peptide was cleaved from the resin by treating with 95~ TFA/5% EDT (3 x 1 hour). ThiS afforded three white solids (70 mg, 61 mg and 42 mg). Hplc (same conditions as above) showed two major peaks again indicating Mtr groups present. The three fractions were combined and re-treated with TFA to give a white solid (149 mg). Hplc showed the product to be essentially homogeneous. FAB-mass spectrometry gave a sharp molecular ion at 1481, this being consistent with a molecular weight 15 of 1480.
Example 3: Preparation o~ Cys-Ile-Pro-Phe-Asp-Leu-Ser-Ala-Thr-Lys-L~s-Asn-Thr-Met-Glu-Met-Tyr-Cys (Peptide 3) and Cys-Ile-Pro-Leu-Asn-Leu-Glu-Ser-Thr-Lys-Arg-Asn-Thr-Met-Asp -Met-Tyr-Cys (Peptide 4) These two peptides comprise amino acid residues 58 to 61 of the VP3 capsid protein of type 1 Sabin poliovirus (Peptide 3) and type 3 Sabin poliovirus (Peptide 4). Both peptides are composed of residues S3 to 68 with Cys residues at each terminus. The peptides were synthesised according to the procedure of Example 2. Hplc showed each product to be essentially homogeneous.
FAB-mass spectrometry gave a sharp molecular ion at 2097 for Peptide 3, this being consisten~ with a molecular 121~7'~

weight of 2096, and at 2134 for Peptide 4, this being consistent with a molecular weight of 2133.
Example 4: Measu,rement of Specific Antibody Responses The specific antibody responses of laboratory rabbits to peptides 1 and 2 were measured in respect to:
1. antibody to uncoupled peptide (1 or 2) detected by enzyme-linked immunoabsorbent assay (E~ISA), and 2. antibody to polioviruses of types 1, 2 and 3 as detected by antigen blocking assays against poliovirus C antigen measured in single-radial-diffusion (SRD) tests in gels.
Coupling of peptide of bovine thyroglobulin 1 ml of O.lM sodium phosphate buffer pH 7.5 was added to a glass vial containing 30 mg of bovine thyroglobulin (BTG, Sigma) or 30 mg of keyhole limpet haemocyanin. The dissolved material was transferred with 1 ml sodium phosphate buffer washing to a second vial containing 10 mg of peptide (1 or 2) to give a final volume of 2 ml peptide-BTG solution. The vial was wrapped in aluminium foil to exclude light. A solution of 2~
glutaraldehyde was made in 0.1 M sodium phosphate buffer pH
7.5 and 200 ul, added to the peptide-BTG solution in four lots of 50 ul, shaking between additions, and then left for 2S 1 hour at room temperature with intermittent shaking. The solution was then dialysed against one litre of phosphate lZ8~7~47 buffered saline (PsS) at 4 overnight, and then against 1 litre of fresh PBS for a further eight hours. Coupled peptide was stored at -70C until required.
Coupled oligopeptides used for immunization of experimental animals Synthetic oligopeptides 1 and 2 were conjugated separately to bovine thyrogobulin (BTG) as described above.
The preparations used for immunization contained 500 ug/ml of peptide 1 or 2 and 1500 ug/ml of BTG suspended in phosphate buffered saline (pH 7.2).
Immunization schedule for experimental animals Young (5-6 months of age) healthy rabbits were injected intramuscularly with an initial dose of 0.5 ml (500 ug) coupled peptide mixed with an equal volume of Freunds complete adjuvant (FCA, Bacto) and subsequently injected with booster doses (0.5 ml) 500 ug coupled peptide according to the following schedule. Serum samples for analysis were collected at intervals up to 76 days after the first injection.
20 Day 0 0.5 ml coupled peptide + FCA serum sample Day 20 serum sample Day 24 0.5 ml coupled peptide + FCA
Day 28 serum sample Day 41 serum sample 25 Day 44 0.5 ml coupled peptide ~2874~7 Day 55 serum sample Day 76 serum sample Enzyme immunoassays (ELISA) for antibody to oligopeptide Enzyme immunoassays were carried out to investigate the immune response of the rabbit to the peptide. Rabbit sera were examined for antibody which bound to oligopeptide linked to polyvinyl plates by glutaraldehyde. The bound antibody was detected b~ the addition of anti rabbit antibodies which were coupled to biotin followed by streptavidin biotinylated horse radish peroxidase complexes. On addition of substrate for the horseradish peroxidase (5-aminosalicylic acid) a colorimetric change takes place, the intensity of which is proportion to the amount of antibody bound to peptide.
Ninety-six well Microelisa plates (Dynatech) were coated with oligopeptide (10 ug/ml). After incubation overnight at 4 centigrade plates were washed x5 with PBS
containing 0.5~ Tween 20 (Koch-Light Laboratories, Colnbrook, Berks). Dilutions of rabbit sera in PBS were addded to wells and incubated for 2 hours at 37C. Plates were washed x5 with phosphate buffered saline containing 0.5~ Tween 20 and donkey anti rabbit Ig linked to Biotin (Amersham International) diluted in PBS added. After 1 hour at 37 degrees centigrade the biotinylated antibody was removed, the plates washed x 5 with phosphate buffered saline containing Tween 20. Streptavidin biotinylated ....

horseradish peroxidase complexes were added and the plates incubated at 37C for 30 minutes. The plates were washed x3 in Pss containing 0.5~ Tween 20 and x2 with Pss and the substrate, 5-aminosalicylic acid (80 ug in 100 ml PBS was added to each well. The plates were incubated at 37 degrees centigrade until colour developed. Optical density was read on a Titertek multiscan set at 492 nm. The machine was 'blanked' using substrate and serum dilutions considered positive if the optical density was greater than that of 1:100 dilution of normal rabbit serum collected from the animal prior to immunization of the peptide.
Antigen blocking assays for antibody to poliovirus anti~en employing single radial diffusion (SRD) in gel The rabbit sera were tested in SRD
antigen-blocking tests to determine their reactivities with C antigen of poliovirus type 1 or 3. The method used was a modification of the autoradiographic SRD method of Schild et al (1980) as described elsewhere (Ferguson et al 1982).
Briefly, [35S]-methionine labelled 80S 'C' peaks of poliovirus antigen from sucrose gradients were mixed with the test monoclonal antibody before adding to wells in agarose gels containing low concentrations of hyperimmune anti-poliovirus type 3 serum. Diffusion or radiolabelled antigen in the gel after 24-48h was detected by autoradiography. Test antibody which reacts with 'C' antigen inhibits its diffusion into the gel compared with ~Z~37447 control antigen treated with phosphate buffered saline alone. The antigen blocking titres are assessed as the dilution of serum which significantly reduces the zone size in comparison with zones produced with control antigen mixed with phosphate buffered saline.
RESULTS
Induction of antibody to homologous peptide The titres of antibody to peptide 2 determined by ELISA assay are shown in Table 5 for representative animals. Twenty one days following the initial immunization with peptide all animals had readily demonstrable antibody to homologous peptide. The titres had increased by day 41 and in later serum samples following booster doses of the oligopeptides. No anti-peptide antibody was detected in prebleeds from any animal. Table 6 shows ELISA titres to peptide 1. This peptide is peptide SlOa of GB-A-2 128 621 plus peptide 2 linked together and the titres of antibody reached with each peptide are also given.
Induction of antibody to poliovi_us a) antigen blocking antibody Antibody specific for empty virions (C antigen) may be detected by antigen blocking assays employing the single radial diffusion test (Schild et al 1980, Ferguson et al 1982). This method was applied to sera obtained from animals immunized with peptides 1 and 2 coupled to BTG.

lZ87~

Table 7 shows the induction of blocking antibodies to poliovirus type 3 C antigen in rabbits injected with peptide 2. Antibody first appeared between day 17 and 31 in 3 out of 4 animals. Antisera from animals immunised with peptide 2 were tested against viruses with mutations in the VPl antigenic sites both of the present invention and of GB-A-2 128 621. The results are shown in Table 8.
Table 9 shows the induction of blocking antibodies to poliovirus type 3 C antigen in rabbits injected with peptide 1.
References Brown et al 1983, J. Chem.Soc.Perkin Trans. I, 1161 et seq Evans et al, 1983, Critical role of an eight amino acid sequence of VPl in neutralization of poliovirus type 3, Nature 304, 459-462.
Ferguson et al 1982, Monoclonal antibodies specific for the Sabin vaccine strain of poliovirus, Lancet II 122-124.
Ferguson et al 1984, Neutralisation epitopes on poliovirus type 3 particles: an analysis using monoclonal antibodies, J. Gen.Virol. 65, 197-201.
Fricks et al 198S, Trypsin sensitivity of the Sabin strain of type 1 poliovirus: cleavage sites in virions and related particles, J. Virol. 54, 856 et seq Leinikki et al 1985, Paralytic poliomyelitis in 1;287~7 Finland, Lancet II 507 MacCallum, Hypogammaglobulinaemia in the United ~ Kin~dom, Medical Research Council special report series, No 310, pps 72-85.
Minor et al 1983, Location and primary structure of a major antigenic site for poliovirus neutralization, Nature 301 674-679.
Racaniello and Baltimore, Cloned poliovirus complementary DNA in infections in mammalian cells, Science 214 916-919.
Schild et al, 1980, J. Gen. Virol. 51 157-170 Stanway et al, 1983, The nucleic acid sequence of poliovirus type 3 Leon 12alb; comparison with type 1, Nucleic Acids Research 11 5629-5643.
Stanway et al, 1984, Comparison of the complete nucleotide sequences of the genomes of the neurovirulent poliovirus P3/Leon/37 and its attenuated Sabin vaccine derivative P3/Leon/12alb, Proc. Natl. Acad. Sci. USA 81 1539-1543.

37~a ~7 Table 1 poliovirus codon 286 287 288 289 290 type 3 Sabin Arg Asn Asn Leu Asp Leon Lys Asn Asn Leu Asp

5 mutant a tExample 1) Arg Asp Asn Leu Asp mutant b (Example 1) Arg Asn Asp Leu Asp mutant c (Example 2) Arg Asn Asn Leu Glu poliovirus codon 286 287 288 289 290 type 2 10 Sabin Lys Asp Gly Leu Thr poliovirus codon 287 288 289 290 291 292 type 1 Sabin Lys Asp Gly Thr Leu Thr Mahoney Lys Asp Gly Thr Leu Thr lZ874~7 _ 42 -Table 2 Reactions of representatives of 342 antigenic mutant viruses with monoclonal antibodies virus ~tr~in 25-1-14 25-4-12 22-4-4 199 194 134 208 175 204 197 165 198 _ _ _ P3 Leon USA/l 9 37 1 r r r r r 2 r r r r r r r 3 r r r r r r r r r r r 4 r r r r r r r r r r rrrr r r r r

6 rr r r r

7 rrrr r r r B r r r r r 9r r r r r r r r r r 1~ r r rrr r r r rrr 11 rr rr rr 12 r rrrrr rr 13 r r r r r r r r 14 r r r rr lSr r rrr r r rr r 16 r r rr r r rr S~bin Le~n 1 2.~ 1 b A r r r r r rr r r r r L r r rrr rr r r r D r r r r r r r r r r r r E r r r r r r r r r r F r r rrr r r rr r rrr r r r r I r r J r rrr r K r r r,r r r r L r r r r r rr ~ r r rr N r r r r O r r r r r r P r r r r r r r r r O r r ~utant viruses were said to be sensitive to antibody if a 1:10 dilution of antibody as ascitic fluid was able to preserve a cell sheet from challenge with 104 TCIDso of virus during incubation at 35 for 2 days.
Results were checked by plaque assay in the presence of dilutions of antibody.
r indicates resistance 12~7~7 Q~ ~
,v oC~ ~, ~ ~
~ 0 ~
,~ u~ r~r- C
O ~\ o ~ ~ . ~
:) ~C
o ~ ~ S
o ~ ~ ~a E~ O
Q ~ ,~
c v u~ ~ ~ ~ E E
C . ~ ~
c c ~~ ~ ~ ~ ~ E E
O ~ o o o E o h ~ ~r ~ ~r ~ ~ ~ co t~ CO
a) ~
S~ ~
`~
o ~ ~ ~ C
_ O OC
Q C R
~ Q
o ~
R
o ~ ~ ~

. 43 ~L2~7~

_ 4 4-Table 4 Location of amino acid substitutions in antiqenic mutants of type 3 poliovirus Mutant Protein Amino acid 1 VP1Argg8 - Gly 2 VP1n287 sp 3 VP3Glu58 - Asn 4 VP3Ser59 - Asn VP3Ser59 - Arg 6 VPlA P290 7 VP3P77 Glu

8 VP3ser79 - Leu

9 VP2hr167 Lys VP2a 166 Ala 11 VP2172 a 12 VP2Glul72 Lys 13 VP2Asnl64 - Lys VP2Asnl64 Thr ~Z~7447 Table 5: Antibody titres of rabbits immunised with BTG
coupled peptide 2 with homologous peptide Rabbit Day 0Day 20 Day 41 237 <100 <100 800 238 <100 <100 1600 ~3g <100 <100 200 240 <lO0 lOO 6400 Table 6: Antibody titres to peptide 1 in rabbits immunised with BTG coupled peptide 1 Rabbit Day 0 Day 17 Day 37 256 <10~ 400 6400 257 <100 1600 12800 25~3 <100 1600 >12800 259 <100 1600 12800 able 7: Antigen blocking antibodies to Sabin type 3 C
antigen in rabbits immunised with peptide 2 Day 0 Day 17 Day 31 Day 53 237 <5 <5 5 40 238 <5 <5 20 40 239 <5 <5 10 5 240 <5 <5 <S 10 able 8: Antigen blocking titres to C antigen of various strains of poliovirus type 3 in rabbits immunised with peptide 2 on day 53 (after 3 doses peptide Difference in sequence from Rabbit Strain SP6 237 238 239 240 Leon VPl 287 20 40 10 5 306 VPl 88-100 ~0 20 10 5 183 VPl 88-100 20 40 10 5 63.1.9 3 changes 10 20 10 10 able 9: Antigen blocking titres against type 3 C antigen in animals immunised with peptide 1 Rabbit Day 0 Day 17 Day 37 Day 64 256 <5 <5 80 20 257 <5 20 >320 1280 258 <5 5 >320 ND
259 <5 <5 10 <5

Claims (41)

The embodiments od the invention, in whixh an exxlusive privilege or property is claimed, are defined aa follows:

1. A synthetic peptide, suitable for use in vaccination against or diagnosis of a disease caused by an enterovirus, which is the peptide coded for by codons 286-290 in the RNA sequence coding for the structural capsid protein VP1 of poliovirus type 3 Sabin strain or by equivalent codons of another enterovirus or is an antigenic equivalent of the said peptide, the antigenic equivalent being:
(i) a said peptide modified by the inclusion therein of one or more changes to the amino acid sequence;
(ii) a first longer peptide which incorporates the sequence of a said peptide or a said modified peptide and which has up to four extra amino acid residues attached to the C-terminal end of the said sequence or up to four extra amino acid residues attached to the N-terminal end of said sequence or up to four extra amino acid residues attached to the C-terminal end and up to four extra amino acid residues attached to the N-terminal end of said sequence;

(iii) a second longer peptide which incorporates the sequence of a said peptide, a said modified peptide or a said first longer peptide, to which sequence is linked directly, by means of a further amino acid residue or by means of a disulphide bridge between Cys residues attached to each sequence, a sequence of up to eighteen amino acid residuea which comprises a hexapep-tide sequence coded for by codons 93 to 98 in said RNA sequence or by equivalent codons of another enterovirus; or (iv) a third longer peptide which incorporates the sequence of a said peptide, a said modified peptide or a said first longer peptide, to with sequence is linked directly, by means of a further amino acid residue or by means of a disulphide bridge between Cys residues attached to each sequence, a sequence comprising residues 58 and 59 of the VP3 capsid protein of an entero-virus of the same type but starting with a residue numbered no lower than 53 and ending with a residue numbered no higher than 68;
each said antigenic equivalent being capable of raising antibodies capable of neutralizing the same strain and type of enterovirus as said peptide to which said antigenic equivalent corresponds and the numbers of the codons being counted from the 5'-terminus of the nucleotide sequence for the VP1 capsid protein.

2. A peptide according to claim 1, suitable for use in vaccination against or diagnosis of a disease caused by type 3 poliovirus, which comprises the sequence A0-A1-A2-Leu-A3 (I) in which (i) A0 is Arg, each of Al snd A2 is independently Asn or Gln and A3 is Asp or Glu or (II), with the others of A0 to A3 being as defined in (i), A0 is Lys or A1 or A2 is Asp or Glu.

3. A peptide according to claim 2, wherein A0 is Arg, both A1 and A2 are Asn and A3 is Asp.

4. A peptide according to claim 2, which comprises the sequence Gly-Val-Asp-Tyr-Arg-Asn-Asn-Leu-Asp-Pro-Leu.

5. A peptide aacording to claim 1, suitable for use in vaccination against or diagnosis of a disease caused by type 2 poliovirus, which comprises the sequence (II):
Lys-Al-Gly-Leu-A3 (II) in which A1 is Asp or Glu and A3 is Thr or Ser.

6. A peptide according to claim 5, which comprises the sequence:
Gly-Val-Asp-Tyr-Lys-Asp-Gly-Leu-Thr-Pro-Leu.

7. A peptide according to claim 1, suitable for use in vaccination against or diagnosis of a disease caused by type 1 poliovirus, which comprises the sequence (III):
Lys-A4-Gly-A5-Leu-A6 (III) in which A4 is Asp or Glu and each of A5 and A6 is independently Ser or Thr.

8. A peptide according to claim 7, wherein A4 is Asp and A5 and A6 are both Thr.

9. A peptide according to claim 7, which comprises the sequence:
Gly-Val-Asp-Tyr-Lys-Asp-Gly-Thr-Leu-Thr-Pro-Leu.

10. A peptide according to claim 1, which further has a Cys residue at one or both ends.

11. A synthetic peptide suitable for use in vaccination against or diagnosis of a disease caused by a poliovirus, which is the peptide coded for by codons 286-290 in the RNA sequence coding for the structural capsid protein VPl of poliovirus type 3 Sabin strain or by equivalent codons of another poliovirus or is an antigenic equivalent of the said peptide, the antigenic equivalent being:
(i) a said peptide modified by the inclusion therein of one or more changes to the amino acid sequence;
(ii) a first longer peptide which incorporates the sequence of a said peptide or a said modified peptide and which has up to four extra amino acid residues attached to the C-terminal end of the said sequence or up to four extra amino acid residues attached to the N-terminal end of said sequence or Up to four extra amino acid residues attached to the C-terminal end and up to four extra amino acid residues attached to the N-terminal end of said sequence; or (iii) a second longer peptide which incorporates the sequence of a said peptide, a said modified peptide or a said first longer peptide, to which sequence is linked directly, by means of a further amino acid residue or by means of a disulphide bridge between Cys residues attached to each sequence, a sequence of up to eighteen amino acid residues which comprises a hexapep-tide sequence coded for by codons 93 to 98 in said RNA sequence or by equivalent codons of another poliovirus;
each said antigenic equivalent being capable of raising antibodies capable of neutralizing the same strain and type of enterovirus as said peptide to which said antigenic equivalent corresponds and the numbers of the codons being counted from the 5'-terminus of the nucleotide sequence for the VPl capsid protein.

12. A process for the preparation of a synthetic peptide as defined in claim 1, which process comprises chemically synthesising the peptide from single amino acids and/or preformed peptides of two or more amino acid residues in accordance with the order of the amino acid residues in the peptide.

13. A process for the preparation of a synthetic peptide as defined in claim 1, which process comprises identifying either (a) the codons in the RNA sequence coding for the structural capsid protein VP1 of an enterovirus which are or which are equivalent to codons 286-290 for poliovirus type 3 Sabin strain, the numbers of the codons being counted from the 5'-terminus of the nucleotide sequence coding for the VP1 capsid protein, or (b) the corresponding codons in a DNA sequence corresponding to said RNA sequence; and producing a said synthetic peptide which comprises the peptide sequence corresponding to the codons thus identified.

14. A conjugate, suitable for use in vaccination against a disease caused by an enterovirus, which conjugate comprises a physiologically acceptable carrier and, linked thereto, a synthetic peptide which is the peptide coded for by codons 286-290 in the RNA
sequence coding for the structural capsid protein VPl of poliovirus type 3 Sabin strain or by equivalent codons of another enterovirus or is an antigenic equivalent of the said peptide, the antigenic equivalent being (i) a said peptide modified by the inclusion therein of one or more changes to the amino acid sequence;
(ii) a first longer peptide which incorporates the sequence of a said peptide or a said modified peptide and which has up to four extra amino acid residues attached to the C-terminal end of the said sequence or up to four extra amino acid residues attached to the N-terminal end of said sequence or up to four extra amino acid residues attached to the C-terminal end and up to four extra amino acid residues attached to the N-terminal end of said sequence;

(iii) a second longer peptide which incorporates the sequence of a said peptide, a said modified peptide or a said first longer peptide, to which sequence is linked directly, by means of a further amino acid residue or by means of a disulphide bridge between Cys residues attached to each sequence, a sequence of up to eighteen amino acid residues which comprises a hexapep-tide sequence coded for by codons 93 to 98 in said RNA sequence or by equivalent codons of another enterovirus; or (iv) a third longer peptide which incorporates the sequence of a said peptide, a said modified peptide or a said first longer peptide, to which sequence is linked directly, by means of a further amino acid residue or by means of a disulphide bridge between Cys residues attached to each sequence, a sequence comprising residues 58 and 59 of the VP3 capsid protein of an entero-virus of the same type but starting with a residue numbered no lower than 53 and ending with a residue numbered no higher than 68;
each said antigenic equivalent being capable of raising antibodies capable of neutralizing the same strain and type of enterovirus as said peptide to which said antigenic equivalent corresponds and the numbers of the codons being counted from the 5'-terminus of the nucleotide sequence for the VPl capsid protein.

15. A conjugate according to claim 14, suitable for use in vaccination against a disease caused by type 3 poliovirus, wherein the synthetic peptide comprises the sequence (I):
A0-Al-A2-Leu-A3 (I) in which (i) A0 is Arg, each of Al and A2 is independently Asn or Gln and A3 is Asp or Glu or (ii), with the others of A0 to A3 being as defined in (i), A0 is Lys or Al or A2 is Asp or Glu.

16. A conjugate according to claim 15, wherein A0 is Arg, both Al and A2 are Asn and A3 is Asp.

17. A conjugate according to claim 15, wherein the synthetic peptide comprises the sequence Gly-Val-Asp-Tyr-Arg-Asn-Asn-Leu-Asp-Pro-Leu.

18. A conjugate according to claim 14, suitable for use in vaccination against a disease caused by type 2 poliovirus, wherein the synthetic peptide comprises the sequence (II):

Lys-Al-Gly-Leu-A3 (II) in which Al is Asp or Glu and A3 is Thr or Ser.

19. A conjugate according to claim 18, wherein the peptide comprises the sequence:
Gly-Val-Asp-Tyr-Lys-Asp-Gly-Leu-Thr-Pro-Leu.

20. A conjugate according to claim 14, suitable for use in vaccination against a disease caused by type 1 poliovirus, wherein the synthetic peptide comprises the sequence (III):
Lys-A4-Gly-A5-Leu-A6 (III) in which A4 is Asp or Glu and each of A5 and A6 is independently Ser or Thr.

21. A conjugate according to claim 20, wherein A4 is Asp and A5 and A6 are both Thr.

22. A conjugate according to claim 20, wherein the synthetic peptide comprises the sequence:
Gly-Val-Asp-Tyr-Lys-Asp-Gly-Thr-Leu-Thr-Pro-Leu.

23. A conjugate according to claim 14, wherein the synthetic peptide further has a Cys residue at one or both ends.

24. A conjugate suitable for use in vaccination against a disease caused by a poliovirus, which conjugate comprises a physiologically acceptable carrier and, linked thereto, a synthetic peptide which is the peptide coded for by codons 286-290 in the RNA sequence coding for the structural capsid protein VPl of poliovirus type 3 Sabin strain or by equivalent codons of another enterovirus or is an antigenic equivalent of the said peptide, the antigenic equivalent being (i) a said peptide modified by the inclusion therein of one or more changes to the amino acid sequence;
(ii) a first longer peptide which incorporates the sequence of a said peptide or a said modified peptide and which has up to four extra amino acid residues attached to the C-terminal end of the said sequence or up to four extra amino acid residues attached to the N-terminal end of said sequence or up to four extra amino acid residues attached to the C-terminal end and up to four extra amino acid residues attached to the N-terminal end of said sequence; or (iii) a second longer peptide which incorporates the sequence of a said peptide, a said modified peptide or a said first longer peptide, to which sequence is linked directly, by means of a further amino acid residue or by means of a disulphide bridge between Cys residues attached to each sequence, a sequence of up to eighteen amino acid residues which comprises a hexapep-tide sequence coded for by codons 93 to 98 in said RNA sequence or by equivalent codons of another poliovirus;
each said antigenic equivalent being capable of raising antibodies capable of neutralizing the same strain and type of enterovirus as said peptide to which said antigenic equivalent corresponds and the numbers of the codons being counted from the 5'-terminus of the nucleotide sequence for the VPl capsid protein.

25. A pharmaceutical composition, useful as a vaccine against a disease caused by an enterovirus, which composition comprises a pharmaceutically acceptable carrier or diluent and, as active ingredient, a synthetic peptide which is the peptide coded for by codons 286-290 in the RNA sequence coding for the structural capsid protein VPl of poliovirus type 3 Sabin strain or by equivalent codons of another enterovirus or is an antigenic equivalent of the said peptide, the antigenic equivalent being:

(i) a said peptide modified by the inclusion therein of one or more changes to the amino acid sequence;
(ii) a first longer peptide which incorporates the sequence of a said peptide or a said modified peptide and which has up to four extra amino acid residues attached to the C-terminal end of the said sequence or up to four extra amino acid residues attached to the N-terminal end of said sequence or up to four extra amino acid residues attached to the N-terminal end and up to four extra amino acid residues attached to the N-terminal end of said sequence;
(iii) a second longer peptide which incorporates the sequence of a said peptide, a said modified peptide or a said first longer peptide, to which sequence is linked directly, by means of a further amino acid residue or by means of a disulphide bridge between Cys residues attached to each sequence, a sequence of up to eighteen amino acid residues which comprises a hexapeptide sequence coded for by codons 93 to 98 in said RNA sequence or by equivalent codons of another enterovirus; or (iv) a third longer peptide which incorporates the sequence of a said peptide, a said modified peptide or a said first longer peptide, to which sequence is linked directly, by means of a further amino acid residue or by means of a disulphide bridge between Cys residues attached to each sequence, a sequence comprising residues 58 and 59 of the VP3 capsid protein of an enterovirus ofthe same type but starting with a residue numbered no lower than 53 and ending with a residue numbered no higher than 68;
each said antigenic equivalent being capable of raising antibodies capable of neutralizing the same strain and type of enterovirus as said peptide to which said antigenic equivalent corresponds and the numbers of the codons being counted from the 5'-terminus of the nucleotide sequence for the VP1 capsid protein.

26. A composition according to claim 25, suitable for use in vaccination against a disease caused by type 3 poliovirus, wherein the synthetic peptide comprises the sequence (I):

A0-Al-A2-Leu-A3 (I) in which (i) A0 is Arg, each of Al and A2 is independently Asn or Gln and A3 is Asp or Glu or (ii), with the others of A0 to A3 being as defined in (i), A0 is Lys or A1 or A2 is Asp or Glu.

27. A composition according to claim 26, wherein A0 is Arg, both Al and A2 are Asn and A3 is Asp.

28. A composition according to claim 26, wherein the synthetic peptide comprises the sequence Gly-Val-Asp-Tyr-Arg-Asn-Asn-Leu-Asp-Pro-Leu.

29. A composition according to claim 25, suitable for use in vaccination against a disease caused by type 2 poliovirus, wherein the synthetic peptide comprises the sequence (II):
Lys-Al-Gly-Leu-A3 (II) in which Al is Asp or Glu and A3 is Thr or Ser.

30. A composition according to claim 29, wherein the synthetic peptide comprises the sequence:
Gly-Val-Asp-Tyr-Lys-Asp-Gly-Leu-Thr-Pro-Leu.

31. A composition according to claim 25, suitable for use in vaccination against a disease caused by type 1 poliovirus, wherein the synthetic peptide comprises the sequence (III):
Lys-A4-Gly-A5-Leu-A6 (III) in which A4 is Asp or Glu and each of A5 and A6 is independently Ser or Thr.

32. A composition according to claim 31, wherein A4 is Asp and A5 and A6 are both Thr.

33. A composition according to claim 31, wherein the synthetic peptide comprises the sequence:
Gly-Val-Asp-Tyr-Lys-Asp-Gly-Thr-Leu-Thr-Pro-Leu.

34. A composition according to claim 25, wherein the synthetic peptide further has a Cys residue at one or both ends.

35. A pharmaceutical composition useful as a vaccine against a disease caused by an poliovirus, which composition comprises a pharmaceutically acceptable carrier or diluent and, as active ingredients a synthetic peptide which is the peptide coded for by codons 286-290 in the RNA sequence coding for the structural capsid protein VPl of poliovirus type 3 Sabin strain or by equivalent codons of another poliovirus or is an antigenic equivalent of the said peptide, the antigenic equivalent being:
(i) a said peptide modified by the inclusion therein of one or more changes to the amino acid sequence;
(ii) a first longer peptide which incorporates the sequence of a said peptide or a said modified peptide and which has up to four extra amino acid residues attached to the C-terminal end of the said sequence or up to four extra amino acid residues attached to the N-terminal end of said sequence or up to four extra amino acid residues attached to the C-terminal end and up to four extra amino acid residues attached to the N-terminal end of said sequence; or (iii) a second longer peptide which incorporates the sequence of a said peptide, a said modified peptide or a said first longer peptide, to which sequence is linked directly, by means of a further amino acid residue or by means of a disulphide bridge between Cys residues attached to each sequence, a sequence of up to eighteen amino acid residues which comprises a hexapeptide sequence coded for by codons 93 to 98 in said RNA sequence or by equivalent codons of another poliovirus;
each said antigenic equivalent being capable of raising antibodies capable of neutralizing the same strain and type of enterovirus as said peptide to which said antigenic equivalent corresponds and the numbers of the codons being counted from the 5'-terminus of the nucleotide sequence for the VPl capsid protein.

36. A method for the diagnosis of a disease caused by an enterovirus, which method comprises contacting a sample obtained from a patient with a synthetic peptide as defined in claim 1 and assaying for the presence or absence of antibody to the enterovirus which has become bound to the peptide.

37. A test kit, suitable for use in the determination of antibody against an enterovirus, which kit comprises a synthetic peptide as defined in claim 1 and means for determining antibody bound to the peptide.

38. The use of a synthetic peptide as defined in claim 1 for vaccination against a disease caused by an enterovirus.

39. The use of a synthetic peptide as defined in claim 11 for vaccination against a disease caused by a poliovirus.

40. A commercial package containing as active pharmaceutical ingredient a conjugate as defined in claim 14, together with instructions for the use thereof in vaccination against a disease caused by an enterovirus.

41. A commercial package containing as active pharmaceutical ingredient a conjugate as defined in claim 24, together with instructions for the use thereof in vaccination against a disease caused by an poliovirus.