NO164246B - ANALOGUE PROCEDURE FOR PREPARING A THERAPEUTIC ACTIVE ANTIGENT PEPTID. - Google Patents

Description

The present invention relates to the production of anti-

gene peptides against infectious diseases and more specifically for the treatment of diseases caused by viruses of the Picornavirus family such as foot-and-mouth disease.

Foot-and-mouth disease is a highly contagious disease of great economic importance that primarily affects animals with cloven hooves. The mortality directly attributable to foot-and-mouth

sick, is generally relatively small, but in young animals the mortality rate can be relatively high. Of greater economic importance is that the disease is so debilitating that infected animals cannot be raised and fed economically. The only recognized effective procedure for removing the infection as soon as it is detected is to slaughter all infected animals, disinfect all localities that have been filled by the animals, and dissolve the corpses in quicklime. As the infection spreads extremely quickly, the economic basis of entire communities or regions can be destroyed by a large outbreak of foot-and-mouth disease.

Vaccines have been produced that immunize against foot-and-mouth disease, primarily by inactivating or weakening the virus. Such vaccines have been found to be effective to some extent, but outbreaks of foot-and-mouth disease have been linked to vaccines in which the virus was incompletely inactivated or insufficiently attenuated. Infections have also been traced back to viruses disappearing from equipment associated with foot-and-mouth research or the production of mouth-

and foot-and-mouth disease vaccines.

Foot-and-mouth disease (FMD) is caused by a Picornavirus

of the genus aphthovirus. There are several viral serotypes of foot-and-mouth disease virus (FMDV), the most common of which is identified by the serotype designations A, 0 and C, and less commonly identified as SAT-1, SAT-2, SAT-3 and ASIA-1.

Among these serotypes, several subtypes and subtype strains have also been identified.' The following are among the identified subtypes and subtype strains: FMDV A, subtype 10,

strain 61 and subtype 12, strains 119, USA and Pirbright;

FMDV 0, subtype 1, strain Kaufbeuren; and FMDV C, subtype 3, strain Indaial.

FMDV has been described in detail, see e.g.

H. L. Backrach, in Beltsville Symposium on Agricultural Research, J. A. Romberger, Ed., Allanheld, Montclair, N.J.

(1977), pp. 3-32; Annual Reviews of Microbiology, J22, 201

(1968). The molecular biology of these viruses has been described, R. R. Rueckert, in Molecular Biology of Picorna viruses, R. Perex-Bercoff, Ed. Plenum, New York, (1979), p. 113. The virus has a molecular size of approx. 7 x 10^ daltons and contains a plus-stranded RNA genome of approx. 8,000 nucleo times. Picornavirus proteins are synthesized in infected cells as a precursor protein that is then processed by cellular and virus-encoded proteases into four major capsid proteins (VP^, VP2, VP3, and VP4) and numerous non-capsid proteins.

When the whole VP3 protein was used to inoculate pigs, it elicited a neutralizing antibody response and protected both pigs and cattle from infection; [J. Laporte, et al., CR. Acad. Sci., 276: 3399 (1973); H.L. Backrach et al., J. Immunol., 115: 1636 (1975), . See also US Patent 4,140,763]. Based on this information, Dennis G. Kleid, et al., Science, 214: 1125-1129 (Dec. 4, 1981) was able to produce a cloned viral protein vaccine for oral

and foot-and-mouth disease that produced an antibody response in cattle and pigs.

It is noted that the literature in this area uses the same names to refer to different capsid proteins. Thus, the indicated researchers in the USA typically refer to the capsid protein which is indicated here and in Europe as VP-^, as the VP^-capsid. However, it is agreed that the capsid protein referred to here as VP-j^, and by others as VP^, is the immunologically active capsid protein.

Recombinant DNA molecules and methods for the production of peptides with specificity1 for foot and mouth viral antigens are described in British patent application GB 2 079 288A, dated 20 January. 1982. See also Boothroyd et al., Nature, 290: 800-802 (1981); Kleid et:al., Science, 214: 1125-1129 (1981), and EPO publication no. 0 068 693 2A corresponding to application no. 82303040.8 submitted 11.6.1982.

K. Strohmaier et al., Proe. 5th Int. Congress Virology, Strasbourg, 1981, have cleaved the VP-^ protein (here indicated as VPThr^ me<^ enzymes as well as cyanobromide and raised neutralizing antibodies using these cleaved peptide fragments. These authors suggested that the amino acid residue sequences at positions 146 to 155 and 200 to 213 from the protein amino terminus induced the production of immunologically important antibodies. These authors also suggested that amino acid residue sequences at positions 141 to 145 and 155 to 161 were among the regions of inactive, non-inducing peptides. This VP^ sequence corresponds to the VP^ sequence previously described in the United States, see explanation by Meloen, A. H., J. Gen. Virol., 45:761-763 (1979).

A paper by Strohmaier et al., J. Gen. Virol., 59:295-306 (1982) discusses in detail the work reported at the above-mentioned congress, and provides a correlation between the various capsid protein nomenclatures used by researchers in this field. This thesis repeats the discoveries reported at the congress, namely that two cyanobromide cleavage products designated CB^ and CB2 and an enzyme cleavage product designated A2 of VP^ corresponding to amino acid residues 55-180, 181-213 and 146-213 from amino end, induced neutralizing antibody. This thesis also reiterated that regions of overlap with other cleavage products, including regions 141-145 and 155-161, apparently had no effect. The authors stated on page 303 that they hypothesized "it is likely that only two small regions are essential for the immunizing potency of the protein".

Poliomyelitis (hereafter polio) and Hepatitis A virus

are also members, i.e. genera, of the Picornavirus family. Successful vaccines against poliovirus types 1, 2 and 3 have been used since 1950, while no successful vaccine against Hepatitis A is known.

One of the distinctive features of Picornavirus is that they contain 4 capsid proteins. The capsid protein designated as VP.^ of polio type 1 has been found to contain an antigenic determinant region capable of inducing the production of antibody that neutralizes the virus, although the specific amino acid determinant regions of the VP^ capsid have not yet been identified . A specific capsid of Hepatitis A virus has not yet been identified as responsible for inducing the production of neutralizing antibody.

Recently, antigens have been obtained in several ways, including derivation from natural materials, coupling of a hapten to a carrier, and by recombinant DNA technology.

Sela, et al., Proe. Nat. Acad. Sei., USA, 68:1450-1455

(July, 1971); Science, 166:1365-1374' (December 1960), Adv. Immun., 5:29-129 (1966) have also described certain synthetic

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antigens.

Antigens derived from natural materials are the countless number of known antigens that occur naturally, such as blood group antigens, HLA antigens, differentiation antigens, viral and bacterial 1 antigens and the like. Considerable efforts have been made in the last century regarding the identification and study of these antigens.

Certain "synthetic" antigens have been prepared by coupling small molecules to carriers such as e.g. bovine serum albumin, in order to provide antigens that will cause the production of antibodies against the linked small molecule. The carrier molecule is necessary as the small molecule itself will not be "recognised" by the immune system of the animal into which it was injected. This technique has also been used in isolated cases to prepare antigens by coupling peptide fragments of known proteins to carriers, as described in the above cited article by Sela et al.

Although this hapten carrier technique has served research well in investigating the nature of the immune response, it has not been of significant use in the production of antigens that will play a role in diagnostic or therapeutic use. There are several reasons for this deficiency.

To select and construct a usable antigenic determinant from a pathogen by this1 technique, one must first determine the entire protein sequence of the pathogen to have a possible chance of success. Because of the difficulty of this work, it has very rarely, if ever, been done.

Classically, vaccines are prepared by introducing killed or weakened organisms into the host together with suitable auxiliaries to initiate the normal immune response to the organism while preferably avoiding pathogenic effects of the organism in the host. The problem suffers from well-known limitations in that it is hardly possible to avoid the pathogenic response due to the complexity of the vaccine which includes not only the antigenic determinant of interest but many related and unrelated harmful materials and where any number of such in some or all individuals can induce an unwanted reaction in the host.

For example, vaccines produced in the classical way may include competing antigens that are harmful to the desired immune response, antigens that include unrelated immune responses, nucleic acids from the organism or culture, endotoxins and components of unknown composition and from an unknown source. These vaccines, developed from complex materials, inherently have a relatively high probability of eliciting competing responses even from the antigen of interest. In addition, such known vaccines against FMDV must be kept refrigerated before use, and cooling in remote areas where the vaccines are used is often difficult to achieve.

Recombinant DNA technology has opened up new avenues for vaccine technology which has the advantage that production starts with a monospecific gene, but much of this advantage is however lost in actual production of antigen in Escherichia coli or other microorganisms. In this procedure, the genetic material is introduced into a plasmid which is then introduced into E. coli. which produces the desired protein, together with other metabolic products, all mixed with the nutrient medium. This problem is complicated by the uncertainty about whether the desired protein will be expressed in the transformed E. coli.

Furthermore, even if the desired protein can be produced, there is some uncertainty as to whether or not it can be harvested, or whether it will be destroyed during the E. coli growth process. It is e.g. it is well known that foreign or altered proteins are degraded by E. coli. Even if the protein is present in sufficient quantities to be of interest, it must still be separated from all the other products of the metabolism of E. coli, including such harmful substances as unwanted proteins, endotoxins, nucleic acids, genes, and unknown or unpredictable substances.

Even if it were possible or became possible by means of advanced but necessarily very expensive techniques to separate the desired protein from all other products of E. coli metabolism, the vaccine would still comprise an intact protein which may include unwanted antigenic determinants, some of which are known to initiate very severe, adverse responses. It is known that certain proteins which can be considered as vaccines contain an antigenic determinant which causes such severe cross-references or side effects that use of the material as a vaccine is prevented.

It is also possible using hybridoma technology to produce antibodies to viral gene products. The hybridoma technology makes it possible to start with a complex mixture of antigens and produce monospecific antibodies later in the process. In contrast, the present invention is the opposite process in that it starts with the final antigenic determinant in high purity so that the necessity of purification of the desired antigenic product is avoided.

Hybridoma antibody is known to have low affinity

and low binding constant, and therefore has limited value.

In the hybridoma technology, one still has to build on the production of antibody with cells that are malignant, with all the accompanying problems with respect to separation techniques, purity and safety.

Hybridoma production is based on tissue culture or introduction into mice, with the obvious result that production is expensive and that there is also variation from batch to batch.

In addition, it is difficult to create a hybrid for molecules that comprise only a small percentage of the complex mixture that you have to start with.

Previous investigations by Arnon et al., Proe. Nat. Acad. Pollock. USA, 68:1450 (1971), Atassi, Immunochemistry, 12:423 (1975) and Vyas et al., Science 178:1300 (1972) have been interpreted by these authors to indicate that short linear amino acid sequences are generally unlikely to induce antibodies which are reactive with the natural protein structure. It was assumed that for most regions of most molecules, antigenic determinants from amino acid residues were well separated in the linear sequence, but adapted to the peptides used to induce antibody, thought to be critical in most cases, even for those antigens that include amino acids near each other in a sequence. Lerner, et al., Cell 23:109-110,

(1981); Nature 287:801-805 (1980), discovered that antibodies to linear peptides react with natural molecules. Detailed biosyntheses thus became unnecessary, uneconomical and obsolete.

Despite the availability of inactivated or weakened virus vaccines against foot-and-mouth disease, there has been a great economic and practical need for, and a great theoretical interest in, the development of a vaccine against foot-and-mouth disease that will be free of the risks that have so far attached manufacture and handling of FMDV which causes the disease. The availability of cloned, viral proteins can be a very significant step forward from the old and highly chance-filled problems.

The cloned, viral protein vaccine area also carries with it a number of disadvantages, limitations and risks. Variations in the biosynthesis system can in themselves cause variation in the expression forms of the proteins and thus affect the purity, yields, strength, etc., of the antigens. In addition, the presence of other proteins and difficult and inefficient separations suggest the chance that vaccines prepared via the cloned viral protein procedure will not be monospecific. Thus, purity, strength and safety are the main problems with the products derived from this technology.

Despite the fact that the general procedure for the production of synthetic antigens, from either a known peptide sequence or from a genome, has been described, and despite

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Although the synthesis of peptides of suitable length for use in antigenic materials is now relatively well known, there is still a very large area within antigen-antibody technology that remains unsolved. Although there are certain guidelines and certain assumptions regarding possible antigenic sequences, the area is still characterized by speculation and by "trial and error" methods. Even with the recognition that a long sequence kia<n> contains antigenically active constituents, there is still great uncertainty and speculation as to whether all or only a portion of the sequence is required to elicit antigenicity, and whether or not a minor portion of the sequence will be of greater or lesser antigenicity.

The present invention relates to an analogue method for the production of a therapeutically active antigenic peptide which has the following amino acid residue sequence written from left to right and in the direction from the amino end to the carboxy end: TyrAsn(Asp or Thr)Gly(Phe)Glu(Thr)Cys( Ser or Asn or Thr)Arg(Lys or Thr)TyrAsn(Ala or Ser or Thr)Arg

(Val or Ala or Asn or Thr)Asn(Gly or Ser)Ala (Asp or Gly)Val(Ser or Gin or X)Pro(Gly or Y) Asn(Z)Leu(Arg or Val)Arg(Ser or Ala )GlyAspLeu(Met or Phe)Gin(Gly)Val(Thr or Ser or,His)Leu(Ile)AlaGln

(Ala or Pro)Lys(Arg or Ala)Val(His)Ala(Val)Arg(Thr or Lys)Thr(Gin or His)LeuPro and optionally Ala, and optionally containing two additional Cys residues attached to each end of the peptides, in which each of the amino-

the residues as well as the symbols X, Y or Z; in brackets indi-

vidually can replace the adjacent amino acid residue immediately

to the left of the parenthesis, and where X and/or Y and/or Z in the peptide amino acid residue sequence independently indicate the absence

of an amino acid residue in the position of the adjacent amino acid residue immediately to the left of the parenthesis, whereby the length of the peptide is shortened by 1, 2 or 3 amino acid residues,

The produced antigenic peptide contains a sequence of approx. 20 amino acid residues corresponding to an amino acid rust sequence from position 130 to 160 from the amino end of the FMDV VP^capsid protein, and more specifically from position 141 to 160. When this peptide is chained to an albus shell hemocyanin carrier as a conjugate and introduced into a effective amount as a vaccine in a host animal, it is capable of eliciting the production of antibody in the host which immunoreacts with foot-and-mouth virus and protects the host against infection caused by that virus.

The synthetic antigenic peptides produced according to the invention can be used together with physiologically acceptable diluents such as water and/or auxiliaries in a vaccine capable of protecting animals against Picornavirus-induced diseases such as foot-and-mouth disease.

The more particularly preferred FMD-related peptide amino acid sequence corresponding to positions about 141 to about 160 from the amino terminus, starting at the amino terminus with the Val(Ser or Gin or X) residue at position 141 of the above sequence.

The analogue method according to the invention is characterized by the fact that a starting amino acid resin is produced by esterification of Boc-AA-OH to er. resin in which AA denotes the relevant amino acid, after which successive coupling of Boc-AA-OH using side chain protecting groups is carried out, the protected polypeptide resin is treated with liquid HF to remove the protecting groups and cleave the polypeptide from the resin, after which the cleaved, unprotected polypeptide is extracted with dilute acid, after which, if desired, the resulting polypeptide is coupled to a protein carrier, and, if desired, the resulting peptide is oxidized. The present invention provides many advantages, in particular when it comes to the use of the peptides produced according to the invention in vaccines against Picornavirus-induced diseases and in diagnosis for determining the presence of these diseases or viruses in animals, including humans.

A striking advantage is that the synthetic peptides can provide part of a vaccine that protects the animals against these diseases.

A particular advantage is that vaccines produced using a synthetic peptide do not need to be cooled before administration to achieve effective vaccination.

Further advantages will be apparent from the following detailed description, examples and claims.

Brief description of the drawings

In the drawings which form part of the present description, show

fig. 1, 8 amino acid residue sequences at amino acid residue positions 130-160 of the foot-and-mouth disease virus VP^ capsid, using the usual three-letter code for each amino acid residue. The sequences are read from left to right and in the direction from the amino end to the carboxy end. The numbers 130, 140, 150 and 160 represent amino acid residue positions relative to the amino terminus of virus of Tubingen type 0, subtype 1, strain Kaufbeuren, (Olk), with the amino acid residue sequences of the remaining viral VP^ capsids aligned by inclusion of one or more hyphens so that the connection between these sequences is more clear. The abbreviations for viruses in addition to Olk are as follows: 01c = type 0, subtype 1, strain Campos; A10 = type A, subtype 10, strain 61; A12 = type A, subtype 12, strain 119, A24 = type A, subtype 24; A2 7 = type A, subtype 27; A79 = type A, subtype 79; and C3 = type C, subtype 3, strain Indaial.

Detailed description of the invention

I. General discussion

The present invention lies in the discovery that

a particular, relatively short synthetic peptide sequence quite unexpectedly and surprisingly is an extremely active antigen. The synthetic peptide contains an amino acid residue sequence of approx. 20 acids in length. The amino acid residue sequence of the peptide corresponds to at least one amino acid residue of an area on the antigenic Picornavirus capsid protein which is located at a distance equal to 60 to 75% of the total amino acid residue sequence length of the antigenic capsid protein as measured from the amino end thereof. The synthetic peptide contains a neutral to positive ionic charge, excluding ionic charges present due to the presence of amino and/or carboxyl end groups.

In addition, synthetic antigens including the later described peptide sequences are monospecific against specific serotypes, subtypes and strains of Picornavirus such as foot-and-mouth disease virus, and are also polyspecific, albeit to a lesser extent, against a majority of the serotypes, subtypes and strains of these viruses .

Synthetic peptides associated with the Picornavirus that causes foot-and-mouth disease (FMD) will be discussed as examples of synthetic peptides that can be produced. Specific amino acid sequences corresponding to amino acid residue positions in the FMDV VP-^ capsid relate only to this virus.

In particular, it has been found that a synthetic peptide containing approx. 20 amino acids corresponding to the amino acid residue sequence of positions around 130 to 160, and especially positions around 141 to 160 from the amino end of the FMDV VP^ protein such as that from Tvibingen type 0, subtype 1, strain Kaufbeuren, have much higher antigenic efficiency and activity than what has been assumed or assumed from previous investigations. A peptide produced according to the invention, alone, in straight chain or cyclic ring structure, as a polymer with peptide units chained with oxidized cysteine residues of adjacent peptides, or as a conjugate chained to a carrier, is a powerful immunological reactor (antigen) for foot and mouth disease, which will be discussed in detail in what follows.

The indications "about position 130 to about position 160" and "about position 141 to about position 160" from the amino end and similar phrases are used here. These amino acid residue positions are determined relative to the reference VP^ capsid protein of type 0, subtype 1, strain Kaufbeuren FMDV.

It should be noted that some researchers in the area such as Kleid et al. in EPO publication no. 0 068 693 A2,

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have shifted the positions of amino acid residues in the 130-160 region of the VP^-(VP^, previously discussed) capsid protein by one amino acid position number towards the carboxy terminus relative to the position numbers indicated here due to the presence of an additional amino acid ( Val) after Asp-53 in the sequence of the type C, subtype 3, VP^ capsid, where the other capsids do not contain any amino acid residues. Consequently, the amino acid at a given position such as 140 appears here as the amino acid at position 141 in the EPO application indicated above. It is thus recognized in the art that there is a difference in one or more amino acid positions when different researchers indicate sequences of the same protein molecule.

Capsid proteins from some foot-and-mouth disease viruses also contain no amino acid residue at one or more positions in the 130-160 range in relation to type VP^ of type 0, subtype 1, strain Kaufbeuren as shown in fig. 1 and indicated by the letters-"X", "Y" and "Z" in the sequence of formula I. In light of the presence of such deletions or omissions, some researchers have indicated amino acid positions as determined from the protein or a DNA molecule encoding it protein, without accounting for the omissions. Other researchers illustrate the relationship between the VP-^ capsids by indicating amino acid omissions with dashes, letters, or other indices and numbering the remaining amino acid residue positions as if the omitted residues were present, which is done here. Thus, there is an insignificant variation within the subject when it comes to specifying amino acid positions.

Thus, the word "around" as used in the above expressions is intended to indicate that the amino acid residue sequence may start or end at an amino acid residue up to 3 residues on either side of the indicated positions to allow for the variation of one to two position numbers as indicated within subject for a given amino acid residue in any particular peptide sequence, and also taking into account the fact that certain amino acid residues are omitted in some VP^ capsid proteins.

As previously stated, there are several types, subtypes and strains of FMDV. For reference purposes, the peptide sequences described herein will be discussed with reference to the VP^ protein from a particular type, subtype and strain; namely Tubingen type 0, subtype 1, strain Kaufbeuren of FMDV, also indicated here as type 0, subtype 1, strain Kaufbeuren and Olk. When using the amino acid sequence of one specific FMDV protein as a reference, other applicable peptide sequences are thus described which contain substituted or omitted amino acid residues at specific locations along the peptide chain.

Peptide sequences from the VP^ capsid protein of 8 FMD viruses at positions around 130 to around 160 are shown in Fig. 1, using the numbering system for the reference protein type Olk. Synthetic, preferably water-soluble, peptides each containing about 20 amino acids, with amino acid residue sequences corresponding or substantially corresponding to the amino acid residue sequences shown in fig. 1 and which meet the unit test condition as specified later, are taken into account.

A preferred peptide having an amino acid residue sequence corresponding to amino acid residue positions of about 130 to 160, read from left to right and in the direction from the amino terminus to the carboxy terminus, is shown in Formula I:

Formula I

wherein each of the bracketed amino acid residues X, Y or Z can independently replace the adjacent; amino acid residue immediately to the left of the bracket; i.e. the amino acid residue closer to the amino terminus;

X and/or Y and/or Z in the peptide amino acid residue sequence independently indicates the absence of an amino acid residue in the position of the adjacent amino acid residue immediately to the left of the parenthesis (closer to the amino end) whereby the peptide length is shortened by 1, 2 or 3 amino acid residues, and

the numbers in parentheses above the above specified amino acid residues in the sequence illustrate positions of the specified amino acid residue from the amino terminus of the VP^ capsid protein of Tiibingen type 0, subtype 1, strain 1 Kaufbeuren FMDV. These figures are given for reference purposes.

The more preferred sequence corresponding to positions around 141 to 160 of the VP^ capsid protein starts at the amino terminus with the Val(Ser or Gin or X) residue at position 141

in Formula I.

Poly-specificity and cross-reactivity among types, subtypes and strains of the FMDV genus is improved by the use of an antigenic peptide whose amino acid-

residue sequence corresponds to an amino acid residue sequence of at least more than one strain, and preferably more than one subtype

or serotype of the FMDV genus. The amino acid residue sequence of such a peptide need not correspond substantially to the amino acid residue sequence of any virus, but nevertheless, when inoculated as a vaccine into an animal host, may elicit the production of antibodies which immunoreact with

a plurality of virus types, subtypes or strains and protects the host against more than one of these viruses.

A polyspecific peptide whose amino acid residue sequence corresponds to an amino acid residue sequence of at least more than one strain of FMDU may have the amino acid residue sequence shown in formula 1 wherein one or more bracketed amino acid residues, X, Y or Z replaces the adjacent amino acid residue immediately to the left of the parenthesis, i.e. the adjacent amino acid residue towards the amino end. Substitutions and omissions in the range of amino acid residue positions around 141 to 155 are preferred to achieve poly-specificity in a peptide with a single amino acid residue sequence. Such a poly-specific peptide can be prepared and used in the same way as any other peptide according to the invention.

The term "substantially corresponding" in its various grammatical forms is used herein and in the claims in relation to the peptide sequences related to Picornavirus to mean the described peptide sequence plus or minus up to 3 amino acid residues at either one or both of the amino and carboxy termini and containing only conservative substitutions in specific amino acid residues along the peptide sequence. The term "equivalent" in its various grammatical forms is used herein and in the claims in relation to peptide sequences related to Picornavirus to denote the described peptide sequence plus or minus up to 3 amino acid residues at either one or both of the amino and carboxy termini and containing conservative as well as radical substitutions in certain amino acid residues because also containing omissions or additions of certain amino acid residues along the peptide sequence.

The term "conservative substitution" as used above is intended to indicate that an amino acid residue has been replaced with another biologically similar residue. Examples of conservative substitutions include substitution of one hydrophobic residue such as Ile, Val, Leu or Met by another, or substitution of a polar residue by another such as between Arg and Lys, between Glu and Asp or between Gin and Asn , and such.

In some cases, replacement of an ionic residue with an oppositely charged ionic residue such as Asp with Lys has been stated to be conservative in the art in that these ionic groups are only believed to provide solubility assistance. As the substitutions discussed here are generally1 made on relatively short synthetic peptide antigens compared to a whole protein, the substitution of an ionic residue with another ionic residue of opposite charge will be considered here as "radical substitution" such as substitutions between non-ionic and ionic residues, and massive residues such as Phe, Tyr and Trp and less massive residues such as Gly, Ile and Val.

The terms "non-ionic" and "ionic" residues are used here in their usual meaning to denote those amino acid residues which normally either carry no charge or normally carry a charge at physiological pH values. Examples of non-ionic residues include Thr and Gin, while examples of ionic residues include Arg and Asp.

The above sequence shown in formula I and the shorter, more preferred synthetic sequence corresponding to positions around 141 to 160 of the FMDV VP^capsid include a large number of individual peptides, each of which, of about 20 amino acid residues in length, is a peptide produced according to the invention. For example, synthetic peptides prepared with an amino-eride starting at position 141 of the capsid can start with a Val-, Ser- or Gln-amino end residue.

When, in addition, the amino-terminal amino acid residue position is represented by X indicating the absence of the amino acid residue immediately to the left of the parenthesis in formula I, the peptide amino terminus may start with the amino acid residues Pro, Gly, or Y of capsid position 142 wherein Y independently denotes the absence of Pro and Gly so that the peptide amino end begins with the amino acid residue corresponding to position 143 in Olk instead of positions 141 or 142. Further investigations of formula I show that the residue in capsid position 143 can be Asn or Z and thereby also be absent.

Thus, a peptide whose sequence corresponds to capsid positions around 141 to 160 may begin at capsid position 141. As previously indicated, peptide sequences corresponding to

a described sequence, have plus or minus up to 3 amino end residues at each end. The above-described peptide whose amino acid sequence does indeed start at position 144 relative to the Olk VP^ capsid protein is among those whose sequences are "minus up to 3 amino acid residues at one or both of the amino and carboxy termini".

Peptides that are encompassed by the invention are included in the region around 130 to 160 of type 0, subtype 1, strain Kaufbeuren and type A, subtype 12, strain 119 FMDV proteins (expressed using type 0 position numbering and denoted respectively as Olk and A12) and the corresponding regions of type C, SAT-1, SAT-2, SAT-3 and ASIA-1. Two examples of such peptides are shown below using Olk virus position numbering, and with a hyphen to indicate an omitted amino acid residue.

Example 1

It has also been determined that the amino acid residue sequence of the capsid region around 141 to around 160 is uniquely and most unexpectedly antigenically active and potent as discussed in detail below.

Peptides with a sequence of about < 20 amino acids, within the region around 130 to 160, and in particular in the region around 141 to 160, to which a Cys or other amino acid as an amino or carboxy terminus may be added to enable binding by covalent binding in a further synthesis step to a support, e.g. an albus shell hemocyanin (KLH)

if a carrier is to be used, one embodiment is

as well as the above-mentioned peptides with variation in peptide length or substitutions or omissions in terms of individual amino acids which do not destroy or significantly change the distinctive and powerful antigenicity exhibited by the FMDV mono-specific and poly-specific synthetic antigenic determinant peptides produced according to the invention.

These sequences, separated from other antigenically active or antigenically masking sequences, constitute an embodiment in another form. Antigens comprising more than one of the preceding antigenically active sequences, separated from antigenically interfering or masking sequences, chemically connected or mixed with each other, constitute another form. Antigens comprising a carrier to which one or more of the preceding antigenically active amino acid sequences are bound constitute a further embodiment.

In view of the present invention it is

important to recognize the following definition of the antigenically active amino acid residue sequences considered as one embodiment. For example is the sequence corresponding to 1 position 141 to 160

of type 0, subtype 1, stem Kaufbeuren from left to right in the direction from amino to carboxy terminus

ValProAsnLeuArgGlyAspLeuGlnValLeuAlaGlnLysVal AlaArgThrLeuPro

when separated from other peptides, gene fragments, amino acids and amino acid sequences which tend to mask or interfere with or cross-react or complicate the antigenic effect of the peptide, a specific embodiment. Although one can thus find the specific

fissed sequence as a 'part of a large protein or larger

peptide containing e.g. about. 30 amino acids or more, such larger materials form no part of the invention because a protein or larger peptide will not exhibit the activity, the unusually and unexpectedly high degree of essentially monospecific antigenic activity exhibited by an approx. 20 amino acid long peptide produced according to the invention.

The term "FMDV mono-specific synthetic antigenic determinant peptide" denotes the particular peptide specified as described above resulting from a chemical synthesis which eliminates the possibility of fragments of genes, proteins or peptides, or any amino acid compound derived directly or indirectly from FMDV and free of peptide or amino acid sequences that could interfere with or alter the monospecific antigenic activity of the specified peptide in eliciting antibody production against FMDV in animals. It should be noted that although the term "mono-specific" is used herein, the individual peptides exhibit poly-specific antigenic activity with a plurality of FMDV types, subtypes and strains. This broad specificity is found in particular where a radical substitution is made in a peptide whose sequence otherwise essentially corresponds to the amino acid residue sequence of a particular FMDV capsid and the radically substituted amino acid residue is a residue found at the substitution position in another viral strain. Use of the term "mono-specific" is thus a short description (in shorthand style) for the broader specificity of the peptides according to the invention.

When the synthetic, antigenic peptides produced according to the invention, alone in the straight chain or cyclic ring form, as a polymer in which adjacent peptide repeating units are bound together with oxidized cysteine residues, or as a conjugate chained to a carrier, are introduced in an effective amount as a vaccine in a animal host, these are typically capable of inducing the production of antibodies in the host that immunoreact with the related Picornavirus and protect the host against infection caused by this Picornavirus. However, a peptide produced according to the invention can be further defined by a unit test of its antigenic characteristics which is independent of the form in which the peptide is finally used, i.e. in the straight chain, cyclic ring form, in polymer form or the chain as a conjugate. According to this unit test, a peptide is in straight chain form and when it is chained to an albus shell hemocyanin carrier as a conjugate and. introduced in an effective amount as a vaccine into a host animal, capable of inducing the production of antibody which immunoreacts with its related Picornavirus and protects the host against that virus. The amounts of peptide and carrier, such as the specific reaction conditions for the conjugate reaction and vaccine preparation, are given in Bittle et al., Nature, 298:30-33 (July, 1982).

A vaccine comprises an effective amount of a peptide antigen which can alone serve as the vaccine when it is present with a physiologically acceptable diluent such as water or saline. The vaccine may include a carrier which may be which

in any of the numerous carriers such as albus shell hemocyanin carrier (KLH), tetanus toxoid, poly-L-(Lys:Glu), peanut agglutinin, ovalbumin, soybean agglutinin, bovine serum albumin (BSA) and the like, to which the FMDV mono-specific synthetic antigenic determinant peptide is chained. A polymer produced by chaining a majority of peptides produced according to the invention through end-to-end chaining of oxidized cysteine end groups may also comprise an exogenous carrier-free vaccine together with a physiologically acceptable diluent. In each case, the peptide acts as the specific antigenic determinant.

The "effective amount" of antigenic peptide depends on a number of factors. Included among these factors are the body weight and species of the animal host to be protected, the carrier when such is used, adjuvant when such is used, the number of inoculations that are desired to be carried out, and the duration of the desired protection for the animal. Individual inoculations typically contain approx. 20 µg of antigenic peptide to 2 mg, exclusive of any carrier to which the peptide may be bound.

When the antigenic vaccine is introduced into the desired host, it initiates the production of antibody in the host to the above antigenic peptide and therefore to the related Picornavirus such as FMDV. Vaccines containing effective amounts of the peptides not only initiate the production of antibodies in the host, but these antibodies are produced in a sufficient amount to protect the animal host against infection with FMDV or other Picornavirus. Protection of the host can be determined by the level of neutralizing antibody produced and/or the neutralizing index, as discussed further below.

Also considered are antigens in which all or part of the entire carrier is antigenic. Thus, a separate carrier part may or may not be used. The synthetic antigen formed by chaining the FMDV mono-specific, synthetic antigenic determinant peptide to an antigen carrier as well as methods for producing such synthetic antigens are specific aspects of the invention.

In general, the synthetic antigen can be formed by producing the Picornavirus-related peptide such as a

FMDV mono-specific synthetic antigenic determinant peptide, which immunologically corresponds or substantially corresponds to antigenic determinants of FMDV, and coupling the synthetic determinant to a pharmaceutically acceptable carrier in a separate synthesis step.

When producing vaccines, the method includes synthesizing FMDV mono-specific synthetic, antigenic determinant peptide as antigen. is the duplicate or substantially the duplicate of specified determinant portion of the FMDV VP^ protein. The synthetic peptide may, but need not always be,

chain to a carrier to form an antigen in which the antigenicity is that of the FMDV mono-specific antigenic determinant peptide and which, when introduced into a host together with a physiologically acceptable diluent, initiates the production of antibody to the FMD virus.

As a method of producing antibody, the vaccine described above is injected into a host, and antibodies formed in the host to the protein antigen are harvested from the host's fluids for use in conventional diagnostic procedures to detect the presence of the protein antibody or as therapeutic agents for passive immunoprophylaxis.

II. Peptide synthesis

Peptides discussed below were synthesized using known procedures. [See e.g. Marglin, A. and Merrifield, R.B., Ann. Fox. Biochem., 39:841-866 (1970)]. The peptides were linked to the protein carrier KLH through a cysteine residue that was typically added to the carboxy end of the peptide unless otherwise stated. The synthetic attachment step of the peptide to the protein support was, unless otherwise indicated, performed by addition of the cysteine sulfur atom to the double bond of the reaction product between the support and N-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS), following the general procedure as is described by Lieu et al., Biochemistry, 18:690-697 (1979).

A low molecular weight, probably cyclic peptide was prepared by synthesizing a peptide with the amino acid sequence of Olk VP^ at positions' 141-160 (Fig. 1) and adding cysteine-(Cys) residues at both the amino and carboxy termini (diCys -peptide). Then became 10 mg. of the diCys peptide (containing Cys residues in non-oxidized form) dissolved in 250 ml of 0.1 molar ammonium bicarbonate buffer in a beaker. The dissolved diCys peptide was then air oxidized by gently stirring the resulting solution for 18 hours. After this time, an Ellman reaction indicated the presence of no free mercaptan. [Ellman,, Arch. Biochem. Biophys., 82:70-77 (1959)].

The solution obtained was freeze-dried. The dried material thus produced, and hereinafter referred to as the cyclic peptide or cyclic ring peptide, has the previously indicated amino acid sequence for positions 141-160 of the Olk VP^ capsid assumed to be linked together at the amino-end to the carboxy-end with oxidized cysteine residues, i.e. with one cysteine residue containing a disulfide bond. Two or more diCys peptides can also be linked together to form the cyclic ring peptide.

Two polymeric peptides were also prepared from the above-mentioned peptide containing non-oxidized Cys residues at both peptide ends (diCys peptide) and bound to these ends with peptidamid bonds. These polymeric peptides are here-

after designated as polymeric peptides A and B.

Polymeric peptide A was prepared from the diCys peptide

by dissolving this peptide in a concentration of 5 mg per ml in the ammonium bicarbonate buffer indicated above. Air oxidation as described above gave a material which had no free mercaptan by the Ellman reaction. The reaction solution did not contain any particulate material after oxidation and was freeze-dried to form polymeric peptide A in dry form.

Polymeric peptide B was prepared in the same buffer with the same oxidation conditions as polymeric peptide A and the cyclic peptide. Here, however, the concentration of diCys peptide used during the oxidation was 2 3.4 mg per 1.2 ml of buffer. No free mercaptan was observed in the Ellman reaction after the oxidation reaction, but a small amount of precipitate present in the reaction mixture was observed. The reaction mixture was freeze-dried to form polymeric peptide B, including the precipitate.

Each of the above prepared dried solids (cyclic peptide and polymeric peptides A and B) was used without further purification. Vaccines were prepared from these dried, solid materials by suspending them in Freund's complete adjuvant at concentrations sufficient to give 100 µg of peptide per inoculation.

III. Immunizations

A. Inoculations

The vaccines used here contained the indicated amount of peptide alone, in straight chain or cyclic form, as a polymer of individual peptides chained through oxidized cysteine residues (cystine) or chained to a carrier. The indicated amounts of peptides refer to the weight of the peptide without the weight of a carrier, when a carrier was used.

The vaccines also contained a physiologically acceptable diluent such as water or saline, and further typically included an adjuvant. Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) are materials well known in the art and are commercially available from several sources. Saponin, a plant-produced glycoside, is also a well-known excipient, as a 5% solution, and was used here together with aluminum hydroxide.

Vaccine stock solutions were prepared with IPA or

CFA as follows: An amount of the synthetic peptide, polymeric peptide or conjugate sufficient: to provide the desired amount of the peptide per inoculation, was dissolved in phosphate buffered saline (PBS). Equal volumes of CFA or IFA were then mixed with the peptide solution to form a vaccine containing peptide, water and adjuvant in which the water-to-oil ratio was 1:1. The mixture was then homogenized to form a vaccine stock solution.

Vaccine stock solutions were prepared with saponin aluminum hydroxide as follows: Aluminum hydroxide in an amount of 10 mg per ml was suspended in an aqueous 0.85% solution of sodium chloride. An amount of the synthetic peptide, polymeric peptide or conjugate sufficient to provide the desired amount of peptide per inoculation after a dilution to 20% was mixed with aluminum hydroxide suspension and allowed to absorb into the aluminum hydroxide particles for 2 to 3 hours. A part of the suspension thus prepared was then diluted with 4 parts of eh previously diluted saponin solution to form the stock solution of the vaccine in which the saponin was present at 0.125% by weight.

Preliminarily, screening assays were performed by enzyme-linked immunosorbent assay (ELISA) as discussed by Bittle et al., Nature, 298:30-33 (July, 1982), in which peptide-antipeptide-antibody immunoreactions were measured. • Viral neutralization index assays, which measure viral inactivation by immunoreactions between elicited antibodies and live virus particles, were also performed as described by Bittle et al. Further details regarding the techniques used and the results obtained here and related work with FMDV can be found in Bittle et al.

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B. Antibodies to peptides homologous to specific FMD viruses

Responses to different peptide regions of VP^ of FMDV type 0, subtype 1, strain Kaufbeuren are given in

Table 1. These data indicate antibody responses in rabbits using conventional protocols and illustrate neutralization of an FMD virus containing an amino acid sequence homologous to that of the peptide used for immunization. (For details see Lerner et al., U.S. Patent Application 248,059 filed Mar. 27, 1981, and Bittle et al., supra).

It should be emphasized that the neutralization indices for the peptides with the amino acid residue sequence of the 141-160 region in Table 1 were each greater than 3.9 <p>g 3.7, while the indices shown for the peptides within the 200-213 region were found to be 3.5

and 3.5. Synthetic peptides with amino acid residue sequences corresponding mainly to the indicated .141-160 and 200-213 amino acid residue positions neutralized both viruses. However, the neutralization indices for the peptides according to the invention whose sequences correspond mainly to the sequence of amino acid residues 141-160 were in reality greater than the indices show. The size of the real one

j

difference in neutralization index is shown in Table 4 for another comparison of the same peptides inoculated into rabbits in which more sensitive neutralization endpoint determinations were made.

It was determined that a single inoculation of the antigens was effective in eliciting neutralizing antibodies in animals and protecting them from infection. Table 2 indicates these results on guinea pigs, carried out using standard

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procedure (see Lerner et al. patent application and Bittle et al., supra). Table 2 shows the effectiveness of the antigens and also shows the markedly surprising antigenicity of the 141-160 region of the VP^ protein. A neutralizing index of

around 1.5 or greater indicates that the animal was protected against the virus.

The above data show that the particularly preferred peptide having the amino acid sequence of amino acid residues 141 to 160 of the Olk VP^ capsid includes amino acid residues on either side of the peptide of positions 146-154 as predicted by Strohmaier et al., supra to exhibit antigenic activity for this capsid protein. In addition, the peptide having the Olk sequence at positions 141-160 contains amino acid residue sequences (141-145 and 155-160) which were predicted by Strohmaier et al., supra, to be inactive, non-inducing peptides.

The results in Tables 1 and 2 illustrate that Strohmaier et al. were not correct in their prediction as to where in the amino acid residue sequence neutralizing antibodies are

would be able to form and not form! The results in Table 3

in the following, in which the particularly preferred peptide according to the invention and with the amino acid residue sequence of position 141-160 of Olk VP^ has been compared with the peptide according to Strohmaier et al. with the sequence of positions 146-155 of Olk VP.^, shows that the peptide according to the invention is 1000 to 100,000 times more active in terms of the production of neutralizing antibodies than the peptide according to Strohmaier et al. An average value of these results also indicates that the peptide according to Strohmaier et al. does not induce the production of sufficient amounts of antibodies to provide protection to the animal host (neutralization indices of 1.1 and 1.5), while the peptide according to the invention provides large

amounts of protective antibodies (neutralization indices equal to or greater than 4.3 and 2.7).

The data in the above table also illustrate that the 32 amino acid peptide with the sequence of positions 130-161 of Olk VP^ is inactive with respect to the production of neutralizing antibody. The data regarding the KLH peptide antigen designated C141-160 show that neutralizing antibody production is not a function of which end of the peptide is attached to the carrier.

C. Cross-reactivity of antigenic peptides with heterologous FMD viruses

The antigenic peptides produced according to the invention are not mono-specific as previously defined. However, these peptides also have varying amounts of cross-reactivity with viral serotypes whose amino acid sequences are heterologous with the specific amino acid sequence of a given peptide. Thus, the peptides are also poly-specific to varying degrees.

The data in Table 4 illustrate the cross-reactivity of antibodies raised against antigenic peptide conjugates with the sequence of amino acid positions 141-160 and 200-213 of Olk VP^

■ used to immunize two rabbits for each sequence. Neutralization indices were determined against the homologous virus (Olk) and the heterologous viruses types A and C. These data show that serotype specificity of sera produced by inoculation with the synthetic antigenic peptide mimics those found with whole virus sera. The cross-neutralization is believed to reflect the sequence homology among different serotypes as shown in Fig. 1.

Table 4 also shows the large difference in neutralizing index against Olk between the synthetic peptides whose amino acid residue sequences correspond mainly to positions 141-160 and 200-213 of Olk. In the above data, the peptide also predicted to be immunologically active by Strohmaier et al., supra, whose amino acid residue sequence corresponds essentially to positions 200-213 of Olk gave neutralization index values similar to those shown in Table 1 for the same peptide under similar conditions. others. However, neutralization indices observed for the peptide of the invention whose amino acid residue sequence corresponds mainly to positions 141-160 of Olk were 1 to 3 units higher, corresponding to a 10 to 1000-fold improvement in neutralization.

D. Antibodies from cystine chain peptides

Stock solutions of vaccines containing 3 cystine-chain peptides (cyclic peptide, and polymeric peptides A and B) were prepared in Freund's incomplete adjuvant as above

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indicated. These vaccines gave concentrations of peptide of lOOyUg per peptide per inoculation.

Preliminary results of an inoculation in guinea pigs indicated a range of neutralization indices (log^Q) of

2.3 to 3.0 for all three vaccines. The average neutralization index was approx. 2.5 which indicated that each of the cystine-disulfide-linked peptides protected the host against Olk FMDV challenge.

A typical neutralization index value for the monomeric, unconjugated peptide whose sequence corresponds substantially to amino acid residues around 141 to 160 of Olk FMDV is approx. 0.5. These results therefore indicate that a carrier is not necessary to achieve protection in animals against foot-and-mouth disease.

IV. Carriers and excipients

A. Alternative Carriers

The above results were obtained using inoculations of a KLH peptide conjugate plus a physiologically acceptable diluent such as water along with adjuvants such as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and/or aluminum hydroxide. KLH is an acceptable carrier for use in animals, but is relatively expensive to use on a commercial scale. The use of alternative carriers including soybean agglutinin, bovine serum albumin (BSA), ovalbumin, peanut agglutinin, tetanus toxoid and poly-L-lysine was also investigated.

The above results were also obtained by chaining the antigenic peptide to the KLH molecule via an additional cysteine (Cys) residue added to the amino or carboxy end of the peptide. The Cys residue was then reacted with the reaction product of KLH and N-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) as discussed in Bittle et al., supra. Both MBS and glutaraldehyde were used as chaining agents in the later discussed results. Conjugation of the synthetic peptide to KLH and BSA with glutaraldehyde was performed using the general method described by Avrameas,

Immunochemistry, 6:13-52 (1969). '

The results shown in Table 5 were obtained by chaining a peptide with the amino acid sequence of

positions 141-160 of the Olk FMDV VP-^ capsid (peptide 65) to the indicated carrier using MBS. Vaccines were prepared in Freund's incomplete adjuvant. Single inoculations containing sufficient conjugate to give 100 µg of peptide were given subcutaneously to each of 6 guinea pigs. The peptide-antibody titers shown are an average of 6 values obtained 4 weeks after inoculation using the ELISA method of Bittle et al., supra.

The above results illustrate that several carriers are practically as active as KLH while bovine serum albumin gave an excellent antibody titer.

Preliminary investigations also showed that the use of peptide 65 and tetanus toxoid with glutaraldehyde as a quenching agent gave a very good antibody response with one inoculation. For these binding reactions, a solution was prepared containing 24.5 mg of peptide 65 and 2{6 mg of tetanus toxoid in 12.5 ml of phosphate-buffered saline (PBS, pH 7.2). This solution was stirred gently while 1.6 ml of a solution containing 0.38% glutaraldehyde in PBS was added. The mixture was stirred for approx. 18 hours at room temperature, was dialyzed against water in dialysis tubes with a cutoff of 12,000 in molecular weight, and was then freeze-dried to form 45 mg of dried conjugate.

B. Excipients

Excipient systems were also investigated using the above peptide 65-chain carriers. The results illustrated in Table 6 show the effects of varying the carrier between KLH and BSA, the linker between MBS and glutaraldehyde, and the adjuvant between Freund's incomplete adjuvant (IFA) and saponin aluminum hydroxide, indicated in Table 6 as saponin. Each of 6 guinea pigs was inoculated subcutaneously with vaccines containing 100 µg of peptide 65 and the adjuvant in two inoculations, 4 weeks later. The results are the mean values for the 6 animals and are given as the results in table 5.

The above data illustrate that saponin-aluminum hydroxide gives a higher and more prolonged antibody response than Freund's incomplete adjuvant (IFA) regardless of whether the peptide was linked with either glutaraldehyde or MBS, and regardless of whether the carrier was KLH or BSA.

The data in subsequent Table 7 show antibody titer responses and neutralization results using the previously described techniques, when peptide 65 linked to KLH was used as the vaccine together with one of two adjuvant systems. The first horizontal row of data for each vaccine contains the neutralization index values (log^Q), while the horizontal row of data below contains the peptide antibody titers obtained by ELISA.

The above-mentioned results illustrate that vaccines containing the three adjuvant systems produced larger amounts of antibodies with a longer duration than the vaccines containing saponin-aluminium hydroxide. There also seemed to be little difference between the two doses administered in saponin-aluminium hydroxide.

The data in Tables 8 and 9 illustrate responses in mixed breeds and pigs to multiple inoculations of vaccine containing the peptide 65-KLH conjugate (MBS-linked) and saponin-aluminum hydroxide. These results illustrate that both animal types responded to the synthetic antigenic peptides by developing antibody at a level considered protective.

In each table, the first horizontal row of data for each animal is neutralization index values (log^0), while the second horizontal row of data is peptide antibody titer values obtained by ELISA.

The above results in Table 8 with cattle illustrate an anamnestic response in that the 6-month booster inoculation triggered memory B-cell production of neutralizing antibodies.

The above results regarding inoculations with synthetic antigenic peptides according to the invention were performed using peptides with only one sequence for each set of data. The data in Table 3 show that some cross-reactivity and poly-specificity was observed.

According to another embodiment, cross-reactivity and poly-specificity is achieved by inoculations using a majority of the peptides produced according to the invention, each of which is mono-specific towards at least one different strain of virus within a genus, or two different serotypes or strains within the genus. Thus, inoculation with a vaccine containing peptides whose amino acid residue sequences correspond mainly to amino acid residue positions around 141 to 160 of both type Olk and type A, subtype 10, strain 61 (A10, 61) confers protection against both type Olk and A10, 61 Similarly, a polymeric peptide such as polymeric peptides A and B, as previously discussed, can be prepared as a copolymeric peptide whose repeating peptide units are present in equal amounts and have amino acid residue sequences corresponding mainly to amino acid residue positions around 141 to 160 of Olk and A10, 61.

V. Synthetic peptides related to Picornavirus

The above-mentioned synthetic peptides can be used alone as a polymer in which the peptide units are linked together with oxidized cysteine residues, or linked to a carrier as a conjugate together with physiologically acceptable diluents such as water or an adjuvant to form a vaccine which, when this introduced into a host in an effective amount, is capable of eliciting the production of antibody that reacts with the Picornavirus to whose capsid protein sequence the peptide corresponds or substantially corresponds, and protect the host against this Picornavirus. This synthetic peptide alone, as a polymer or conjugate, can also be used as discussed previously or in the following, for the 20 amino acid residue-containing peptides whose sequences correspond or substantially correspond to the amino acid residue sequence of positions around 130 to 160 from the amino end of the FMDV VP, capsid.

Upon examination of the active, antibody-inducing regions of the antigenic Picornavirus capsid, it is noted that a determinant region of FMDV VP^ against which neutralizing antibodies can be formed corresponds to amino acid residue positions around 130 to 160 from the amino terminus of the capsid. The VP^ capsid includes a total of approx. 213 amino acid residues from the amino terminus to the carbpxy terminus using Olk virus VP^ as a reference protein.

The region of the protein at which the neutralizing antibody determinant begins is thus located approx. 60% (130/213 x 100% = 61%) of the long amino acid residue sequence of this protein from the amino terminus. This neutralizing antibody-producing determinant region ends around amino acid residue position 160 which represents approx. 75% of the amino acid residue sequence from the amino end. For the more preferred peptides corresponding mainly to amino acid residues around 141 to 160 of the FMDV VP-^ capsids, the neutralizing antibody-producing determinant region is within the region located at a distance from the amino terminus equal to 66 to 75% of the total amino acid residue sequence.

Examination of the amino acid residue sequences in FIG. 1

and of the readily available pK 3. data shows that the number of residues in each sequence that will carry a positive ionic charge at physiological pH values (Arg, Lys and His) is numerically superior to the number of residues that will carry a negative charge at this pH value (Asp and Glu) for all sequences except one. This one sequence of FMDV type A, subtype 10, strain 61 (A10, 61), carries a neutral ionic charge. However, it should be noted that the particularly preferred region of the A10 capsid corresponding mainly to amino acid residues around 141 to 160 carries a net positive charge.

The synthetic antigenic peptides produced according to the invention typically carry a net neutral or positive charge, excluding any ionic charge caused by terminal amino and/or carboxyl groups. Preferably, these peptides carry a net positive ionic charge. Such peptides are also preferably water-soluble. However, it appears that the net neutral to positive charge on the synthetic antigenic peptide is not as necessary for the peptide's antigenicity as the fact that the amino acid residue sequence of the peptide corresponds at least to a region of the antigenically neutralizing antibody-eliciting capsid that is between 60 and 75% of the length of this sequence from the amino terminus.

WE. Methods and known technique in

Methods and materials that are distinctive for the present i

invention, is described with reference to the particular procedure under consideration. In general, the laboratory techniques, methods and materials used are those normally used in molecular biology and biochemistry in general.

In particular, reference is made to Methods in Enzymology, Colowick, S.P. and Kaplan, N.O., editors, Academic Press, New York; Methods in Immunology and, Immunochemistry,

Academic Press, Handbook of Biochemistry and Molecular Biology, Chemical Rubber Publishing Company, and Cell Biology: A Comprehensive Treatise, Goldstein and Prescott, Academic Press, N.Y., N.Y. for a description of general materials and techniques of interest.

VII. Conclusion

Lerner et al. has worked with FMDV for a long time, and i

for a time they assumed they had identified the optimal, specific, antigenic determinant peptide fragment for FMDV

to later find that the putative antigenically active part did not elicit the production of antibodies to FMDV or elicited antibody production at such low levels as to be of little or no practical value. The applicants are clear that Strohmaier et al., supra, have drawn certain conclusions regarding antigenically active parts of their ^VPi^ FMDV serotype 0 gene and that Kelid et al. had determined the nucleotide sequence of the VP^ (VP^) FMDV serotype A, subtype 12 gene [Science, 214:1125-1129 (1981)]. It was of course impossible to determine from the nucleotide sequences which peptide fragment or fragments would be antigenic and in particular it was impossible to predict or even guess which peptide fragments would have optimal antigenicity for FMDV virus. As shown above, the antibody-eliciting region of the Olk VP-^ capsid reported by Strohmaier et al. was found to be much less active than the relatively longer region required here which includes regions

as by Strohmaier et al. was predicted not to be able to elicit protective antibodies.

A number of peptides were synthesized, bound to carriers, e.g. KLH, and the resulting antigens were injected into animals. Antibodies from the animals were then challenged with FMDV to determine whether the antigen was antigenically effective in eliciting antibodies against the infectious organism.

It was a totally unexpected discovery that such a relatively small peptide fragment in the region of capsid positions around 130 to 160, e.g. a 20-residue long peptide, such as the sequence corresponding to positions around 141 to 160,

was extremely antigenic. It is of course impossible to determine whether or not there may be other and possibly even more antigenic nucleotide sequences in the FMDV gene,

although there is no reason to predict that such will exist. A 20 amino acid residue sequence from the VP^ capsid 130-160 amino acid residue sequence according to this discovery appears rather unpredictably and completely surprisingly to be the optimal and probably final FMDV mono-specific, synthetic, antigenic determinant peptide plus/minus one or two (possibly three ) amino acid residues.

It is currently not known how much one can deviate

from the exact peptide without losing the highly unexpected activity and efficacy of the vaccine or antigen of which the determinant is the FMDV mono-specific synthetic antigenic determinant, however, it is known from experiments that (1) the peptide can be extended by a few amino acid units, (2) that at least one or two, possibly up to four or five substitutions can be made, and (3) that the peptide sequence can be shortened insignificantly, probably by two or three, possibly four, without losing its distinctiveness. Such insignificant deviations are in principle known to be possible without deviating from the described concept and the discovery that has been made. Such minor variations are considered equivalent variants of the invention.

Our results clearly show that a simple inoculation of the synthetic peptide, which consists of approx. 20 amino acid residues in the 130-160 region, e.g. region 141-160, elicits sufficient virus-neutralizing antibody to protect against an injection of the virus. The protection provided by the peptide is several times greater than the best results obtained by immunization with the capsid protein VP^, regardless of whether this is produced by bursting virus particles or by development in E. coli cells. It has been postulated that a small, free peptide may be able to adopt a structure close to that adopted by the virus particle, a situation that is unlikely when this is inhibited by neighboring amino acid residues in a misfolded VP^.

An alternative explanation is that immunodominant regions

of VP^ can be buried in the virus and is irrelevant for the neutralization.

A clear advantage of the synthetic peptide produced according to the invention is its activity in eliciting a protective antibody response with a single inoculation.

This good response to a simple inoculation is very important because successful immunization against foot-and-mouth disease depends on the vaccines being sufficiently active to give a protective response with an inoculation. Preliminary work with cattle and pigs has shown that the synthetic peptide can elicit an antibody response sufficient to protect these species against the disease.

IN

Industrial application

The therapeutic use of the antigens produced according to the invention, and the vaccines and antibody preparations thereof, are of great industrial and economic value. Animals such as pigs and cattle can be protected from the ravages of Picornavirus-induced diseases such as foot-and-mouth disease, thus increasing the supply of food and protein for humanity.

Claims (3)

1. Analogy method for the preparation of a therapeutically active antigenic peptide having the following amino acid residue sequence written from left to right and in the direction from the amino end to the carboxy end: TyrAsn(Asp or Thr)Gly(Phe)Glu(Thr)Cys(Ser or Asn or Thr)Arg(Lys or Thr)TyrAsn(Ala or Ser or Thr)Arg (Val or Ala or Asn or Thr)Asn(Gly or Ser)Ala (Asp or Gly)Val(Ser or Gin or X)Pro( Gly or Y) Asn(Z)Leu(Arg or Val)Arg(Ser or Ala)GlyAspLeu(Met or Phe)Gin(Gly)Val(Thr or Ser or His)Leu(Ile)AlaGln (Ala or Pro)Lys( Arg or Ala)Val(His)Ala(Val)Arg(Thr or Lys)Thr(Gin or His)LeuPro and optionally Ala, and optionally containing two further Cys residues attached to each end of the peptides, in which each of the amino acid residues as well as the symbols X, Y or Z in parentheses can individually replace the adjacent amino acid residue immediately to the left of the parenthesis, and where X and/or Y and/or Z in the peptide amino acid residue sequence independently indicates from honored by an amino acid residue in the position of the adjacent amino acid residue immediately to the left of the parenthesis, whereby the length of the peptide is shortened by 1, 2 or 3 amino acid residues, characterized in that a starting amino acid resin is prepared by esterification of Boc-AA-OH to a resin in which AA denotes the relevant amino acid, followed by successive coupling of Boc-AA-OH using side chain protecting groups, the protected polypeptide resin is treated with liquid HF to remove the protecting groups and cleave the polypeptide from the resin, after which the cleaved, unprotected polypeptide is extracted with dilute acid, after which, if desired, the obtained polypeptide is coupled to a protein carrier, and, if desired, that the obtained peptide is oxidized. 2. Method according to claim 1 for the production of a polypeptide with an amino acid sequence, from left to right, and in the direction from the amino end to the carboxy end as follows: characterized in that corresponding starting materials are used. 3. Method according to claim 1 for the production of a polypeptide with an amino acid sequence, from left to right, and in the direction from the amino end to the carboxy end as follows: characterized in that corresponding starting materials are used.