NO853153L - SYNTHETIC POLYPEPTIME RESPONSIBLE FOR A PART OF THE HEATABLE ENTEROTOXIN OF ESCHERICHIA COLI, PREPARATIONS AND METHODS THEREOF - Google Patents

Description

Cross-reference to related applications

The present description is a continuation of

US Application No. 559,469 filed on December 12, 1983, which is a continuation of US Application No. 455,265 filed on January 3, 1983.

Known technique

Acute diarrheal diseases due to changing colonization in the small intestine by enterotoxigenic strains of Escherichia coli (E. coli or ETEC) are a major health problem of global scale.

cane for both humans and domestic animals. These organisms, along with rotavirus and Campylobacter jejuni (C_. jejuni), are the principal cause of the often fatal acute diarrhea that is

among children living in underdeveloped countries and among newborn animals, especially lambs and piglets. ETEC strains are also the common cause of acute diarrhea among people from temperate zones who travel to the tropics, and may be responsible for sporadic or epidemic episodes of diarrhea in children and adults living in either temperate or tropical environments.

prevails,

The sick judgment produced by ETEC is conveyed by release of

t o enterotoxins, either alone or together. The low-molecular-weight, heat-stable toxin (ST) formed by ETEC strains of human or porcine origin has recently been purified. Preparations of ST have a relatively high half-cystine content, induce secretion by stimulating guanylate cyclase and are haptenic as determined by their ability to induce an antitoxin-

response in animals immunized with the toxin linked to a high molecular weight carrier.

The high-molecular-weight, antigenic heat-labile toxin (LT)

has been purified to homogeneity. Its subunit structure has been characterized as (1) five B subunits that bind the holotoxin (complete toxin) to specific GM^ganglioside receptors on the mucosal surface, and (2) a single A subunit that stimulates intracellular adenylate cyclase activity, thus eliciting fluid - and electrolyte secretion.

Immunization with either biological LT, the biological

LT B^subunit or the biological ST toxin induces an anti-tQ;xlnr_es,p_ons i. in used for immunization. Thus, immunization with LT whole toxin or its B subunit confers protection against viable heterologous strains producing this toxin alone (LT + /ST — ) or together with ST (LT + /ST + ), but not against those producing only ST (LT /ST<+>). Similarly, immunization with biological ST confers protection against direct challenge with viable heterologous ST-producing strains (LT /ST+ and LT<+>/ST<+>), but not against LT-producing strains.

However, the biological toxins or the B subunit are not suitable for immunization when given alone in view of their toxicity, their inability to provide protection against strains producing the other toxin form, and the fact that the high molecular weight carriers that have been used for to render the haptenic biological ST molecule immunogenic, is typically unsuitable for human use. Therefore, the most practical approach to protection against ETEC-induced diarrhea would be an immunization program that provides protection against heterologous ETEC serotypes that produce one or both of the LT or ST enterotoxins and that does not use an irrelevant -used carrier molecule,

US Patent 4,411,888 to Klipstein and Klipstein et al., J. Infec. Haze. 147:318-326 (1983), describes i.a. development of a vaccine prepared by conjugating natural or synthetic ST toxin to the biological B-subunit of LT toxin (LTB) by means of a carbodiimide reaction. As a result of this reaction, synthetic ST achieves immunogenicity when coupled to the high molecular weight LTB carrier, LTB maintaining most of its immunogenicity, while both cross-linked materials lose most of their toxic (secretion-inducing) properties. Rats immunized with the vaccine thus prepared have been shown to be highly protected against challenge with either LT or biological ST, as well as against challenge with viable ETEC strains producing these toxins,

The LTB preparation used for this vaccine was obtained via recombinant methods. Although this method provides a useful result, it is relatively cost effective as the resulting recombinant LTB must be extensively purified to remove toxic materials before it can be formulated into a useful vaccine in combination with ST. Recombinant LTB must also be coupled to ST with an equivalent ligand such as soluble carbodiimide, glutaraldehyde or dimethylsuberimidate which may be vaBre_juakji_epjtable; in the._s.last_able for mulation for human use. Finally, the size of the LTB may necessitate the use of an excess of ST in the preparation of the LT/ST immunogen formulation, increasing the price accordingly.

Richard A. Lerner et al. have shown that a synthetic polypeptide whose amino acid residue sequence corresponds to that of part of a protein can be used to elicit antibodies that recognize the intact protein. See Sutcliffe et al. Science 219:660-666 (1984). Synthetic polypeptides prepared after the first work by Lerner et al., have been successfully used for the production of the previously described synthetic ST molecule.

The nucleotide sequences encoding the LTB proteins of human- and swine-infecting (LT^ and LT^) E. coli have been determined. Translation of the nucleotide sequences into amino acid sequences leads to proteins whose largest form can contain 124 amino acid residues. See Dallas and Falkow, Nature 288:499-501 (1980) and Yamamoto et al., J. Bacteriol. 155:728-733 (1983).

The 124 residue protein includes 21 amino acid residues at the amino terminus in what is sometimes referred to as a "signal peptide". Reported amino acid residue position numbers for ( ■ —- ..

LTB in the literature can therefore vary by 21 positions, depending on whether the authors included or excluded the 21 residue signal peptide in their position numbering nomenclature.

Brief description of the invention

The present invention has several aspects. One aspect contemplates a synthetic polypeptide containing 10 to 35 amino acid residues corresponding in sequence to the amino acid residue sequence from position 35 to position 95 of the amino terminus of the B subunit of the heat labile enterotoxin of Escherichia coli (LTB); where the position numbers include the 21 residue signal polypeptide of the B subunit. Preferably, a synthetic polypeptide contains 15 to 30 residues corresponding in sequence to position 55 to position 85 from the amino terminus of the B-subunit, again wherein the position numbers include the 21 residue signal polypeptide. HelsJ:-, the synthetic polypeptide contains 20 to 25 amino acid residues corresponding in sequence to - position 60 to position 85 from the amino end of the B-subunit n of the heat-labile enterotoxin, again including the 21-residue signal polypeptide of this subunit.

Another aspect of the invention contemplates a composite LT/ST polypeptide containing 25 to 55 amino acid residues comprising 2 amino acid residue sequence peptides linked together. One of these amino acid residue sequences corresponds in sequence to at least the carboxy-terminal 14 residues of the heat-stable enterotoxin Escherichia coli (ST), while the other sequence lasts from position 35 to 95 from the amino end of the B-subunit of the heat-labile enterotoxin of Escherichia coli (LTB) in which the latter position numbers include the 21 residue signal polypeptide with the B subunit.

In preferred practice, the first-mentioned amino acid residue of this polypeptide d includes an 18-residue sequence of the heat-stable enterotoxin of E. coli and the above-described more preferred sequence corresponding to part of the B-subunit of the heat-labile enterotoxin of E. coli. Preferably, the polypeptide includes the 18-residue sequence of the heat-stable enterotoxin and the above-described 20 to 25-residue sequence corresponding to part of the B-subunit of the E. coli heat-labile enterotoxin. The two sequences that make up a composite LT/ST polypeptide are preferably linked together by a peptide bond between the carboxy-terminal residue of one sequence and the amino-terminal residue of the other sequence.

The above-mentioned polypeptide, here indicated as a composite LT/ST polypeptide, can also include 1 to 4 further amino acid residues chained to one or both ends, as well as 1 to 2 further amino acid residues between the two polypeptide sequences that make up the composition. Jl yer of diss e further residues carry an acidic or basic side chain capable of carrying an ionic charge, by. pH. for the jejunum..(-ca—pH.~6-.5). When more than one of these additional residues is present, each of the side chains of an additional amino acid residue is capable of carrying the same ionic charge at the jejunum pH. Preferred additional amino acid residues that can be chained to one or both of the ends and between the polypeptide components are selected from the group consisting of lysine-(Lys) and arginine-(Arg) residues, or are selected from the group consisting of aspartic acid-(Asp) and glutamic acid -(Glu) residues.

A composite LT/ST polypeptide according to this embodiment of the invention can also be described in general, written from left to right and in the direction from the amino end to the carboxy end, as a compound corresponding to the formula:

in which

X, Y and Z, when present, are additional amino acid residues to the sequences which are (a) selected from the group consisting of Lys and Arg residues, or (b) selected from the group consisting of Asp and Glu residues;

"n" and "m" are integers with a value of 0, 1, 2, 3 or 4, so that the respective X and Y are absent when one or both of "n" and "m" have the value 0, while the respective X and Y residues are present when one or both of "n" and "m" has a value different from 0, where the average number of X and Y residues present per polypeptide, is equal to the values for X and Y;

"r" and "s" are integers having a value of 0, 1, or 2, such that the respective Z is absent when one or both of "r" and "s" have a value of 0, while the respective Z is present when "r" and "s" are present, where the average number of Z residues per composite LT/ST polypeptide molecule or repeating unit is equal to the value of "r" or "s",

provided that when one of "r" or "s" is greater than 0, the other of "r" or "s" is 0 and the respective Z if "r" or "s" is 0 is absent;

"o" and "p" are integers having the value 0 or 1, so that the corresponding A is absent when one or the other of "o" and "p" has the value 0, provided that "o" and "p" not both have the same value;

A is a polypeptide containing 10 to 35 amino acid residues corresponding in sequence to the amino acid residue sequence of position 35 to position 95 from the amino end of the B subunit of the heat labile enterotoxin of Escherichia coli in which the position numbers include the 21 residue signal polypeptide of the B subunit; and

B is a polypeptide containing up to 18 amino acid residues corresponding in sequence to at least the carboxy-terminal 14 residues of the heat-stable enterotoxin of Escherichia coli.

Also contemplated by the invention are polymers comprising a plurality of polypeptide repeating units containing 10 to 35 amino acid residues corresponding in sequence to the amino acid residue sequence of positions 35 to 95 from the amino terminus of the B subunit of the heat-stable enterotoxin of Escherichia coli, wherein the position numbers includes the 21 residue signal peptide of the B subunit. When these polymers, dissolved or dispersed in a physiologically acceptable diluent, are introduced as an inoculum in an effective amount into a host animal, they are capable of eliciting the formation of antibodies which immunoreact with the natural enterotoxin B subunit.

In one embodiment, the repeating units of polymers according to the invention are linked together with cystine disulphide bonds formed by oxidation of Cys residues added to the repeating unit end; in that the Cys residues are present at both the amino end and the carboxy end of the repeating polypeptide units before oxidation. Such repeating units are in unoxygenated form designated as diCys-LT polypeptides. The polymer is a linear homopolymer when no Mohorar repeating units are present during the polymer-forming oxidation.

An arbitrary three-dimensional network copolymer is

the result when oxidative polymerization is carried out using

generation of both the above-mentioned diCys-LT polypeptide and a second repeating polypeptide unit whose amino acid residue sequence corresponds to at least the 14 carboxy-terminal amino acid residues of E. coli heat-stable enterotoxin (ST polypeptide) as time-

stated above. If network polymers are desired, at least 3, and preferably all 6 Cys residues of the ST polypeptide are to-

present. It is also preferred that a complete, 18-residue ST polypeptide sequence is used.

A net-ty_exkp_oJyme r comprising a majority of co-

sa tte LT/ST^polypgp time-repeating units are taken into consideration according to a further aspect of the invention. The polypeptide repeating units, written from left to right and in right-

ning from the amino end to the carboxy end, individually corresponds to the formula for the above-described compound LT/ST poly-

peptide. The repeating units of a particularly preferred network polymer, written from left to right and in the direction from amino end to carboxy end, correspond individually to the formula:

wherein X and Y, when present, are amino acid residues which are (a) selected from the group consisting of Lys and Arg residues, or (b) selected from the group consisting of Asp and Glu residues; and

"n" and "m" are integers with a value of 0, 1, 2, 3 or 4, so that the respective X and Y are absent when one or both of "n" and "m" have a value equal to 0, while the respective X and Y residues are present when the one or

both of "n" and "m" have a value different from 0, where the average number of X and Y residues present per repeat-

end polypeptide unit, is equal to the values of X and Y;

The ^mino acid residues in parentheses are each an alternative to

the immediately preceding amino acid residue in the sequence of the formula; and the repeating units are linked together by intramolecular, interpolypeptide cystine bonds formed between oxidized Cys residues of the repeating units. r — -

The invention also includes inocula usable for the formation of antibodies for use in diagnostic o methods and t, j systems, or as vaccines capable of protecting A f animals including humans against the heat-labile, or heat-labile and heat-stable endotoxins of E ■ coli. The inoculum contains an effective amount of an LTB polypeptide conjugate, composite LT/ST polypeptide conjugate or network polymer as described herein, in a physiologically acceptable diluent. When the inoculant is introduced in a unit dose into a host

animals such as by injection or by oral administration, it is capable of eliciting the formation of antibodies that immunoreact with at least the B subunit of the heat-labile entero-

toxin of JEL CO-li. In more preferred practice, the antibodies thus elicited immunoreact with both the B subunit of heat-labile enterotoxin and also with heat-stable enterotoxin

toxin of JL. coli. When such an inoculum is used as a

vaccine to immunize a host animal such as human, pig

or a laboratory animal such as a rat, the host is protected

against heat-labile enterotoxin-producing _E_. coli, and for-

gradually also against heat-stable enterotoxin-producing

E.coli.

Brief description of the drawings

In the drawings which form part of the present description, show: fig. 1 the translated amino acid residue sequence of B- subunit of porcine-(LT ) and human (LT, ) heat-labile entero-

p. h

toxins of Escherichia coli written from left to right and in the direction from the amino end to the carboxy end. Peptide bonds s<p>m chains of amino acid residues are depicted by bond lines.

The amino acid residues of the 21-residue signal peptide sequence are shown with d lowercase letters, while the remainder of the main part of the protein is shown: with uppercase letters. A forward slash separates the signal peptide sequence from the remaining sequence. Dots in the LT^ sequence (below the LT P sequence) indicate that the identical residue is present in both sequences. Residues that differ between the sequences are indicated. Numbers above the LTp sequence indicate residue positions measured from the amino terminus. The sequences were described by Dallas and Falkow, Nature 288:499-501 (1980) and by Yamamoto et al., J. Bacteriol. 152:506-509 (1982) and Yamamoto et al., J. Bacteriol. 155:728-733 (1983).

Fig. 2 is a diagram showing the antigenicities of 3 of the examined LT^-related polypeptides as measured by the B/B ELISA technique described in the Materials and Methods section, and is expressed as a percentage of the antigenicity of the natural LT^B- subunit. The ordinate shows the percentage of

native LT^B subunit antigenicity. The abscissa illustrates the sequence position of the LT^B subunit protein. Antigenicities of LT 3? B-related polypeptides alone are shown by rectangles enclosing diagonally hatched hatches, the thickness of the enclosed hatches being indicated for the purpose of ease of reading, and not to express experimental error in the measurements. The antigenicities of the LT^B-related polypeptides peptide bound to the amino-terminal residue of an ST Ibh polypeptide and oxidatively polymerized, is illustrated by the rectangular columns with dashed lines.

Fig. 3 is a diagram illustrating the results of immunization of rats by primary parenteral immunization

(IP) followed by two oral (PO) immunizations. The ordinate shows the percentage of reduced intestinal secretion, while the abscissa reflects the total oral dose in LT^B subunit (B) antigen units. Graded antigen unit doses were introduced using vaccines containing as active ingredient (immunogen) either natural LT^B subunit

(•—0; natural B ), or the monomeric form of a LT B-

h p subunit-related polypeptide, peptide-linked to an 18-residue ST Ibh polypeptide (composite LT/ST polypeptide) alone

(A; BFRAG~ST alone) or conjugated to porcine immunoglobulin G (pig) (0—0; BFRAG~STxpig) . Rats were challenged with viable LT<+>/ST~ E. coli strain PB 258. Results are expressed as the mean +SEM reduced secretion in immunized rats compared to similarly challenged control groups. Numbers above or below the data points indicate the mucosal IgA antitoxin titers for those points. Fig. 4 are diagrams illustrating the results of immunization of rats by primary parenteral (IP) immunization followed by two oral (PO) immunizations. Graded antigen unit doses were introduced using vaccines containing as active ingredient (immunogen) a synthetic 18-residue SJL_lbh^polypeptide, chemically cross-linked to the natural Xlge_LTh_^iSiAen called [•—• ; ST (S) XB ] , and a network polymer with the composite LT/ST polypeptide according to fig. 3 as repeating unit [0 0; B_,_. -ST] in the left diagram, and the same network polymer [0 0; BFRAG~ST] and natural-^like LT^B subunit [•—• ; natural B^]in the right diagram. Challenges were made with viable E. coli LT+/ST strain Tx 452 in the left diagram, and with viable E. coli in T,T<+>/ST strain PB 258 in the right diagram. The ordinates for both diagrams are as described for fig. 3. The abscissas show the total PO dose in ST(S) and natural LT^B-subunit antigen units for respectively. left and right diagrams. The number of milligrams (mg) of immunogen corresponding to the antigen units of the abscissa is also shown. Other results are shown as in fig. 3. Fig. 5 is a diagram (right part) illustrating the results of immunization of rats given four weekly PO immunizations (PO/PO) of vaccines containing as active immunogen the network polymer containing the composite LT/ST polypeptide according to fig. 3 as repeating units [Aog A; vaccine], the natural LT^B subunit [•—•;

B subunit], or the above-described synthetic ST cross-linked to the natural LT^ B subunit [0 Q) ; ST(S)XB]. Challenges and the results obtained are as described for fig. 3, with the exception that the number of mg of the immunogen in the vaccines (mg VAC) is shown on the abscissa, as is the number of antigen-

devices manage.

The present invention entails several advantages. One

advantage of the invention is that it provides a uniform, completely synthetic vaccine against both heat-labile and heat-stable enterotoxin-producing strains of Escherichia coli.

Another advantage is that the uniform vaccine does not induce substantially any intestinal secretion in the immunized host, i.e. the vaccines prepared according to the invention exhibit substantially none of the harmful secretion-inducing activity of each of the enterotoxins.

Another advantage of the invention is that its LT B subunit-related polypeptides are synthetically produced by standard synthetic laboratory procedures, and are thereby free from the presence of unwanted biological contaminants.

A further advantage of the invention is that its polypeptides are relatively cheap to produce and purify compared to biological or recombinant DNA technology.

Further advantages will be apparent from the following detailed description.

The present invention comprises a synthetic polypeptide containing 10 to 35 amino acid residues corresponding in sequence to position 35 to position 95 from the amino end of the B-subunit of the heat-labile enterotoxin Escherichia coli, in which the position numbers include the 21-residue signal peptide of the B-subunit, Conjugates, compounds LT/ ST polypeptides and polymers comprising such polypeptides, as well as grafts comprising an effective amount of a conjugated polypeptide, composite LT/ST polypeptide or polymer, antibodies elicited by the graft and methods related thereto, are also contemplated.

I. LTB<p>olypeptides

The translated amino acid residue sequences of the B subunit of LTp and LT^ are shown in fig. 1. The amino acid residue sequence of the B subunit of the heat labile enterotoxin of Escherichia coli from sequence position numbered 35 to sequence position numbered 95 (wherein the position numbers include the 21 residue signal polypeptide) written as a single sequence, from left to right in the direction from the amino terminus to the carboxy terminus, is represented by formula I in the following:

wherein amino acid residues in parentheses are alternatives to the respective immediately preceding residue in the sequence of the formula, i.e. the residue to the left of each residue in parentheses. The numberings above specific residues indicate amino acid positions in the sequence, measured from the amino end, and including the signal peptide sequence.

It should be noted that the sequence of formula I contains two alternative amino acid residues. These residues are located at positions 64 and 67 from the amino end. Unless otherwise indicated, a reference herein to a polypeptide according to the invention which includes an amino acid residue sequence corresponding to a part of LTB (an LTB polypeptide) will include both LT^ and LT polypeptide sequences as a composite sequence; i.e.

an amino acid residue sequence that includes (a) either a Lys or a Met residue at position 64, and (b) either a Glu residue or an Ala residue at position 67. Thus, an LTB polypeptide that may include the four sequences shown in the following from position 64 to 67, be included with reference to an LTB polypeptide or with reference to

LTB:

The data discussed in the following illustrate that

a synthetic polypeptide containing 10 to 35 amino acids-

residues corresponding in sequence to positions 35 to 95 of the B subunit of the heat-labile enterotoxin (LTB) are useful as an immunogen for inducing the production of anti-

substances that can be used in diagnostics for the presence of the heat-labile enterotoxin (LT) produced by_E_. coli. preferential

way, the polypeptides according to the invention are used as an immuno-

ge n in inoculum. In such applications, it is preferred that the polypeptides contain 15 to 30 amino acid residues and that the sequence of the polypeptides corresponds to the LTB sequence of still-

ing 55 to position 85, Preferably a polypeptide containing 20 to 25 residues is used, and where the sequence of this polypeptide corresponds to the sequence from position 60 to position 85

by LTB. Such a polypeptide is typically used as a conjugate of the polypeptide coupled to a carrier. Unconjugated polypeptides

times are also useful in diagnostics as an antigen or competitive ligand for anti-LTB polypeptide or anti-natural LT antibodies.

II. Composite LT/ST polypeptides including LTB sequences

A particularly applicable embodiment of the invention is

a composite polypeptide containing 25 to 55 amino acid residues and comprising two amino acid residue sequences linked together by a peptide bond between the carboxy-terminal residue of the first sequence and the amino-terminal residue of the second sequence. The two amino acid residue sequences of the composite polypeptide include a synthetic polypeptide as previously described, whose amino acid residue sequence corresponds to position 35 (*>—$T to % 5 from the amino end of LTB. The other sequence corresponds to at least the carboxy-terminal 14 amino acid residues of the heat-stab jle enterotoxin of Escherichia coli (hereafter generally designated as ST), Such a polypeptide is designated hereafter as a composite LT/ST polypeptide.

The peptide bond between the two amino acid residue sequences that make up the composite LT/ST polypeptide can be formed between the amino-terminal residue of an ST sequence and the carboxy-terminal residue of the LTB sequence, or between the amino-terminal residue of the LTB sequence and the carboxy-terminal residue of ST - sequence. The polypeptide sequences that make up a composite LT/ST polypeptide can also be said to be linked head-to-tail.

It is particularly preferred that the amino acid sequence corresponding to the heat-stable (ST) part of the composite LT/ST polypeptide includes the 18-residue (18-mer) sequence of the ST molecule.

At least two types of ST have been identified by their physical properties. The first type, known as ST I (also indicated as STa), is soluble in methanol and is active in the lactating mouse model. The other type, ST II (also designated as STb) is methanol-insoluble and not active in the lactating mouse model, but is active in ligated pig ileum loops. ST I polypeptides are of interest here and will be the only ST polypeptide type referred to here.

Among the ST I polypeptides, at least two similar polypeptides, or determinant regions of these polypeptides, have been identified, and their amino acid residue sequences have been investigated. These two types of ST I are here indicated as (i)

ST Ia which was first found in an __E_. coli strain from bulls and where part of this is also encoded in pig strains, and (ii) the one designated ST Ib from a human isolate of

E.coli.

The nucleotide sequence encoding the ST Ia polypeptide,

has been determined. Translation of the nucleotide sequence into a polypeptide amino acid sequence leads to a polypeptide containing 72 amino acids terminated at the carboxy terminus with a tyrosine group [So et al., Proe. Nati. Acad. Pollock. USA, 77:4011-4015 (1980)]. The ST Ib polypeptide is reported to contain only 18 amino acids [Chan et al., J. Biol. Chem. 256:7744-7746 (1981)], and these 18 residues are homologous to the 18-carboxy-terminal residues encoded by the ST Ia nucleotide sequence.

v

Recent investigations indicate that the originally reported amino acid residue sequence for ST Ib was wrong. The originally indicated carboxy-terminal asparagine-(Asn) residue is now believed to be a tyrosine-(Tyr) residue, while the originally indicated Tyr at position 11 from the amino end of the 18-mer is now

is believed to be an Asn residue. For ease of discussion, polypeptides whose sequences correspond to the original sequence reported by Chan et al., supra, will be designated herein as ST Ibh, while polyp,e.p^tides corresponding to...the revised__sequence..,^_ will be' indicated as ST Ibp.—-.

The 18 amino acids of the ST Ibh polypeptide (18-mer) show high homology with amino acids numbered 55 to 72 of the polypeptide of ST Ia. The homologous, virtually identical region of ST Ibh is illustrated below together with the indicated sequence of ST Ia and that of ST Ibp, starting at amino acid number 55, from left to right and in the direction from the amino end to the carboxy end of ST Ia- the polypeptide:

As can be seen, the 18 residue sequences of ST Ia and ST Ibp are identical.

Examination of the 3 (2 different) 18 amino acid residue polypeptide sequences indicated above also shows that 6 half-cystine (Cys) residues are also present. Oxidation of these half-cystine residues to cystine residues containing intramolecular, intrapolypeptide disulfide bonds in the native enterotoxin is believed to provide the observed heat stability to this material.

However, it should further be noted that although cystine disulfide bonds are known to be present in natural ST, it is not known which pairs of half-cystine esters combine to form the 3 disulfide bonds present in the natural ST molecule . These 3 disulfide bonds can theoretically be formed from 15 different combinations of the 6 Cys residues present.

Staples et al., J. Biol. Chem. 255:4716-4721 (1980) has shown that the disulfide bonds in biological ST are necessary for the biological activity of the toxin. Thus, it was shown that a chemical reduction during the formation of half-cystins or permauric acid oxidation to cysteic acid destroyed the biological activity of the toxin. In addition, Chan et al., supra, have described that the first 4 residues from the amino terminus of the homologous 18-amino acids of the above sequence of ST Ibh are not required for biological activity. Thus, biological activity was obtained from the amino acid-containing polypeptide comprising the above-mentioned carboxy-terminal 14 amino acids and their disulfide bonds.

Aimoto et al., Biochem. Biophys. Res. Chem. 112:320-326 (April 15, 1983), have reported the synthesis of the carboxy-terminal 14 amino acid residues of a material they have designated as ST^. This synthetic molecule was indicated to have a biological activity 2 to 5 times that of native ST^ on a molar basis, using a lactating mouse assay.

In an oral presentation on August 29, 1982 by Duflot et al, Proceedings European Peptide Symposium:683-686, published in Berlin in June 1983, these researchers described the synthesis of porcine and human ST 18-mer polypeptides with their Cys mercapto groups blocked (S-blocked) with acetamidomethyl groups. These amino acid residue sequences were claimed to be identical to the sequences reported by So et al., supra, for ST Ia and by Chan et al., supra, for ST Ibh. However, the 7th amino acid residue from the amino end of the sequences described by Duflot et al. was a glycine residue (Gly), while this residue in the previously described sequences is a glutamic acid residue (Glu).

Duflot et al. indicated that immunization of mice or rabbits with their S-blocked porcine ST toxin coupled to tetanus toxoid or ovalbumin elicited antibodies that recognized the natural or the synthetic toxins equally. Essentially no biological activity in the lactating mouse assay was reported for the S-blocked porcine synthetic polypeptide toxin. These authors suggested that the lack of biological activity was due to the absence of intramolecular disulfide bonds in the S-blocked molecule, which is consistent with the previous report by Staples et al., supra.

The ST polypeptides contemplated herein include at least the carboxy-terminal 14-residue of ST Ibh and ST Ibp (ST Ia). This polypeptide can also include up to 4, preferably 3 or fewer, and preferably 2 or fewer, alternative amino acid esters to the 6 Cy s residues of the ST sequence. Where Cys residues are present, in addition up to 4, preferably 3 or fewer, preferably 2 or fewer, sulfur atoms of the Cys residues present may be alkylated.

A useful ST polypeptide contains at least one intramolecular cystine disulfide bond. Accordingly, the presence of one or more alternative amino acid residues to a Cys residue, and of one or more alkylated Cys mercaptan groups, is subject to the proviso that the ST polypeptide is capable of forming at least one intramolecular cystine disulfide bond. This caveat is discussed in detail below.

A useful 14-residue ST polypeptide sequence corresponding to the carboxy-terminal 14 residues of ST, written from right to left and in the direction from the amino terminus to the carboxy terminus, corresponds to formula II.

wherein the two specific amino acid residues in parentheses (Asn and Tyr) are each an alternative to the immediately preceding amino acid residue in the sequence of the formula;

a, b, c, d, e and f (a-f) and g, h, i, j, k and 1 (g-1)

are integers each having a value of 0 or 1, provided that if the value of any of a-f or g-1 is 0, the corresponding r\ R^, R3, r1 , R^ or R^ (R1 ^ )-group 3. JO C Q 6 X cl — i

,, _7 „8 ^9 10 _11 ,, _12 ,_7-12xr

or R , R, , R., R. , R, or R, (R , )-group absence-

g h 1 1^6 ^ 1 9-1

end, and when an R --group is absent, forms sulfur-^—g

atom of the Cys residue with an absent R ^ group eii_cy-stin-dis sulfide bond, while if the value of any of

1 — 6 7-12

a-f or g-1 is 1, the corresponding R or R n -

1-6 g group present, wherein the Ra_^ groups are individually the same or different groups attached to the sulfur atom of the Cys-r ester and are selected from the group consisting of hydrogen, an alkyl group containing 1 to 4 carbon atoms such as methyl, ethyl, isopropyl and sec-butyl, and a substituted alkyl group containing 2 to 4 carbon atoms such as carboxymethyl, carbamoylmethyl, carboxyethyl and carbamoylethyl;

7-1*>

Rg_^^r^like. or various alternative^^minpsy acid residuesSfaJMiy,e-2>, immediately preceding the Cys-res"t shown in the formula, and is selected from the group consisting of amino acid residues with a neutral side chain such as alanine (Ala) and serine (Ser); and

at least two of a-f and two of g-1 are 0, and two non-adjacent Cys residues are present, provided that the synthetic ST polypeptide portion of the composite LT/ST polypeptide has the capacity to form at least one intramolecular cystine disulfide bond formed from at least two Cys residues present.

In monomeric form, it is the above stated, at least

one disulfide bond, an intramolecular, intrapolypeptide cystine disulfide bond formed between at least two Cys residues present in the ST polypeptide, When the ST polypeptide is in a polymeric form containing a plurality of LT polypeptide-containing repeating units, the above may, in the at least one disulfide bond being an intramolecular, intrapolypeptide cystine disulfide formed between at least two Cys residues. present in

each ST polypeptide repeating unit. At least one disulfide bond may also be an intramolecular interpolypeptide cystine disulfide bond formed between one of at least two Cys residues present in the first ST-containing repeating unit and another of at least two Cys residues present in a second ST-containing repeating unit , or one of the Cys residues may be present in another polypeptide repeating unit. It therefore appears that an intramolecular cystine disulphide bond is present in both the monomeric and polymeric compound forms of ST. In the monomeric ST-containing composition, this cystine disulfide bond is an intrapolypeptide bond, while in a polymer that includes an ST polypeptide sequence in the repeating unit, the disulfide may be an interpolypeptide or an intrapolypeptide bond.

I,enjn is a preferred embodiment of the monomeric and polymeric forms of a composite LT/ST polypeptide containing a synthetic ST polypeptide part, with reference to the above-mentioned ST synthetic polypeptide of formula II, is

"e" 0 when "a" is 0,

"d" is 0 when "b" is 0, and —D c "f" is 0 when "c" is 0; and rtu.Lt^each of "g" and "k" is 0 when "a" is 0,^ each of "h" and "j" is 0 when "b" is 0,'and each of "i" and "1" is 0 when "c" is O.i

J

The sequence shown in formula II without the two specific alternative amino acids and substituted and alternative R groups corresponds to the carboxy-terminal 14 amino acid residue sequence of ST Ibh. The 14 amino acid residues comprising amino acids 59-72 of ST Ia (the carboxy-terminal 14 residues of ST Ibp) differ from the sequence illustrated in formula II without its alternative amino acids and R groups at position 65 in which an asparagine (Asn) residue replaces tyrosine-( Tyr) the residue at position numbered 11 from the amino end of ST Ibh (residue-Position 8 from the carboxy end), and at position 18 from the amino end of ST Ibp (the carboxy end) in which a tyrosine residue replaces the shown asparagine residue,

Thus, the Tyr residue immediately to the right of the fourth Cys residue from the amino end (Tyr-6 5 in ST Ia) can be replaced with the Asn residue indicated in brackets in formula II. Conversely, the carboxy-terminal Asn can be replaced by a Tyr, as shown by the parenthesis of the final Tyr residue.

It is particularly preferred that at least one of the four amino-terminal amino acid residues present in the sequence of the 18-residue ST Ibh and ST Ibp molecules is also present in the natural position sequence in the synthetic ST part of a composite LT/ST polypeptide, or in any other portion containing an ST polypeptide. It is even more preferred that all four of these additional amino acids are present in the synthetic ST portion in the same natural positional sequence as they are present in ST Ibh and ST Ibp.

The preferred 4 additional amino acids at the amino terminus of an ST polypeptide correspond to amino acid position no. 55 to 58 of ST Ia (position 1 to 4 of ST Ibp) and are identical to these 4 amino-terminal amino acids in ST Ibh. The 4 amino acid polypeptide (4-mer) which is additionally present at the amino terminus of the synthetic ST of formula II has, in a preferred embodiment, a sequence taken from left to right and in the direction from the amino terminus to the carboxy terminus as shown in formula

III:

Formula III

AsnThrPheTyr

Thus, a preferred ST polypeptide has a sequence of 18 residues which are identical to the sequences of ST Ibh or ST Ibp (ST Ia) as shown previously. A useful ST polypeptide may also include an Asn residue at the carboxy terminus and an Asn residue at position numbered 11 from the amino terminus of ST Ibh. Conversely, both residues can be Tyr residues. The

4 applicable ST 18 residue sequences thus defined are shown below in formula IV, written as previously defined.

The 4 sequences of formula IV can be written as a single sequence using alternative residues indicated in parentheses. The combined ST polypeptide sequence, written as previously indicated, and using the amino acid residue sequence of ST Ibh as the basis for the alternative residues, is represented by formula V:

wherein the specific amino acid residues in parentheses are an alternative to each of the immediately preceding residues shown in the formula.

Unless otherwise indicated, a reference to ST or an ST polypeptide will refer to an ST-related polypeptide amino acid residue sequence that includes the alternative amino acid residues. This is the case for 14-residue polypeptides,

18 residue polypeptides and those containing 15-17 residues.

The preferred 18-residue ST sequence comprising

_l-6,,7-12 14_

R,a_£ and Rg_^ groups and alternative amino acid residues, written from left to right and in the direction from the amino end to the carboxy end, are represented by formula VI:

wherein R a-x ^, R g*-" and the specific amino acid residues in parentheses are as previously indicated.

The monomeric, composite LT/ST polypeptide contains at least one intramolecular, intrapolypeptide disulfide bond, preferably two intramolecular, intrapolypeptide disulfide bonds and preferably 3 intramolecular, intrapolypeptide disulfide bonds. The disulphide bonds are assumed to be formed between the pairs of Cys residues of R^ and Rg, and and R^ as well as between the Cys residues of R3 and R^ when a-f have the value 0.

However, the Cys residues of R and R' as well as the Cys residues ap R and R^ are adjacent pairs. Consequently, composite LT/ST polypeptides containing one disulfide bond can have essentially the same secondary structures and antigenicities regardless of whether a single disulfide bond is formed between Cys-1 5 2 5

the residues of R and R or of R, and R . Similar results

a J e b 3 e

relates to secondary structures formed due to disulfide-14 2 formation between the Cys residues of R& and R^ rather than R^ and R^ and the like.

A composite LT/ST polypeptide that is synthesized before

the oxidative formation of an intramolecular, intrapolypeptide-disulfide bond contains at least two Cys residues so that the 7-12 value of at least two of g-1 is 0 and the corresponding Rg_^ ~ groups are absent. In light of the similarity of the secondary structure provided by the formation of an intramolecular, intrapolypeptide cystine disulfide bond between one of two adjacent Cys residues and another Cys residue, it is added

hold that at least one pair of non-adjacent Cys residues from the 7-12 Cys residues preceding the Rg_^ groups is present.

This pair is selected from the group consisting of Cys residues preceding R7,R*?and R9,k\°;R7,R?ogr}1;and R9, R10

■^2 gn in j g n k 13

and R^ . Expressed by the amino acid residue positions in ST parts of ST-containing groups, the pairs of non-adjacent Cys residues

in synthetic ST moieties selected from the group consisting of those numbered 5 or 6 and 9 or 10, 5 or 6 and 14, and 9 or 10 and 17 from the amino terminus of an ST 18 residue polypeptide moiety.

The composite LT/ST polypeptides and network polymers containing the composite LT/ST polypeptides as repeating units are antigens to sera (antibodies) elicited by both the LT B subunit (as well as by the entire LT toxin).

and biological, natural ST. Such anf^l^^ni^ijbejt^r are discussed in detail in Results, section VI, below.

Broadly speaking, the percentage of antigenicity of a composite LT/ST polypeptide containing a synthetic ST sequence as well as an LTB sequence is, however, a relative measure of the amount of anti-biological ST antibody that recognizes (binds to) a synthetic ST compared to biological ST recognized by the same anti-biological ST antibodies, and the correspondingly measured anti-LTB antibody that recognizes (binds to) a synthetic LTB polypeptide part compared to LTB recognized by the same anti-LTB antibodies . Antigenicity calculations are based on the weight of antigens used, and are independent of whether the investigated antigen is in monomeric or polymeric form.

Preferred LTB polypeptides, composite LT/ST polypeptides and network polymers containing LTB polypeptide repeat units such as composite LT/ST polypeptide repeat units exhibit at least 5% of the antigenicity of the natural LT B subunit. Composite LT/ST polypeptides and network polymers containing composite LT/ST polypeptide repeating units also preferably exhibit at least 10% of the aQtigenicity of native ST.

Suitable a^t^iger^sxite^t and ijm^ncjjejii^ also been found for synthetic ST-containing molecules in which the sulfur atoms of Cys residues comprise parts of bonds different from cystine disulfide bonds. Because of this fact, the Cys residues of the above sequence of formulas II and VI for synthetic ST portions of composite polypeptides are shown as attached to Ra_^ groups whose identities are indicated hereinafter.

It should be noted, however, that because at least one intramolecular, intrapolypeptide cystine disulfide bond is required for antigenic activity in the monomeric synthetic ST, and biological activity when desired, not all 6 of the R^j"^ groups other than hydrogen may exist present in a synthetic ST polypeptide. Rather, a maximum of only 4 of these groups can be present in each molecule. Where thus

e.g. The Cys residues of R 1 and R 5 are combined during formation

a e

of an intramolecular, intrapolypeptide cystine disulfide bond, the values of "a" and "e" are equal to 0, the R"<*>" and R^ groups are from-2 3 4 6 ^ e

being and only R^, Rc, R^ and Rf may be present in a monomeric, synthetic ST molecule. Where "a" and "e" are 0, "g" and "k" are equal to 0, and Rg and R^° are absent.

To account for the presence of 1, 2 or 3 intramolecular disulfide bonds of cystine residues formed among the 6 Cys residues, each of the R groups 1-6 has also been labeled with an index letter a-f. Each index letter represents an integer with a value of 0 or 1. For more preferred embodiments, the caveat is added that "e" is 0 when "a" is 0, "d" is 0 when "b" is 0, and " f" is 0 when "c" is 0, with the further proviso that at least one of "a", "b" or "c" must be 0, and with the further proviso that a disulfide bond is present between the respective pairs of Cys -residues for which an index value of 0 requires that another index value must also be 0.

Each of the R 1 , f groups present in the synthetic ST may be hydrogen. In such a case, the Cys residue is

1—6

to which R 3. — x ^-group"~en is bound, unsubstituted accordingly

hydrogen is a normal group bonded to the sulfur atom of a Cys residue. The presence of hydrogen bonded to the sulfur atom of

a cysteine is indicated here by the designations Cys or CysH.

The R 1 groups can also be alkyl groups containing 1 to 4 carbon atoms. Examples of such R 1 — 6^ groups are

cl—X

methyl, ethyl, propyl, i-propyl, n-butyl, sec.-butyl and the like. The 1-6 Ra_^ groups can further be substituted alkyl groups containing 2 to 4 carbon atoms in which the substituents include hydroxy, carboxy and carboxamido. Examples of such substituted alkyl groups are 2-hydroxyethyl, 2-hydroxy-propyl, carboxymethyl (-CH2CO2H), carboxamidomethyl (-CH2CONH2), carboxyethyl (-CH2CH2CO2H) and carboxamidoethyl (-CH2CH^CONH2).

The R ^ groups may be present separately in a co-

a 1-6

set LT/ST polypeptide molecule, or mixtures of RSlx,. groups may be present in a polypeptide or polypeptide repeating unit. When all index letters a-f in a monomeric ST polypeptide-containing unit have a value of 0, the R"'"^ groups are absent, and 3 intramolecular, intrapolypeptide-cystine-disulfide bonds are present in an oxidized polypeptide.

The index letters a-f can also all have the value 0, and the 1-6 Ra ^ x^ groups are absent in a network polymer containing ST polypeptide repeating units in which intramolecular, interpolypeptide-cystine-disulfide bonds between and/or among synthetic ST polypeptide-containing parts of repeating units are present. Intramolecular, intrapolypeptide cystine disulfide bonds within the synthetic ST-containing portions of repeating unit parts may also be present in a network polymer.

On average, the ST polypeptide repeating units in a network polymer contain at least two such interpolypeptide cystine bonds per repeating unit. Accordingly, in preferred practice, at least 4 of a-f and 4 of g-1 have a value equal to 0 for such polypeptide repeating units, and at least 4 R"*" ^ groups and 4 corresponding R ^ groups are absent due to the formation of at least two interpolypeptide cystine disulfide bonds, and typically one intrapolypeptide bond.

Antigenicity and immunogenicity can also be obtained using composite LT/ST polypeptides containing at least one intramolecular intrapolypeptide cystine disulfide bond between pairs of Cys residues such as those shown in formulas II and VI as attached to R and R , R . 2 and R 1 , or R 3

6 and R^, corresponding to the positions numbered 5 and 10, 6 and 14 and 9 and 17 from the amino end of the ST Ibh or ST Ibp polypeptide parts, when the Cys residues not included in the disulfide bond are replaced with the same or different alternative amino acid residues, such as Ser -remains.

The preferred alternative amino acid residues to the Cys residues of formulas II and VI contain neutral side chains and thus impart no ionic charge to the synthetic polypeptide when the synthetic polypeptide is dissolved in an aqueous solution of physiological pH values; i.e. that the preferred alternative amino acid residues are free of ionic charges when they are part of a composite LT/ST polypeptide and are in aqueous solution. The amino acid residues alanine (Ala) and serine (Ser) are examples of preferred alternative amino acids that can be used to replace Cys residues.

The alternative amino acid residues to the non-disulfide-bonding Cys groups are illustrated in the formulas II and VI above by the bracketed groups R 7 , R,8,

9 10 1112 ~o

R^, R^, R^, and R^, each of which may replace the preceding Cys residue (the adjacent Cys residue toward the amino terminus), and wherein the indices g-1 are integers having the value 0 or 1. In preferred synthetic ST polypeptides are if "a" is equal to 0, "g" and "k" are equal to 0; if "b" is 0, "h" and "j" are each equal to 0; and if "c" is equal to 0, then "i" and "1" are each equal to 0.

The description given above in relation to the groups 16 7 12

R 3. -RX _ and R -Rx, , also apply to composite LT/ST polypeptides containing ST polypeptide moieties including the carboxy-terminal 14 amino acid residues shown in formula II, and the more preferred synthetic ST-containing composite LT/ST polypeptide containing 18 amino acid residues whose sequence is shown in formula VI. This description also applies to composites

LT/ST polypeptides whose amino acid residue sequences correspond to

the carboxy-terminal 14 residues of ST and additionally includes at their amino terminus 1, 2 or 3 residues from the carboxy terminus of the sequence illustrated in formula III linked to the amino terminus of the 14-residue polypeptide in their naturally occurring sequences as shown. Thus, the above description also applies to a composite LT/ST polypeptide whose amino acid residue sequence corresponds to the 14 carboxy-terminal ST residues plus a further Tyr, PheTyr or ThrPheTyr peptide bound to the amino end of the 14 residue ST sequence.

A composite LT/ST polypeptide according to the invention can have its LTB polypeptide part bound at the amino-terminal or carboxy-terminal end of the ST polypeptide. The term "composite LT/ST polypeptide" is intended to include both arrangements of the LT polypeptide portion relative to the LT polypeptide portion.

As previously stated, the LT and ST polypeptide portions are linked via a peptide bond. In particularly preferred embodiments, the two polypeptide parts of a composite LT/ST polypeptide are peptide-bonded directly to each other. Thus, the carboxy-terminal residue of one polypeptide part is peptide-bonded to the amino-terminal residue of the other polypeptide part. l 1 in.ng-. Composite polypeptides containing both amino-terminal and carboxy-terminal LTB polypeptides are illustrated in the following.

In the previously seen forms, the ST and LTB polypeptide parts are separated by an electrically bonded amino acid residue which is typically added to the body of ^the- composite polypeptide, a conjugate of which it may be a part of, or a network polymer of which a composite LT/ST polypeptide is a repeating unit. The ^g_ST_ pply peptide moieties are not present in the LTB or ST sequence,

and e^L^d^^s-o^mjT^Lr^idejcjeder which provides an ionic ladiiijvg^jti]^^ compound LT/ST poiypepticT at the pH of the gut, dismal pH 6.5. Such amino acid residues thus contain acidic or basic side chains and are examples

fused at Asp or Glu, and Lys or Arg.

When two additional amino acid residues separate the LTB and ST polypeptide parts of a composite LT/ST polypeptide,

the two residues need not be the same, but these residues provide the same ionic charge to the composite LT/ST polypeptide at pH 6.5. Thus, two Glu residues, two Asp residues or one Glu and one Asp residue can be used together. In a similar way, two Lys residues, two Arg residues or an Arg-

and a Lys residue are used together. However, a Glu and an Arg or an Asp and Lys are not used together.

The amino- and/or carboxy-terminal ends of a composite LT/ST polypeptide (a) as a conjugate when linked to a carrier, (b) free or (c) preferably as a repeating unit of a network polymer, may also include 4 additional amino acid residues that provide ionic charge

(providing acidic or basic side chains) as previously described. Preferably, two such external

lower residues, and again these residues are preferably those containing basic side chains, i.e. Arg and Lys.

A composite LT/ST polypeptide according to the invention can thus be described by formula VII in the following:

Formula VII

wherein X, Y and Z when present are amino acid residues which are (a) selected from the group consisting of Lys and Arg residues, or (b) selected from the group consisting of Asp and Glu residues;

"n" and "m" are integers with a value of 0, 1, 2, 3, or 4, such that the respective X and Y are absent when one or both of "n" and "m" have a value equal to 0, while the respective X and Y residues are present when one or both of "n" and "m" has a value different from 0, where the average number of X and Y residues present per poly-

peptide equals the values of X and Y;

"r" and "s" are integers with a value of 0, 1, or 2 such that the respective Z is absent when one or both of "r" and "s" have a value equal to 0, while the respective Z is present when "r" and "s" are present, where the average number of Z residues per composite LT/ST polypeptide molecule or repeating unit is equal to the value of "r" or "s", provided that when one of "r" or "s" is greater than 0, the other of "r" or "s" is 0 , and the respective Z if "r" or "s" is 0, is absent;

"o" and "p" are integers with the value 0 or 1, so that the corresponding A is absent when one or the other of "o" and "p" has a value equal to 0, provided that "o" and "p " not both can have the same value;

A is a polypeptide containing 10 to 35 amino acid residues corresponding in sequence to the amino acid residue sequence from position 35 to position 95 from the amino terminus of the B subunit of the heat labile enterotoxin of Escherichia coli, wherein the position numbers include the 21 residue signal polypeptide of the indicated B subunit ; and

B is a polypeptide containing up to 18 amino acid residues corresponding in sequence to at least the carboxy-terminal 14 residues of the heat-stable enterotoxin of Escherichia coli.

The polypeptide sequence indicated as "A" in formula VII may be any of the LTB polypeptides indicated herein. As previously stated, however, includes preferred

LTB amino acid residue sequences 15 to 30 residues corresponding in sequence to position 55 to position 85 from the amino terminus of the LT B subunit. Preferably, the LTB sequences contain 20 to 25 amino acid residues corresponding in sequence to position 60 to position 85 from the amino terminus of the LT B subunit.

Examples of LTB polypeptides are illustrated in section Results (VI) below.

The polypeptide sequence indicated as "B" in formula VII may be any of the ST polypeptides described herein. As previously indicated, the ST polypeptide portion contains it

18 residue sequence of either ST Ibh or ST Ibp which can also be defined as the carboxy-terminal 18 residues of an ST polypeptide as the 72 residue sequence encoded by the ST Ia genome ends with the same 18 residue sequence as ST Ibp. In addition, the preferred ST polypeptide portion contains at least 4 of a-f and 4 g-1 which is 0. Preferably, the ST polypeptide portion is free of 1-6

R a—x^ groups bound to Cys-mercaptans (sulfur atoms), and

in the final polypeptide or polypeptide repeating unit used, essentially all the Cvsn residues are present in ox ydroform as cystine residues which form intramolecular, intrapolypeptide disulfide bonds in monomeric composite LT/ST polypeptides, or which form intramolecular, intrapolypeptide and interpolypeptide disulfide bonds in network polymers containing composite LT/ST polypeptides as repeating units. S^åjhadjss^j^ preferably all of a-f and g-1 equal to 0,

Monomeric composite LT/ST polypeptides are typically produced by air oxidation of a reduced, composite LT/ST polypeptide-containing oxidation medium containing the polypeptide in a concentration of less than 0.5 mg per ml (mg/ml). Preferably, the concentration is 0.1 to 0.25 mg/ml. Lower concentrations can be used, but typically give too little of the monomeric, oxidized compound LT/ST polypeptide for it to be useful for most purposes. Reduced amounts of monomeric polypeptide are produced to concentrations above 0.25 mg/ml,

Oxygen present in the surrounding air is preferably used as an oxidation agent. Other oxidants such as hydrogen peroxide and ferricyanide ion can be used, but are not preferred.

The oxidation is carried out with careful or no stirring until free mercaptan groups can no longer be detected by the Ellman test. [Ellman, Arch. Biochem. Biophys. 82:70-77 (1959)]. They are typically carried out over a period of 1 to 24 hours and are generally completed after 8 hours where 3 intramolecular, intrapolypeptide-cystine-disulfide bonds are formed. 2 or 1 intramolecular, intrapolypeptide bond is typically formed in less than 8 hours.

The reduced, composite LT/ST polypeptide is dissolved or dispersed in an oxidizing medium with a pH value of 7.5 to 10.5. Air oxidations are preferably carried out at a pH value of 7.0 to 9.5. Preferably, the oxidation is carried out at a pH value of 7.5 to 8.0. The oxidation is typically carried out at ambient room temperature, but is preferably carried out at a temperature of from 0 to 25°C.

After the oxidation reaction has ended, the oxidized compound LT/ST polypeptide is collected as in the case of lyophilization.

The dried material thus collected can be used

as it exists or can be purified such as by column chromatography using an ion exchange resin or a gel exclusion matrix as the stationary phase.

Previous studies have indicated that the intramolecular, intrapolypeptide cystine disulfide bonds in the monomeric synthetic ST are found to form between the first and fifth, second and fourth, and third and sixth Cys residues from the amino terminus; and these Cys residues correspond to the residues of ST Ibh numbered 6 and 10, 5 and 15, and 9 and 17 from the amino terminus. These Cys residues also correspond to the Cys residues bound to R and r\ 9 436a e

Rcog R^, and Rcog R^, whose positions from the carboxy end in the 18-residue ST polypeptide are analogous to the carboxy-terminal positions in the 14-residue polypeptide shown in formula II.

The degree of intramolecular intrapolypeptide cystine disulfide bond formation for monomeric synthetic ST polypeptides was found to be in the order of the Cys residues of R,2

4 3 6

and R,, followed by the Cys residues of R and R^, and then followed by the Cys residues of Ra and Rg. Using the numbering from the amino terminus of ST Ibh, the order of disulfide bond formation between the Cys residues was numbered 6 and 10, then 9 and 17, followed by 5 and 14.

The primary and secondary structure of the monomeric synthetic ST Ibh was therefore from the amino end to the carboxy end:

in which the lines connecting the Cys residues represent the intramolecular, intrapolypeptide cystine disulfide bond formed between these residues.

In light of the identity of ST-related amino acid sequences, the similarity between the ST antigenicities exhibited by the ST-containing composite LT/ST polypeptides according to the invention and the previously prepared unsubstituted ST monomeric polypeptides, it is assumed that the secondary structures of the ST parts of monomeric composites LT/ST polypeptides are essentially similar to previously prepared ST polypeptides. Accordingly, for example, composite LT/ST polypeptides containing 3 intramolecular, intrapolypeptide-cystine bonds are believed to have the primary and secondary structures represented by formulas VIIIa and VIIIb.

wherein "LTB-" is any of those previously described

LTB polypeptides peptide-bonded to the amino terminus of ST Ibh or to the carboxy terminus of ST Ibp, and the lines connecting the Cys residues represent the intramolecular, intrapolypeptide cystine disulfide bonds formed between these Cys residues.

Similarly, an exemplary composite LT/ST polypeptide containing two intramolecular, interpolypeptide 7-12 cystine disulfide bonds, and which are free of Rg_i groups, is believed to have primary and secondary structures represented by general formula IX

in which LTB-, the lines connecting the Cys residues, and the Asn- and Tyr-1 5 residues in parentheses and Ra and Rg are as previously described.

An illustrative composite LT/ST polypeptide containing a single intramolecular, intrapolypeptide cystine-7-12 disulfide bond, and free of Rg_]_' groups, is believed to have primary and secondary structures represented by formula X:

wherein LTB-, the line connecting the Cys residues, the Asn- and Tyr-13 5 6 residues in parentheses and R a , R c , R e and R^ xer as previously described.

It should be noted that the LTB polypeptide may be attached to the carboxy terminus of the ST sequence portion of a composite LT/ST polypeptide, that up to two additional residues giving charged side chains at pH 6.5 may be present between the LTB and ST portions of the composite polypeptide, and that from 1 to 4 additional acid or base side chain-containing residues providing the same ionic charge at pH 6.5 may be attached at the ends at one or both of the LTB and ST portions of the composition.

III. Polymers containing LTB polypeptide repeating units

Polymers containing a previously described LTB polypeptide repeating unit are particularly preferred embodiments according to the invention.

Polymers have several advantages. Used as an immunogen, ktewer_ firstly, the polymers are not a carrier protein. Second, the polymers generally exhibit none of the biological activity of either the naturally occurring L.T.B subunit or ST..toxins...n&r,_ s%_e.r._e.n„^ nnbef a11et polymer-repe.ter.ende_en±Le.t. When both LTB and ST polypeptide repeating units are present in a polymer, thirdly, the immunogen units of the polymers are typically improved over similar monomeric materials with the same sequences, particularly for the ST portion of the polymers.

A. Linear polymers

Linear polymers comprise a group of LTB polypeptide repeating unit-containing polymers under consideration. Such materials contain a majority of the previously described LTB polypeptides as repeating units. In reduced, monomeric form, each LTB polypeptide includes an additional Cys residue peptide-linked to its amino terminus, and an additional Cys residue peptide-linked to its carboxy terminus. A polypeptide thus constructed is designated as a diCys-LT poly-j<pe>pii<d-..>

Ox<y>dation of a diCys-LT p olypeptide written in the ej^erXøX gend gives a Xiii.e.er_polymer containing a majority^ of LTB^pjDlypeptide repeats^ . The repeating units of such a polymer are bound together by intramolecular, interpolypeptide-cystine-disulfide bonds formed by the oxidation of the terminal ^ys residues peptide-bonded to the LTB polypeptide repeating units.

When the polymerization medium contains only diCys-LT polypeptides, a homopolymer is formed. If another polypeptide containing only two mercaptan-containing Cys residues such as a previously described ST polypeptide containing 4 Ser residues as alternatives to 4 of the Cys residues is mixed into the polymerization medium, a copolymer is formed.

Preliminary studies have been carried out during application

of a polymer produced by oxidation of a diCys-LT polypeptide whose LTB polypeptide repeating units correspond in the amino acid residue sequence to positions 58 - 83 of LTB (diCys-LTg_gj-). The results of these studies indicated an immunoreactivity similar to that obtained with a composite LT/ST polypeptide of the same LTB sequence as described below.

B. Network polymers

Network polymers containing a previously described LTB polypeptide and ST polypeptide repeating units are particular embodiments of the invention. These polymers are designated as "network polymers" because it is believed that the polymers are extensively cross-linked due to their preparation by oxidation of ST-containing polypeptides that preferably contain 6 Cys residues that can form both intramolecular intrapolypeptide cystine disulfide bonds and ijitjjrjpol^R and thus form a cross-linked, three-dimensional network.

(1) Composite LT/ST polypeptide repeat units

A network polymer according to the invention contains a majority of the previously described composite LT/ST polypeptides as repeating units. The repeating units are linked together by intramolecular, interpolypeptide-cystine-disulphide bonds provided by the oxidized cysteine (Cys) residues.

A network polymer is produced from a reduced (cysteine-containing) form of a previously described composite LT/ST polypeptide that is oxidized with molecular oxygen in ambient air.

A composite LT/ST polypeptide is oxidized to a concentration of more than 0.5 mg/ml, and preferably to a concentration of 1 to 5 mg/ml or higher, to the limit of the solubility of the composite LT/ST polypeptide in the oxidation medium.

Again, the oxidation is carried out with gentle stirring or without stirring, and the contact between the solution (oxidizing medium) and oxidizing air is maintained for a period of from 1 hour to 24 hours, preferably around 8 hours,

or until there are no free mercaptone groups as measured by the previously described Ellman test. Except for the concentration of the reduced form of a composite LT/ST polypeptide, the oxidation to form a monomeric composite LT/ST polypeptide or to form a network polymer with a plurality of oxidized composite LT/ST polypeptides as repeating units is essentially identical.

Molecular weights of the network polymers according to the invention can vary widely. Average molecular weights range from 20,000 daltons (20 kd) to over 1 million daltons.

(2) LTB polypeptide and ST polypeptide repeating units

Another embodiment of a network polymer according to the invention contains a plurality of first _djjCy-s--LT repeating units as previously described, as well as a plurality of other, ST polypeptide repeating units as previously described, wherein the ST polypeptide includes at least the 14 carboxy-terminal residues of ST as illustrated in formula II, and preferably the 18 residues shown in formula VI, and is free of a peptide-linked LTB polypeptide. The polymer is thus a copoJ_yjner_^if the former and other polypeptide repeating units are linked together by cystine-disulfide bonds formed by oxidation of the Cys residues present in each of the repeating units.

The two repeating units may be present in the polymer in a molar ratio of 10:1 to 1:10. Preferably, the repeating units are present in a molar ratio of 5:1 to 1:5. Preferably, the molar ratio of diCys-LT to ST repeat units is from 1:1 to 3:1, since a prejiripped LTB polypeptide such as that corresponding to positions 58 to 83 of the LT PB subunit has been found to be halj/jDaj^ The polymers are typically as or more immunogenic than the natural ST.

It is preferred that the ST polypeptide repeating units 1 - 6

are free of R ..-groups bound to Cys-mercaptans, and that

a—x

substantially all of the Cys groups are present, as discussed herein for the preparation of other polymers containing an ST polypeptide repeat unit, such as for composite LT/ST polypeptide repeat units, due to the desirability of having at least one intramolecular, intrapolypeptide cystine disulfide bond, and two_Cys residues are required for linear polymerization. Thus, two Cys residues are available for the formation of the crosslinks in the network polymer.

These network copolymers are prepared from the reduced forms of the repeating units by following the steps discussed below in section IV for polymer preparation.

(3) Additional LTB polypeptide-containing network polymers

A further group of LTB polypeptide-containing polymers are those copolymers which include repeating units comprised of (i) a diCys-LT polypeptide and (ii) a composite LT/ST poly peptide^ These polymers can be purified ~following the oxidation procedures described in the following, by contacting molecular oxygen present in ambient air, with an oxidation medium (solution or dispersion) containing the reduced forms of a diCys-LT polypeptide and a composite LT /ST polypeptide. The repeating units of such a copolymer are also linked together through cystine-disulfide bonds of the oxidized Cys residues present in the reduced polypeptides.

These copolymers may contain the LTB polypeptide repeating unit provided by a diCys-LT polypeptide and the LT/ST repeating unit provided by a composite LT/ST polypeptide in a molar ratio of 0.25:1 to 5: 1. Preferably, the molar ratios used are 1:1 to 2:1, in the order indicated. The molar ratio between total LTB polypeptide-containing repeating units and ST polypeptide repeating units in such a polymer is thus 1.25:1 to 6:1, and preferably from 2:1 to 3:1.

De±_skaJ^bj^ejrJ<ej3 ^J^^ LTB pj^y.PÆP±ijl^hoJidi^ need to be the same. Preferably, however, both are the same and correspond to a previously described, particularly preferred LTB polypeptide sequence. IV. General synthetic procedure for the oxidation of ST-containing polypeptides and for the preparation of polymers A general synthetic procedure for the preparation of a monomeric material comprising an ST polypeptide such as a composite LT/ST polypeptide, and for the preparation of polymers containing LTB polypeptide repeats units based on the results discussed later and several other determinations, are as follows: (1) a monomeric first polypeptide containing Cys residues in a reduced form is prepared in the substantial absence of oxidizing agent. The first polypeptide includes the amino acid residue sequence of an LTB polypeptide according to the invention, such as a diCys-LT polypeptide or a composite LT/ST polypeptide, with or without the previously discussed additional amino acid residues, and is mainly free of intramolecular cystine-disulfide bonds. (2) The first polypeptide thus prepared is dissolved or dispersed in an aqueous oxidation medium to a concentration of 5 mg/ml, preferably to a concentration of less than 2 mg/ml, and most preferably to a concentration of 1 mg/ml to 0.5 mg/ml for the preparation of a polymer; and to less than 0.5 mg/ml, and preferably 0.25 to 0.1 mg/ml for the production of a monomeric composite LT/ST polypeptide. The pH value of the oxidation medium in which the first polypeptide is dissolved or dispersed is preferably alkaline and less than 10.5, and preferably 7.5 to 10.5. (3) The first polypeptide-containing oxidation medium is then brought into contact with molecular oxygen in the air as an oxidizing agent. The pH value of the medium during the oxidation is preferably 7.0 to 9.5, more advantageously from 7.5 to 9, and preferably from 7.5 to 8.0. The medium is preferably brought into contact with ambient air, with or without gentle stirring. (4) The contact between the oxidizing medium and the air is maintained for a period of from 1 to 24 hours, and preferably from 2 to 8 hours, during the formation of at least one intramolecular, intrapolypeptide or interpolypeptide cystine disulfide bond from Cys-(CysH) residues present . For monomeric, self-assembled LT/ST polypeptides, the at least one cystine-disulfide bond is an intramolecular, intrapolypeptide bond, while for a polymer, the at least one cystine-disulfide bond is an intramolecular, interpolypeptide bond. It is preferred that each ST polypeptide-containing repeating unit of a polymer forms an average of approx. two interpolypeptide cystine disulfide bonds so that polymers with more than two compound LT/ST polypeptide repeating units are formed,

In preferred practice in the case of monomeric composite LT/ST polypeptides, the disulfide bond is formed between the Cys residues preceding the pairs R7 and Rf1, R^ and R^° and 9 12 R. and R^ of formulas II and VI, which correspond to the positions of the residues numbered 5 and 14, 6 and 10 and 9 and 17 from the amino terminus of the ST Ibh or ST Ibp molecule. In more preferred practice, the contact between molecular oxygen and the solution is maintained for a period of time sufficient to form two disulfide bonds, preferably between the above-mentioned pairs of Cys residues, and preferably for a period of time sufficient to form three disulfide bonds, again preferably between the above indicated pairs of Cys residues.

The oxidation is preferably carried out at a temperature of from 0 to 25°C. (5) After the end of the oxidation reaction, the synthetic, monomeric, composite LT/ST polypeptide or polymer is typically collected, such as by lyophilization, and purified by column chromatography.

V. Immunizations and antibodies

A polypeptide conjugate, composite LT/ST polypeptide conjugate or polymer according to the invention will, when introduced into a mammalian host as a unit dose inoculum with an effective amount of polypeptide conjugate, sanimense LT/ST poly-Pspjy^dkpnjAiga-t^eXlej^poXymex in a physiologically acceptable diluent, be i^j^n^d^tJJ^å-Jto a v-_a n£i s±p f f,e r™^ subunits alone, or with the LT B subunit as well as an ST polypeptide, and preferably protect .host animals. a courage

in vivo infection induced by J_. coli that secrete these toxins.

The "effective amount" of polypeptide conjugate, composite LT/ST polypeptide conjugate or polymer in a unit dose depends on a number of factors. Included among these factors are the body weight of the immunized animal and the number of inoculations it is desired to use. Individual unit dose inoculations typically contain 10 µg to 5 mg of polypeptide, composite LT/ST polypeptide or network polymer per kg body weight of the mammalian host. Commonly used unit doses typically contain 0.5 mg per kg body weight of the host. Inoculation methods and amounts in rabbits and rats, with the aim of generating antibodies and providing protection against challenge with viable ETEC, are described below.

Polypeptides that are not repeating units of a polymer are administered as a conjugate of a polypeptide-hapten covalently bound to a carrier. Useful carriers used herein include porcine immunoglobulin G (PIG),

tetanus toxoid (TT) and albus shell hemocyanin (KLH). Additional useful carriers include bovine serum albumin (BSA), human serum albumin (HSA), peanut agglutinin, ovalbumin, curcubin, poly L-(Lys:Glu) and the like.

Physiologically acceptable diluents are well known in the art. Examples of such diluents are distilled or deionized water, normal saline solutions and phosphate-buffered saline solutions (PBS).

The immunizing material or inoculum can be introduced into the host orally or by intravenous, subcutaneous or intraperitoneal injection or the like, using known methods. Adjuvants such as Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA), alum, tetanus toxoid and the like, such as are well known in the immunological arts, may also be included in the inocula as part of the physiologically acceptable diluent, Booster injections may also given if desired, to build up a desired antibody titer in the host's serum.

Exact doses depend on the animal and the polypeptide conjugate, composite LT/ST polypeptide conjugate or polymer used, and can be determined using known challenge techniques.

The term "inoculum" is used here to indicate any such inoculating material. As such, the term includes vajcsins--which are useful in humans and other mammals to effect in vivo protection against enterotoxin-producing E. coli strains. A given vaccine and inoculum may be identical where non-human mammalian hosts are involved, but typically differ where humans are the intended hosts. The reason for this difference is that adjuvants such as CFA are not used in humans, and another adjuvant Plå is used if any adjuvant is to be present in a

human vaccine.

The term "unit dose" denotes physically separated

units suitable as unit doses for animals, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect in conjunction with the necessary diluent or carrier. The specifications for a new unit dose according to the invention

is dictated by and is directly dependent on (a) the distinctive characteristics of the immunogen and the particular therapeutic effect to be achieved, and (b) the limitations of the art in the composition of such active materials for therapeutic use in animals.

Antibodies to a polypeptide, composite LT/ST polypeptide or polymer according to the invention can be used in assays or to treat ETEC infections. The antibodies can be used directly as whole, intact antibodies, or can be processed to form Fab or F(ab')2 parts, all of which are biologically active. The term "antibody" denotes a whole, intact antibody or the idiotype-containing polyamide part of the antibody which is biologically active and which is capable of immunoreacting with or binding to its antigenic ligand, i.e. an intact LT toxin, its B subunit and/or natural ST.

To produce antibodies, an immunizing inoculum is introduced as previously described into the host animal as by injection. The host is maintained for a sufficient period of time for antibodies to be elicited, usually 1 to 4 months. The desired, formed antibodies are then attached from host fluids. The whole antibodies thus formed can be used directly, or these can be cleaved with pepsin or papain, as is well known, to form FCab'^-

or Fab parts that can be used. The antibodies produced in this way can also be used as therapeutic agents for passive immunoprophylaxis,

WE. Results

A. Antigenicity of LTB polypeptides

A series of polypeptides were synthesized containing different lengths of amino acids from different regions of the 124 amino acid sequence of the LTB subunit. 3 exemplary polypeptides that had more than 5% of the antigenicity of native B subunit as determined by B/B ELISA (described in the Materials and Methods section) are shown schematically in Fig. 1. Values determined by GM^/B ELISA (also described in the section Materials and methods) were very similar to those obtained by B/B ELISA in each case.

2 LTB polypeptides were sequentially synthesized together from the amino terminus of the 18 amino acid residue sequence of ST Ibh forming composite LT/ST polypeptides. Oxidation of the composite LT/ST polypeptide to form a network polymer with composite LT/ST polypeptides as repeating units increased the antigenicity of the B-subunit polypeptide portion in that the B-subunit antigenicity of the sequence corresponding to positions 35 to 55 of LTB increased to about. 49%, and that the sequence corresponding to positions 58 to 83 rose to 95%

of that of native B subunit (Fig. 1). The sequence of the two assembled LT/SJL_polypeptides is shown below, from left to right and in the direction from the amino end to the carboxy end, represented by formulas Xla and Xlb: Formula Xla

AsnThrGlnlieTyrThrIleAsnAsoLysIle

,/ ST

LeuSerTyrThrGluSerMetAlaGlyLys|ksnThrPhe TyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsn

Formula Xlb

MetValllelleThrPheMetSerGlyGlu ThrPheGlnValGluValProGlySerGlnHisIleAsp

Lr9l SC

SerGlnLys&snThrPheTyrCysCysGluLeuCysCysTyr ProAlaCysAlaGlyCysAsn

The composite LT/ST polypeptide of formula Xlb was selected for further evaluation alone, attached to a carrier as a conjugate and as a repeating unit of a network polymer.

B. Immunogenicity of monomeric/composite LT/ST polypeptides

The immunogenicity of the composite LT/ST polypeptide

of formula Xlb was first determined in its monomeric form by immunization of rats with this material given either alone or conjugated to a high molecular weight carrier, porcine immunoglobulin G (PIG). The polypeptide was conjugated to PIG using glutaraldehyde as a coupling agent, using a polypeptide/PIG ratio of 2.35 to 1 by weight

basis. The resulting conjugate contained 58% composite LT/ST polypeptide by weight. The antigenicity of the composite LT/ST polypeptide was not affected by the conjugation reaction so that 1 milligram (mg) of the conjugate contained 580 B subunits.

Rats were given intraperitoneal (IP) primary immunization with 200 AU of either native B subunit or the combined

put LT/ST polypeptide, followed by two oral (PO)

boosters with graded AU doses. As shown in fig. 3, the conjugated polypeptide elicited the same titers of intestinal IgA antitoxin against the B subunit and provided only a marginally lower degree of protection against challenge with the viable LT+/ST strain than was obtained with native B subunit,

As expected, immunization with low molecular weight, unconjugated composite LT/ST polypeptide failed to elicit an antitoxin response or to confer protection. These promising results with the conjugate led to the preparation and characterization of the properties of the composite LT/ST polypeptide in polymerized form.

C. Properties of a network polymer containing co-

inserted LT/ST polypeptide repeat units

(1) Antigenic! the site

The antigenicity of another network polymer with repeating units corresponding to the sequence of the compound

LT/ST polypeptide of formula Xlb, 57% of that of native B subunit as determined by GM-^/B ELISA and 54% as determined by B/B ELISA? its ST antigenicity was 47% of synthetic ST as determined by ST/ST ELISA (described in the Materials and Methods section).

(2) Toxicity

The network polymer exhibited a negative response in the Chinese hamster ovary (CHO) assay [Guerrant, Infect. Immune. 10:320-327, (1974)] at the maximum tested dose of 100 micrograms (µg) which is 5 million times more than the minimal dose of LT [20 nanograms (ng)] required to elicit a positive response by this provision. The network polymer exhibited a negative response in the lactating mouse assay [Giannella, Infect. Immune. 14:95-99,

(1976)] at the maximum tested dose of lOOyUg which is 20,000 times more than the minimum effective dose of 5 ng of natural or synthetic monomeric ST in this assay.

Administration of 1 mg of the network polymer failed i

to induce fluid secretion in ligated rat ileum loops [Klipstein, Infect. Immune. 40:924-929 (1983)]. This value is 5.9 million times greater than the ED50 (half the dose that produces maximum secretion) of LT [170 picogram (pg)]

and 500,000 times greater than the ED^Q dose of ST (2 ng) in this assay.

(3) Immunogenicity

The results of immunization of rats with the network polymer as an immunogen in a vaccine were compared with those obtained by immunization with either native B subunit or synthetic ST chemically cross-linked to native B subunit. These results are shown in fig. 4.

Based on ELISA determinations, the network polymer-containing vaccine was considered to contain 500 AU of both ST and B subunit. All rats received IP primary immunization with 200 AU followed by two PO booster immunizations with graded AU doses,

A total PO dose of 4,000 ST AU of the network polymer-containing vaccine induced the same level of intestinal

nal IgA ST antitoxin titers and of the same degree of protection against challenge with the viable LT/ST<+> strain that was raised

achieved with a total PO dose of 3,000 ST AU in the chemically bound synthetic ST/natural B-subunit conjugate [ST(S)XB]. One^

however, a total PO dose of 6,000 B-subunit AU of the network polymeV-containing vaccine was required to provide the equi--!' valent antitoxin response and degree of protection against challenge

ring with the LT /ST strain as 2,000 AU of natural B-subenhejJ. a These observations indicated that the immunogenicity of the ST polypeptide portion of the network polymer immunogen was approximately the same as that of synthetic ST alone, but the immunogenicity of the LTB polypeptide portion of the network polymer immunogen was between one-half and one-third of the immunogenicity

of the natural B subunit.

The immunogenicity of the network polymer-containing

vaccine was also determined in rats immunized exclusively PO,

given at 4 week intervals. Rats typically differ from rabbits in that they require larger PO doses of toxins or cross-linked toxoid vaccines in the absence of parenteral primary immunization [Klipstein and Engert, Infect. Immune. 31:252-260

(1981); Klipstein et al., Infect. Immune. 40:924-929 (1983);

and Klipstein et al., Infect. Immune. 40:888-893 (1983)].

The total PO dose that was required to form intestinal

IgA antitoxin titers to 128 and provide strong protection in rats,

Was 4,000 AU of synthetic ST (chemically cross-linked to

natural B subunit) and 5,000 AU of B natural subunit,

or roughly double what was needed with the IP/PO solution. This is shown by the data in fig. 5.

Based on this observation plus the fact that the immunogenicity of the LTB polypeptide portion of the network poly-

the lake was approx. half the immunogenicity of native B subunit, rats were immunized with a total PO dose of 20 mg of synthetic vaccine containing 10,000 AU of ST and B subunit. This elicited the same antitoxin response and degree of protection as was achieved by immunization with synthetic ST or natural B subunit.

The observation that the LTB polypeptide portion of the network polymer immunogen was less immunogenic than native B-

subunit, was surprising as previous results indicated that the antigenicity, as determined by ELISA, closely matched the immunogenicity as determined by the response to immunization in laboratory animals [Klipstein et al., Infect. Immune. 40:924-929 (1983); and Klipstein et al., Infect. Immune.

44:268-273 (1984)]. The reason for this discrepancy is uncertain, but it is feared that the relatively lower immunogenicity was due to the fact that the amino acid residue sequence of the LTB polypeptide portion of the immunogen was based on that described for the porcine B subunit [Dallas and Falkow, Nature 277:406- 407 (1979)], while immunized rats were challenged © , . with a human LT-producing _E_. coli strain. J^l

First, the amino acid residue sequence of %^ porcine and human B subunits differs only by 6 amino acids where the

enestce_f.orskj.ell in the area from sjti j_li_ng_5J3__til_stj.Ili ng 83__e.r_ at ^4^-s^till.ingen that contains en_Met i,. The B subunit._from_ swine jD_g ^en^.Light i^djeri^umane ,. B subunit [Yamamoto et al.,

J, Bacteriol. 152:506-509 (1982)]. Although pork and

human LT contains shared and distinct antigenic deter-

minants [Honda et al., Infect. Immune. 34:337-340 (1981)]

where the differences lie in the B subunits [elements, Infect.

Immun, 38:806-809 (1982)], it has secondly become

reported that immunization with either human or porcine

native B subunit provided similar levels of protection against homologous or heterologous LT toxins and viable strains producing these toxins [Klipstein et al., Infect.

Immun, 40:924-929 (1983); and Klipstein et al., Infect. Immune. 43;811-816 (1984)].

It seems more likely that the observed discrepancy is due to other factors. One of these factors may be the configuration of the polymeric form of the synthetic vaccine, as

kyl s te r k t_-.PAYi.r.ker^ its iiimiunogeni^ity^ [Klipstein et al.,

Infect. Immune. 44:268-273 (1984)], and the configuration

of the B-subunit polypeptide containing amino acids from still

ing 58-85, was affected depending on whether it was alone or

bound to ST, or whether the entire immunogen was in monomeric or polymeric form.

A second possible factor may be suboptimal solubility properties of the vaccine immunogen. The network polymer becomes insoluble at pH values of less than 7.0 which is marginally more alkaline than the contents of the intestines in some cases. j

Preliminary j^iÆX-Jiax^v ist that immunogejTlai.te-t&n—of f^- J [ the synthetic Vaccine can økj^__ypd—a-ci-roå-rd Pi-h-r^ri' ng a v Hpnrp simultaneously with bicarbonate to øjce__alkalini the teat in the intestine. In addition, as discussed herein, the amino acid residue sequence of the synthetic composite LT/ST polypeptide can be affected by the addition of acidic or basic side chain-containing residues not present in the LTB or ST polypeptide sequences that provide an ionic charge at the pH of the intestines (approx. pH 6.5) to help dissolve the synthetic immunogen in the intestinal tract.

D. Additional antigenicity studies

The data in Table 1 below illustrates the antigenicities of synthetic polypeptides corresponding to positions of the LTB protein sequence from position 35 to position 95 from the amino end, and includes the 21 residue signal peptide in the numbering. The data are for porcine LTB polypeptides:

(a) alone; or

(b) as a composite LT/ST polypeptide repeat

unit' of a network polymer (polymer). Antigenicities were measured using B/B and GM^/B ELISA assays for LTB polypeptides and ST/ST ELISA assay described below in the Materials and Methods section.

The above results illustrate the antigenic efficiency of a polypeptide and various network polymers containing composite LT/ST polypeptides as repeating units.

E. Jmmuno<g>enisity/ antigenicity studies

Additional antigenicity studies were also performed with antibodies raised against immunogens composed of LT^B polypeptides conjugated to tetanus toxoid (TT) and albus shell hemocyanin (KLH) as carriers. The immunogenic conjugates dispersed in a physiologically acceptable diluent were introduced here as a vaccine in unit doses in rabbits as host animals,

The hosts were maintained for a sufficient period of time to elicit LT B polypeptides over a period of 4-5 weeks. The animals were then bled, and antibody-containing antiserum to the immunogenic polypeptides was obtained.

Half of the maximal binding titers for each antiserum were then determined by ELISA using either the polypeptide immunogen or the natural LT P B subunit as antigen. Details of the procedures are given in the Materials and methods section. The results of these determinations using tetanus toxoid as carrier are shown in Table 2.

^"Results are reported for antiserum dilutions

(titer), e.g. 1:1280, at which half maximal binding was observed. Two rabbits were used for each determination. Titers separated by a semicolon (;), indicate that titers from both animals differed significantly. Titers separated by a hyphen (-), indicate that half of maximal binding was observed between the two dilutions indicated.

<2>"Amino acid residue sequence corresponding to the specified still-

ings from the amino terminus of the LT PB subunit. The specific amino acid residue sequences used can be obtained under

view to fig. 1 or table 6 in subsection I below.

<3>The antigenicity of the immunogen towards the polypeptide.<4>The antigenicity towards natural LT^B subunit.

^Repetition of the immediately preceding provision

using antiserum from a second preparation of immunogen.

The antigenicity of an oxidized, composite LT/ST polypeptide monomer containing the indicated LT PB polypeptide that was peptide-linked to the amino terminus of the 18-residue ST-Ibh polypeptide, and linked as a conjugate to TT.

The results shown in Table 2 illustrate that poly-

the peptides and composite LT/ST polypeptides according to the invention

Eisen are immunogenic and are also antigenic. J3i sse polypeptides 1 are the immunoae by_a±_dÆ—e-r—:-^—s-fe-a-nd-4-i-l—jL.fx.emka.Ll_ e dannels.e of antibodies as m immunorea. aers with (binding.S—t_L-U—-de-t— n natural L. T^^--Ji^_sjibejihjetprc^ The polypeptides are anti-genij^ke^yed^that they immunoreact with antibodies that they have elicited.

A similar study was carried out using conju-

gates containing KLH as a carrier for the composite LT/ST polypeptide, but using only one rabbit as host. The results of this study are shown in table 3 below.

As can be seen from the above results, the use of KLH as a carrier similar to T.T.~.foxJaeder to^E^J^nTs^ of an£Ts"tof fejie^fr_emka_lt for the immunizing polypeptide, while it will reduce the antigenicity of the antibodies against the natural LT^B subunit.

It should be noted that the results in tables 2 and 3 differ from those shown in table 1 or in fig. 2 in a meaningful way. The results in Tables 2 and 3 show a direct interaction between antibodies elicited by the polypeptide conjugate and composite LT/ST polypeptide conjugates according to the invention with the natural LT^B subunit protein molecule. The antigenicities illustrated in table 1 and fig. 2, shows the immunoreactivity of antibodies raised against the native LT^B subunit protein with a polypeptide or composite LT/ST polypeptide.

It is known that antibodies formed against an intact

protein, frequently does not bind to polypeptides that correspond in amino acid residue sequence to antigenic determinants of the protein. See e.g. Shinnick et al., Ann. Fox. Microbiol. 37:425-446 (1983). A failure to expel bond-

ing, or a low binding titer in GM, ELISA-

/B or B/B

the investigations can thus only indicate that antibodies develop

called by the whole natural LT^B subunit does not recognize a polypeptide or composite LT/ST polypeptide of the invention.

Details regarding immunization systems and ELISA

studies are indicated in the Materials and methods section.

F. Vaccine reproducibility

A further study was carried out to determine the reproducibility of the results obtained during use

of vaccines produced using a network

polymer according to the invention dispersed in a physiologically acceptable diluent. The network polymers used were two preparations of polymer containing a majority of composite LT/ST polypeptides linked together with intramole-

chiral, interpolypeptide cystine disulfide bonds provide

brought by the Cys residues of the ST Ibh polypeptide sequence portions of the composite material. The LT polypeptide part of the combined

placed the LT/ST polypeptide corresponding in sequence to position 58

to 83 of the LT B subunit.

P

The results for a polymer preparation and vaccine, hereinafter referred to as Lot 001, are discussed in Section C below, and are shown in FIG. 4 and 5. This material^.^Sl-vakM has also previously been referred to in fig. 4 and 5 as the B^... -ST-<3> vaccine, The second preparation of this polymeric immunogen is hereafter designated as Lot 002. This sample exhibited antigenicities of 53 and 53% of natural LTB antigenicity at B/B and GMj /B determinations, and 48% of ST antigenicity in the ST/ST determination.

The lack of toxicity exhibited by Lot 001 has been discussed previously. Lot 002 showed a negative response in the lactating mouse assay at a dose of 100 µg in each of three mice. Lot 002 failed to induce fluid secretion in ligated rat ileal loops at a dose of 1000 µg in each of two rats. Both preparations therefore mainly showed no biological activity.

Vaccines prepared containing each Lot 001 and Lot 002 were used to immunize rats. The immunization system used consisted of a primary parenteral injection of 600 µg of immunogen in the vaccine followed by two P0 boosters of 4 mg each of each of the two samples.

When tested for medium intestinal IgA, the host antitoxin titers were 64 against the ST and 128 against the LT B subunit for both vaccines. Protection of the thus immunized rats against challenge with viable ETEC strains producing either ST (ST<+>/LT~) or LT (ST~/LT+) enterotoxin is shown in subsequent Table 4.

As can be seen from the data in Table 4, the vaccines were essentially identical in terms of reduction of secretion and in terms of protection of the immunized hosts against the challenge strains of E_, coli.

The vaccine prepared from network polymer Lot 002 was also administered at a higher concentration than is normally used to determine whether any adverse side effects could be observed in the host animals. Here, intraperitoneal injections of 15 mg were given to each of two guinea pigs weighing 200-250 g each, and to each of two mice weighing 12-15 g. The weight and general condition of the animals were studied over a 7 day period. The guinea pigs increased their weight by 40.1 and 47.6 g, while the mice increased their weight by 5.3 and 8.8 g. No adverse side effects were observed in any of the animals.

G. Antigenicity/immunogenicity with varied LT polypeptide chain length

A further study was performed using a series of composite LT/ST polypeptide coupled to tetanus toxoid (TT) as a carrier to determine the effect of the polypeptide chain length of the LTB polypeptide portion of a composite LT/ST polypeptide, the LTB polypeptide being from the region of position 58-83 of the LT B subunit, including the 21 residue signal peptide sequence. These studies were carried out as described previously in section VI E, and in table 2. The results are shown in table 5 below.

The results stated above illustrate that all a\T~de

11-36 residues and with amino acid residue sequences corresponding to positions 58-83 of the LT polypeptide portion were equally effective immunogens in generating antibodies that immunoreact with the natural LT 3 B subunit.

Immunoreactions of antibodies elicited by the above-mentioned composite LT/ST polypeptide conjugates were also examined against ST Ibh and ST Ibp. Although the ST polypeptide portions of the compositions remained the same, improved titers were also observed with composite LT/ST polypeptides containing 20-25 residues in the LT polypeptide portions. The antibodies formed immunoreacted with both ST Ibh and ST Ibp, where the titers against ST Ibh were several times greater than against ST Ibp.

H. Additional polypeptides

A composite LT/ST polypeptide was prepared containing the residues of positions 48-57 (numbered to include the signal peptide) of the LT 2 B subunit peptide linked to the amino terminus of ST Ibh. (The position numbers of this polypeptide correspond to positions 27-36 if the signal peptide amino acid residues are not included). The polypeptide was then oxidized at concentrations of 0.1 mg/ml, 1.0 mg/ml, and 5.0 mg/ml in 0.5 M ammonium bicarbonate buffer using dual oxidation procedures, and at concentrations of 0.1 mg/ml and 0 .2 mg/ml in 0.1 M ammonium acetate at pH 8.

The 0.1 and 0.2 mg/ml concentrations were thought to contain mostly monomeric composite LT/ST polypeptides. The oxidation at 1.0 mg/ml was thought to yield mostly network polymer containing the composite LT/ST polypeptide as repeating units. The 5.0 mg/ml preparation contained essentially only polymer.

Antigenicities determined using the ST/ST assay discussed in the Materials and Methods section yielded antigenicities of: (a) 31.5 and 68.1% for the 0.1 mg/ml oxidations in ammonium bicarbonate buffer; 3 2.9 and 41.9% for oxidations at 0.1 and 0.2 mg/ml in ammonium acetate buffer; (c) 103.3 and 350.0% for the 1.0 mg/ml oxidations in ammonium bicarbonate; and (d) 98.4 and 26 2.5% for the 5.0 mg/ml oxidations in ammonium bicarbonate. The percentages were based on the antigenicity exhibited by monomeric ST Ibh polypeptides which have an antigenicity substantially similar to that of native ST Ibp.

A similar study was performed using another composite LT/ST polypeptide. The sequence of this polypeptide corresponded to position 58-83 (including the signal peptide) of the LT p B subunit peptide-linked directly to the amino terminus of ST Ibh. (The sequence positions of this polypeptide correspond to position 37-62 without the signal peptide residues).

Oxidations in ambient air as above were carried out

at 0.185 mg/ml, 0.25 mg/ml, 0.50 mg/ml and at 0.523 mg/ml in 0.5M ammonium bicarbonate• Oxidations were also carried out at 0.25 mg/ml and 1.25 mg/ml in 0.1M ammonium acetate at pH 8.0. Antigenicities measured as described above were 205, 446,

208, 236, 42 and 13%.

The results of the above two studies illustrate the usability of all the oxidation conditions studied. These results also indicate that materials with improved antigenisi te r b.l.e_o.ppjiådd—y-ed—ox-yd-a-s4Q-n er__i_ ammo n i um - bicarbonate compared to ammonium acetate as an oxy-da.s4©-ns- -'-— buf f ersyst^emT

Composite LT/ST polypeptides containing an LTB polypeptide corresponding in amino acid residue sequence to position 35-55 of the LT^B subunit peptide-bonded directly to the carboxy terminus of an ST Ibh polypeptide, and corresponding in sequence to position 58-83 of the LT^B subunit peptide-linked to the amino-terminus of an ST Ibh polypeptide and further comprising two additional Lys residues peptide-linked to the amino-terminus of the LTB polypeptide were also prepared.

The former of these polypeptides (ST—LT-j^^)

is thus consistent with a compound of formula VII wherein: "n", "o", "r", "s" and "m" are equal to 0;

"p" is equal to 1;

B is a polypeptide containing 18 amino acid residues corresponding in sequence to the carboxy-terminal 18 residues of a heat-stable enterotoxin of Escherichia coli; ie, the ST Ibh polypeptide; and

A is a polypeptide containing 21 amino acid residues corresponding in sequence to the amino acid residue sequence of still-

ing 35-55 from the amino terminus of the B-subunit of the heat-labile enterotoxin of Escherichia coli, including the signal polypeptide, i.e. the LT^ B-subunit.

The other ay these polypeptides (LysLysLTj-^g^-ST) correspond to a compound of formula VII wherein: "n" is equal to 2;

"p" is equal to 1;

"r", "s", "o" and "m" are equal to 0?

X is Light;

Y and Z are absent;

A is a polypeptide containing 27 amino acid residues corresponding in sequence to the sequence of position 58-83 from the amino terminus of the B subunit of the heat-labile enterotoxin of E. coli/comprising the signal peptide; and

B is the ST polypeptide discussed immediately above.

Initial studies with a network polymer with a majority of LysLysLt5g_g3—ST repeating units indicated that solubility was improved over similarly prepared polymers made from a composite LT/ST polypeptide with the same sequence of LTB and ST polypeptides but lacking the additional Lys -remains; and the immunoreactivities for the two polymers were essentially similar.

The remaining composite LT/ST polypeptides for which specific results as conjugates or as repeating units of network polymers are discussed herein correspond to compounds of formula VII wherein: "n", "r", "s", "p" and " m" is equal to 0;

X, Y and Z are absent;

"o" is 1;

A is a polypeptide containing 8-31 amino acid residues corresponding to the amino acid residue sequence of position 35 to position 95 from the amino terminus of the B subunit of the heat labile enterotoxin of E. coli, wherein the position numbers include the 21 residue signal peptide; and

B is a polypeptide containing 18 amino acid residues corresponding in sequence to the carboxy-terminal 18 residues of a heat-stable enterotoxin of E. coli, as previously discussed.

I. Sequences of engineered LTB polypeptides

In subsequent table 6, the sequences of LTB-containing polypeptides that are referred to here are indicated. Each polypeptide is indicated by an abbreviation indicating the index sequence position numbers counted from the amino terminus of the LT B subunit, including the 21 residue signal peptide, and where the polypeptide sequence corresponds to only an LTB polypeptide or to a composite LT/ST polypeptide. A composite LT/ST polypeptide with an LTB polypeptide at the amino terminus is indicated by the abbreviation LT, while a polypeptide with an ST polypeptide at the amino terminus is abbreviated ST — —LI. The amino acid residue sequence corresponding to each abbreviation is shown to the right of the abbreviation and is written from left to right and in the direction from the amino end to the carboxy end. Each ST polypeptide sequence shown below is that of ST Ibh.

VII. Materials and methods

A. Enterotoxin production

Native LT toxin B subunit was purified by chromatographic techniques [elements et al., Infect. Immune. 29:91-97

(1980)] from batch cultures of E. coli strain pDF 87, a transformed K-12 derivative carrying the B subunit plasmid of human E. coli strain H-10407 [elements et al., Infect. Immune. 40:653-658 (1983)]. Synthetic ST Ibh whose sequence is based on the 18 amino acid residue sequence described by Chan and Gianella for human ST [Chan and Gianella, J. Biol. Chem. 256:7744-7746 (1981)], was prepared as described herein by solid phase techniques; this polypeptide alone and unbound to an LTB polypeptide has the same biological properties and immunogenicity as natural ST, but differs in thin-layer chromatographic and electrophoretic properties. Additional synthetic ST-containing molecules were prepared by similar techniques. The synthetic ST was chemically cross-linked to native B subunit using a water-soluble carbodiimide for the conjugation reagent as described in Klipstein et al., J. Infect. Haze. 147:318-326 (1983). The amounts of natural and synthetic immunogen used in vaccines were based on their protein concentrations determined by the method of Lowry et al., J. Biol. Chem. 193:265-275 (1951).

B. Synthesis of polypeptides

Polypeptides were prepared comprising different lengths of amino acid residues from different regions of the porcine heat-labile enterotoxin B subunit (LT 2 B) sequence described by Dallas and Falkow, Nature 288:499-501 (1980). These polypeptides were produced either individually or synthetically at the amino- or carboxy-terminal ends of an ST amino acid residue sequence to form a composite LT/ST polypeptide.

Polypeptides were prepared by the solid phase methods generally described in Houghten et al., Int. J. Pept. Prot. Res. 16:311-320 (1980) and Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1983), and modified as described herein. One

Sam II Automated Peptide Synthesizer (Bioresearch Inc.,

San Raphael, CA) or a Beckman model 990B peptide-

synthesizer (Beckman Instruments Co., Berkeley, CA) was used for the synthesis.

The following discussion is particularly concerned with presentation

of composite LT/ST polypeptides as the synthesis of such polypeptides also describes the procedures for LTB polypeptides that are not parts of a composite LT/ST polypeptide. DiCys polypeptides were also prepared as described in the following

the following with the exception that blocked Cys residues were the first and last residues of the polypeptides.

Typically, the synthesis started with benzhydrylamine resin

(0.57 milliequivalents per g; meq/g) to which a protected carboxy-terminal amino acid residue of a specific sequence such as α-O-benzyl-BOC-aspartic acid was coupled to form the carboxy-terminal ST asparagine after cleavage with hydrogen fluoride. This and subsequent couplings were performed with a 10-fold molar excess of amino-terminal amine-protected amino acid (N-t-butyloxycarbonyl; BOC) using dicyclohexylcarbodiimide as the coupling peptide bond-forming reagent. A 2 molar excess of both reagents can also be used.

Commonly used amino acid side chain protecting groups

were used and are as follows: 0-(p-bromobenzyloxycarbonyl) for tyrosine and lysine, 0-benzyl for threonine, benzyl ester for

in glutamic acid and aspartic acid, S-methoxybenzyl for cysteine, dinitrophenyl for histidine and tosyl for arginine when these are present.

For couplings including protected asparagine, the

an equimolar amount of N-hydroxybenzotriazole was added to the reaction mixture, and dimethylformamide (DMF) was used as a solvent. The couplings were typically found to be greater than 99% complete by the picric acid test according to Gisin Anal. Chem, Act, v58:298-249 (1972). Protected amino acids were recrystallized from suitable solvents before use to form single spots by thin layer chromatography.

The histidine protecting group was removed prior to cleavage from the resin by thiolytic cleavage with 1% thiophenol in DMF.

The amino-terminal BOC group was also removed prior to cleavage from the resin with a final trifluoroacetic acid deprotection step to prevent acetylation of the methionine during the hydrogen fluoride cleavage procedure.

The blocked amino acids were coupled serially to the resin. The amino-terminal BOC group of the last linked amino acid was removed before the addition of the next blocked polypeptide until the desired polypeptide was synthesized, chained (bound) to the resin. The resin-bound polypeptide was then cleaved to form the resin and the polypeptide.

Cleavage of polypeptides from their resins and removal of the remaining protecting groups was accomplished by treating the polypeptide resin [(0.5 g (g) ] with 0.5 milliliters (ml) of anisole and 20 ml of anhydrous hydrogen fluoride (or by treating the polypeptide resin with twice the weight of anisole and 40 times its volume relative to the weight of anhydrous hydrogen fluoride) at 4° C. for 1 hour. After evaporation of the hydrogen fluoride first with a stream of N 2 and finally with suction, the residue was extracted with anhydrous ether three times to remove the anisole and was dried under suction followed by vacuum.

The vacuum-dried material was first extracted with

5% aqueous acetic acid (3 times, 50 ml each time) followed by extractions using 50% aqueous acetic acid

(4 times, 50 ml each time). The first extraction removed low molecular weight polypeptides and tyrosine used in some preparations to protect the Cys mercaptan groups. The second extraction separated the free polypeptide from the resin. After dilution with water to a concentration of 10-20% acetic acid, the resulting solution was lyophilized to form a monomeric, unoxidized, first polypeptide.

The crude polypeptides were mixed with a threefold molar excess of dithiothreitol to keep them in the reduced state and were partially purified by Sephadex^3-50 gel filtration (Pharmacia Fine Chemicals, Piscataway, N.J.) with 0.1 molar (M) ammonium bicarbonate (pH 8.0) as eluent. The first fraction in the hollow volume of the column

(65% by weight of a preparation) was lyophilized or used immediately after collection to prevent uncontrolled oxidation when sulfhydryl groups were present. The amino acid composition of such materials was confirmed and found to be -10% of theoretical.

Preparations of synthetic LTB polypeptides that were not bound to a synthetic ST polypeptide were typically used after the gel filtration purification indicated above. Preparations of composite LT/ST polypeptides were further processed as described below.

In one preparation, a 44 amino acid residue composite LT/ST polypeptide sequence containing B subunit residues 58 - 83 was prepared, linked directly to a synthetic 18 amino acid residue sequence of ST by a peptide bond between the carboxy-terminal Lys residue of LTB residue 83 and the amino-terminal Asn -remains of the synthetic ST part. The monomeric composite LT/ST polypeptide, used as a repeating unit, was oxidized by bringing molecular oxygen (02) present in ambient air into contact with an aqueous oxidation medium containing the reduced LT/ST composite polypeptide , typically present, at 1.0 milligrams (mg) per ml in 0.1M ammonium bicarbonate (pH 8.0) under slow stirring at 22°C for 20 hours to form a network polymer of composite LT/ST polypeptide repeat units linked together by interpolypeptide cystine disulfide bonds. Free sulfhydryl groups were negligible as determined by the Ellman reaction [Ellman Arch. Biochem. Biophys. 82.70-77 (1959)]. Network polymers containing other LT polypeptide sequences were prepared in a similar manner unless otherwise indicated. Preparations of composite LT/ST polypeptides under formation of oxidized monomers was typically performed at 0.1 to 0.25 mg/ml using similar oxidation conditions.

Oxidations to form useful monomeric and network-polymeric composite LT/ST polypeptides have also been performed using 0.5M ammonium carbonate and 0.1M ammonium acetate, the pH of the oxidizing media has also been varied between pH 6.0 and 10.0.

Partial purification of this polymeric material was accomplished by passage through a Sephadex® G-50 (Pharmacia) column equilibrated with 0.1 M ammonium bicarbonate (pH 8.0). The hollow volume was collected and lyophilized. A sort column of

Cr)

SephacrylS^ S-200 (Pharmacia) exhibited a single peak for a network polymer thus prepared with a molecular weight of approx.

2 2,000 daltons.

C. Determinations of toxic properties

The toxic properties of the network polymer immunogen discussed in section VI C(2) were tested by (i) the Chinese hamster ovary (CHO) tissue culture assay for LT activity [Guerrant et al., Infect. Immune. 10:320-327

(1974)], (ii) the lactating mouse determination with regard to ST secretory activity [Giannella, Infect. Immune. 14-95-99

(1976)] and (iii) instillation into ligated rat ileal loops for secretory activity [Klipstein et al., Infect. Immune. 40:924-929 (1983)].

D. Determination of antigenicity

The antigenicity of the synthetic polypeptides was determined by double "sandwich" enzyme-linked immunosorbent assays (ELISA) using for the solid phase/second antibody, either GM^ganglioside (Sigma Chemical Co,, St. Louis, Mo.)/anti- LT B-subunit hyperimmune antiserum (GM^/B), or dual "species" hyperimmune antisera to B-subunit (B/B), and dual "species" hyperimmune antisera to synthetic ST (ST/ST) as previously described [Klipstein et al., J. Infect. Haze. 147:318-326 (1983) and Klipstein et al., Infect. Immune. 44:268-273 (1984)].

Briefly, antibody from an animal species raised against LTB or ST, or the ganglioside was adsorbed onto a microtiter plate well as a solid support, and any excess unbound material was removed. A suitable antigen (LT polypeptide, ST or LTB) was then mixed with the solid support, maintained in contact for a sufficient period of time for binding to occur between the antibody or ganglioside and the antigen, after which any excess of unbound antigen was removed by rinsing. Then an antibody from another animal species (or a first species for the GM^/B assay) was raised against LTB or ST, mixed with the solid carrier-bound antigen. After maintaining the mixture for a period of time sufficient for the mixed antibody to immunoreact, any excess unimmunoreacted antibody was removed by rinsing. The amount of the last added antibody that immunoreacted with the solid carrier-bound antigen was then determined, and a comparison was made between the binding of the last added antibody to the investigated polypeptide and to the control antigen, for example natural LT B subunit.

E. Conjugates of LT and composite LT/ST polypeptides linked to carrier tetanus toxoid

Composite LT/ST polypeptide preparations were weighed out and mixed with an equal amount of tetanus toxoid (TT). The polypeptide and TT were dissolved in phosphate-buffered saline (PBS) (pH 7.2) and diluted to a final concentration of 2 mg/ml vehicle.

The glutaraldehyde cross-linking agent (GA) was first prepared as a 25% stock solution. 200 microliters of the stock solution was mixed with 13 ml of PBS to form a GA working solution, the GA working solution was kept chilled. The working solution was mixed in an amount of 124 microliters (µl) per

ml carrier polypeptide solution.

The GA working solution/carrier polypeptide solution mixtures were stirred for 18 hours (hr) at ambient room temperature. This reaction mixture was then dialyzed against distilled or deionized water for 6 hours and then lyophilized.

The dried, lyophilized material was believed from experiments to include 90% recovery of the carrier. The linked, cross-linked polypeptide typically constitutes 40% by weight of the total recovered material. Similar preparations are described in Klipstein et al., J. Infect. Haze. 147:318-326

(1983).

F. Conjugates of LT and composite LT/ST polypeptides

coupled to albus shells heiaocyanin as a carrier

The LTB polypeptides according to Table 2 were also coupled to albus shell hemocyanin (KLH) carrier to form conjugates. An amino-terminal Cys residue was added to those polypeptides whose sequence does not contain such a residue.

KLH was dialyzed against 10 mil phosphate buffer (pH 7.2), and its concentration was adjusted to 20 mg/ml before use. Solutions of polypeptides to be coupled were prepared at a concentration of 5 mg/ml. Coupling concentrations of 4 mg KLH to 5 mg polypeptide were used.

A desired volume of the KLH solution used above was

selected, and 55 µu/l of the above-mentioned phosphate buffer was added

set per 4 mg KLH, A solution containing m-maleimido-benzoyl-N-hydroxysuccinimide ester (MBS) dissolved to 6 mg/ml in DMF was then added to a volume of 85 µu/l per 4 mg of

KLH forming an MBS:KLH molar ratio of 40:1. The resulting reaction mixture was then stirred for 30 min-

otter at ambient room temperature.

After 30 minutes, the reaction solution was run through

a Sephadex® G-25 (Pharmacia) column containing a layer

volume of approx. 15 ml. The buffer used for preparing the column was 50 mM phosphate at a pH value of 6.0.

1 ml fractions were collected from the eluted column.

Each fraction was read at a wavelength of 280 nanometers

to determine the fractions in which the resulting MBS-

activated KLH (KLH-MB) was located. The KLH-MB-containing fractions were then collected. A recycling on

80% by weight of KLH-MB is assumed in this procedure.

The collected, pooled KLH-MB-containing solution was

then used as a stock solution which was used for coupling to each of the polypeptides. A lot of that time-

previously discussed polypeptide-containing solution (5 mg/ml) was added to a quantity of the recovered KLH-MB-containing solution

ning while forming reaction mixtures containing 4 mg

KLH-MB.

The pH value of the reaction mixtures was adjusted to

7.0-7.5 with NaOH or HC1, and was then stirred at ambient room temperature for 3 hours. The reaction solutions were then frozen and stored frozen until use.

G. Polypeptide conjugates as immunogens and antigens

Immunization and ELISA systems used in relation to the material and results indicated in Tables 2 and 3 were as follows.

Rabbits were injected subcutaneously (SQ) on day 0 with a vaccine containing 1 mg of the above prepared TT or KLH conjugate dispersed in approx. 0.75 milliliters of complete Freund's adjuvant (CFA) and 0.75 ml of phosphate-buffered saline (PBS) with a pH value of 7.2. Animals received booster SQ injections of a vaccine containing the same amount of conjugated polypeptide or composite LT/ST polypeptides contained in 0.75 ml Freund's incomplete adjuvant plus 0.75 ml PBS on day 14, followed by a second intraperitoneal (IP) booster injection of the second vaccine on day 21. Serum samples from the immunized rabbits were obtained from the ear vein on

day 28 and 35 after first immunization.

Two rabbits were used for each TT conjugate, while

one rabbit was used for each KLH conjugate determination.

The natural LT 2 B subunit antigen was dissolved in a TEAN buffer (tris, EDTA, sodium azide, sodium chloride) at pH 4.0 to form a stock solution containing 1 mg/ml. This stock solution was then diluted with a carbonate/bicarbonate buffer of pH 8.0 to form a second stock solution containing native LT-β subunit at a concentration of 10 picomoles/50 µl.

50 µl of the second stock solution was placed in the wells of a microtiter plate, forming 10 picomoles of the antigen in each well. The plate was then incubated in a moist chamber for 18 hours at ambient room temperature.

The wells were then washed three times with a PBS solution containing 0.07% 'Polysorbate' 20 (Tween® 20;

ICI USA, Inc., Wilmington, DE), followed by two washes with deionized water. 100 µl of 1% bovine serum albumin (BSA) in PBS (1% BSA/PBS) was then added to each well to block sites of non-specific binding, and the BSA-containing solutions were maintained (incubated) in the wells for 1 hour at 37°C.

Solutions containing unbound BSA were shaken off

the plate. Without first drying the plate, 9 Cyul of 1% BSA/PBS was added

was added to each well of the top row of wells, while 5C<y>ul 1% BSA/PBS was added to each well in the rest of the plate. Then lCyul antiserum was added to each of the wells in the top row, and the mixture thus obtained

ing was mixed. 5 Gyul of antiserum-containing solution from each well in the top row was then added and mixed with 5 Gyul of 1% BSA/PBS in the lower well, making further two-fold dilutions. Similar serial doubles

in dilutions were continued for the rest of the wells in the plate. in

t After all dilutions had been made, the diluted antiserum containing wells were incubated at 37°C for 1 hour. The liquid in the wells was then removed, and the wells were rinsed as previously described, i

Peroxidase-labeled goat anti-rabbit serum (Zymed Laboratories, Burlingame CA) was diluted 1:3000 on a volume basis with 1% BSA/PBS. 50 µl of the resulting diluted antibody-containing solution was added to each well. The

thus treated wells were maintained (incubated) for 1 hour at 37°C and were then washed as previously described.

A solution of ortho-phenylenediamine (OPD) was prepared by dissolving 1 tablet of OPD substrate (Pittman-Moore, Inc.,

Washington Crossing, NJ) in 6 mL of deionized water to which 1 drop of 3% hydrogen peroxide was also added. 100 µl of the resulting solution was added to each of the microtiter plate wells. Color development in the wells was developed at ambient room temperature and was stopped after 20 minutes by the addition of 25 µl of 4 N sulfuric acid to each well. The amount of dye present in each well was determined by measuring the optical density of the liquid at 492 nanometers.

The amount of antibody in the rabbit serum that was required

to saturate half of the LTB antigen on the plate, there was

after calculated using standard techniques.

H. Immunization procedures

Immunization procedures for the data shown in Figs. 3-5,

were as follows: Sprague-Dawley rats (150 to 175 g) were given primary immunization intraperitoneally (IP) using Freund's complete adjuvant followed by two booster immunizations given orally (PO) at 4-day intervals. Immunization PO was given via an intragastric tube 2 hours after PO administration of cimetidine (Tagamel®; Smith Kline & French Laboratories, Carolina, P.R.) at a dose of 50 mg/kg 50 milligrams/kilogram (mg/kg) body weight to remove stomach acid secretion.

Doses of the chemically cross-linked ST--B subunit conjugate and that the network polymer-containing composite LT/ST polypeptide repeating units are expressed in antigenic units (AU) per mg. Such doses (in AU) were derived by determining, by ELISA procedures, the percentage antigenicity of the conjugate relative to the natural toxin and by multiplying this value by 1000. One AU has the equivalent antigenicity of 1 microgram (^ug) of undiluted natural toxin.

I. Challenge Procedures

The above immunized rats were challenged 4 more times

6 days after the final booster immunization by drip in a single 10 centimeter (cm) wrapped loop of disal-

9

ileum for 18 hours of 0.1 ml of a culture containing 10 viable organisms per ml of E. coli LT /ST+ strain TX 452 (078:H12) of E. coliLT<+>/ST~ strain PB258 (015:H~)

as previously described [Klipstein et al., J. Infect. Haze. 147:318-326 (1983)) and Klipstein et al., Infect. Immune. 40:924-929 (1983)], Each datum was determined in 4 to 6 immunized rats, and the reported results are for the mean value within standard deviation of the mean (SEM) percent decreased secretion in immunized rats compared to the value in 5 of similarly challenged, unimmunized control animals. Reduced secretion of less than 50% represented a significant (P less than 0.00l) difference as determined by

Student<1>'s test for two independent means, between values in immunized and control animals.

J. Antitoxin response

Intestinal secretory IgA antitoxin titers against

ST and B subunit were determined by double sandwich ELISA assays as previously described [Klipstein et al., J. Infect. Haze. 147:318-326 (1983)]. The reciprocal values for mean titers in each group are shown in fig. 3, 4 and 5 as number indications above or below the zero points. VIII. Diagnostic determination systems and methods

The LTB polypeptides, composite LT/ST polypeptides and polymers containing a plurality of LTB polypeptide repeating units, as well as antibodies and antibody combining sites (receptors) formed against the above-described LTB polypeptides, composite LT/ST polypeptides and polymers, and methods according to the invention, can also be used for diagnostic tests such as immunoassays. Such diagnostic techniques include e.g. enzyme immunoassay, enzyme-multiple immunoassay techniques (EMIT), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay, either single or double antibody techniques, and other techniques in which either the receptor or the antigen is labeled with a - visible mark or indicating devices. See generally Maggio, Enzyme Immunoassay, CRC Press, Cleveland, Ohio

(1981); and Goldman, M:, Fluorescent Antibody Methods, Academic Press, New York, N.Y. (1980). Further specific examples of such determination methods and systems which are applicable for carrying out these methods are discussed in the following.

A method for determining the presence of native LT enterotoxin or its B subunit in a body sample is contemplated herein. In a general method, a body sample such as a faeces sample is provided to be determined, and the bacteria present there are preferably cultured. It in cultured bacterial samples or the faeces sample itself is mixed with receptor molecules containing antibody-combining sites induced by an LTB polypeptide-containing molecule according to the invention such as one of the previously described network polymers. The mixture is maintained for a predetermined period of time sufficient for the receptor molecules to immunoreact with enterotoxin present in the culture. The degree of this immunoreaction is then measured to determine where widely enterotoxin molecules were present or absent in the examined body sample.

An illustrative diagnostic system in package form which

constitutes an embodiment of the invention which is applicable for the detection of LT enterotoxin present in a body fluid, contains receptor molecules according to the invention elicited by a polypeptide according to the invention in a container. Ex-

examples of such receptors are antibodies, mainly whole antibodies, or antibody combining sites such as formed Fab and F(ab'.2antibody parts. This system includes

also includes indicator devices for signaling the presence of an immunoreaction between receptor and antigen.

Typical indicating agents include radioisotopes

1v5 131

such as 'I and I, enzymes such as alkaline phosphatase, horseradish peroxidase, |3-D-galactosidase and glucoseoxidase, and fluorochrome dyes such as fluorescein and rhodamine.

The indicating agents can be chained directly to the receptor

according to the invention. The indicating agents can also be linked to a separate molecule such as to a second antibody,

or to an antibody combining site or to Staphylo-

coccus aureus (S, aureus) protein A which reacts with

(binds to) the receptor according to the invention. Specific

125

examples of separate molecular indicating agents are I-

labeled S, aureus protein A and peroxidase-coupled goat anti-rabbit antibodies.

The indicating agents enable the immunoreaction

the product can be detected and packaged separately from the receptor when they are not linked directly to a receptor according to the invention

else. Mixed with a bacterial culture sample, the receptor molecule immunoreacts with the LT enterotoxin to form a

immunoreactarit, and the indicating agents present then signal the formation of the immunoreaction product.

One embodiment of an LT enterotoxin diagnostic method is an immunofluorescence assay. In such a determination, a bacterial cell culture spot is fixed on a clear microscope slide as a solid support. A sample of (receptors) antibodies formed according to the invention, e.g. formed in rabbits, are brought into contact with the glass using well-known techniques. The receptors and bacterial cells are maintained in contact for a period of time sufficient for the receptors to immunoreact with LT.

After washing away any unimmunoreacted receptor according to the invention, any non-specific binding site on the glass is blocked with a protein such as bovine serum albumin (BSA), if desired. A second reagent (an amplification reagent) such as complement, or anti-immunoglobulin antibodies, e.g. guinea pig complement, can then be mixed and maintained in contact with the receptor-bound cells for a period of time sufficient for a reaction to take place (incubate).

After this second incubation, any unreacted amplification reagent is removed by rinsing, leaving the amplification reagent bound to the first-mentioned antibodies on the slide. A third reagent (indicators), e.g.

an antibody such as goat anti-guinea pig complement is then contacted with the glass and its bound materials,

and maintained in contact for a period of time sufficient for a reaction to take place with bound amplification reagent (incubate). The third reagent is labeled by being linked to a fluorochrome dye such as by reaction with fluoroscein isothiocyanate (FITC), rhodamine B isothiocyanate (RITC), tetramethylrhodamine isothiocyanate (TRITC), 4,4<1->diisothiocyanostilbene-2 ,2<1->disulfonic acid (DIDS) and the like which are well known in the art.

Any unreacted third reagent is washed off after this third incubation, forming fluorochrome-labeled goat anti-guinea pig complement antibodies that bind to the complement on the slide. The presence of fluorochrome dye-labeled third reagent can be detected using fluorescence microscopy and thereby signal the presence of infection by an LT-producing strain of E. coli.

This determination can also be carried out without the use of the second and/or third reagent. Here, anti-LT receptors according to the invention are bound directly to fluorochrome dye, and this receptor containing indicating agents is used directly.

A preferred diagnostic system, preferably in package form, applicable for carrying out the above-mentioned determination, includes, in separate packages, (a) receptors (antibodies) according to the invention which immunoreact with the natural LT endotoxin, (b) a second, amplifying reagent such as complement, such as guinea pig complement, anti-immunoglobulin antibodies or S. aureum protein A which reacts with the receptor, and (c) indicating agents which (i) can be chained directly to the enhancing agents, (ii) can be part of a separate molecule such as an antibody or antibody fragment that reacts with the enhancing reagent, or (iii) may be linked directly to the anti-LT receptor,

in which case the kit need only contain a single immunoreactive reagent. The indicating agents indirectly signal the immunoreaction of the receptor molecule and an LT endotoxin, preferably via the mediation of the amplification reagent.

Receptor molecules and separate indicating agents in any diagnostic system described herein, as well as that

amplification reagent described above, can be provided in solution such as a liquid dispersion or as a substantially dry powder, e.g. in lyophilized form. Where the indicating agents are a separate molecule from the enhancing agent, it is preferred that the indicating agents are packaged separately. Where the indicating agents are an enzyme, the enzyme's substrate can also be provided in a separate package of the system. A solid support such as the microscope slide described above, or one or more buffers and acetone, may also be included as separately packaged elements in this diagnostic determination system.

The packages and containers discussed here in relation to diagnostic systems are those commonly used in diagnostic systems. Such packages contain glass and plastic (e.g. polyethylene, polypropylene and polycarbonate), bottles, ampoules, plastic and piast foil-laminated envelopes, etc.

The use of whole, intact, biologically active antibodies is not necessary in many diagnostic systems such as the immunofluorescence assay described above. Rather, only the immunologically active, idiotype-containing, antigen-binding and recognizing receptor site, i.e. the antibody combining site, of the antibody molecule can be used. Examples of such antibody combining sites are those known in the art as Fab and F(ab') 2 antibody fragments which are produced by proteolysis using papain and pepsin, which is well known in the art.

Another diagnostic embodiment of the invention is particularly applicable for competitive determinations, and this system, preferably in package form, includes a first reagent and a second reagent in separate containers. Buffer salts in solid or liquid form, a microtiter plate, indicating agents and the like can also be contained in their own containers.

A first reagent comprises an antigenic LTB polypeptide according to the invention such as a network polymer containing composite LT/ST polypeptide repeating units as antigen. The second reagent comprises receptors such as antibodies that immunoreact with the natural LT B subunit such as those previously described. Means to indicate the presence ay an immunoreaction between the antigen and receptors, signaled by additional, antireceptor, receptors chain

125 to a label such as a radioactive element such as I, a fluorescent dye such as fluorescein or an enzyme such as peroxidase. The indicating agents are either contained in a separate container such as peroxidase-linked goat anti-rabbit antibodies with another separate container for its substrate (eg o-phenylenediamine), or may be part of the second reagent such as where a radioactive element is bound to the receptor molecules. They indicate-

End funds can also be added separately.

Mixing predetermined amounts of the first and second reagents in the presence of a predetermined amount of a sample to be examined such as a faecal sample or a bacterial culture from a faecal sample produces a degree of immunoreaction signaled by the indicating agents . The degree of immunoreaction is different from a known degree of immunoreaction when the enterotoxin or its B subunit is present in the examined sample.

In common practice, an LTB polypeptide-containing antigen is bound as a first reagent to a solid support such as the walls of a microtiter plate or a nitrocellulose dip slide. The non-specific binding sites on the solid support are bound by an unrelated protein such as BSA using known techniques.

Predetermined quantities of the bacterial culture and other reagents are mixed in an aqueous medium. This mixture is maintained for a period of time sufficient for the second reagent to immunoreact with the LT endotoxin that may be present.

Next, the solid carrier-bound first reagent and the bacterial culture- and second reagent-containing aqueous medium are mixed to form a solid/liquid phase mixture. This mixture is maintained for a period of time sufficient for the second reagent present in the liquid phase to immunoreact with the solid phase-bound first reagent.

The solid phase and the liquid phase are then separated to form a solid phase-bound immunoreagent. The amount of solid phase-bound immunoreagent is then measured, and the degree is compared to a degree of immunoreaction that occurs when a known amount of LT enterotoxin is similarly determined.

The use of whole, intact, biologically active antibodies is again not necessary in many diagnostic systems such as the competitive determination described above. Rather, only the biologically active idiotype-containing moiety of the antibody molecule that binds to the antigenic LTB polypeptide may be needed, as discussed above.

The preceding description is intended to illustrate the invention and not to begiriise it. ø^?es—utrer r~å~~a v v' l^^ £' xrar7Tpp^ rTffi-eJ-S-ens—i?afflme.

Claims (25)

1. Synthetic polypeptide containing 10 to 35 amino acid residues corresponding in sequence to position 35 to position 95 from the amino end of the B-subunit of the heat-labile enterotoxin of Escherichia coli in which the position numbers include the 21-residue signal polypeptide of the specified B-subunit. 2. Synthetic polypeptide according to claim 1, characterized in that the indicated polypeptide contains 15 to 30 amino acid residues corresponding in sequence to position 55 to position 85 of the indicated B subunit. 3. Synthetic polypeptide according to claim 1, characterized in that the polypeptide contains 20 to 25 amino acid residues corresponding in sequence to position 60 to position 85 of the specified B subunit. 4. Synthetic, composite polypeptide containing 25 to 55 amino acid residues, characterized in that said composite polypeptide comprises two amino acid residue sequences bound together by a peptide bond, which two sequences are (a) a sequence of amino acid residues corresponding to at least the carboxy-terminal 14 residues of a heat-stable enterotoxin of Escherichia coli and (b) a sequence of amino acid residues corresponding to residue positions 35 to 95 from the amino terminus of the B subunit of the heat labile enterotoxin of Escherichia coli wherein the position numbers include the 21 residue signal polypeptide of said B subunit. 5. Composite polypeptide according to claim 4, characterized in that specified two sequences are linked together with a peptide bond formed between the amino-terminal residue of specified heat-stable enterotoxin sequence and the carboxy-terminal residue of specified heat-labile enterotoxin B-subunit sequence. 6. Composite polypeptide according to claim 5, written from left to right and in the direction from the amino end to the carboxy end, corresponding to the formula wherein the amino acid residues in parentheses are each an alternative to the immediately preceding amino acid residue in the sequence of the formula. 7. Synthetically composed polypeptide, written from left to right and in the direction from the amino end to the carboxy end, corresponding to the formula: wherein X, Y and Z when present are additional amino acid residues which are (a) selected from the group consisting of a Lys and an Arg residue, or (b) selected from the group consisting of an Asp and a Glu residue ; "n" and "m" are integers with a value of 0, 1, 2, 3 or 4, so that the respective X and Y are absent when one or both of "n" and "m" have the value 0, while the respective X and Y residues are present when one or both of "n" and "m" has a value different from 0, where the average number of X and Y residues present per polypeptide is equal to the values ay X and Y; "r" and "s" are integers with a value of 0, 1, or 2, such that the respective Z is absent when one or both of "r" and "s" have a value of 0, while the respective Z is present when "r" and "s" are not 0, where the average number of Z residues per compound polypeptide molecule is equal to the value of or "s" is greater than O, the second of "r" or "s" is 0, and the respective Z if "r" or "s" is 0 is absent; "o" and "p" are whole numbers with the value 0 or 1 such that the corresponding A is absent when one or the other of "o" and "p" has a value equal to 0, provided that "o" and "p" not both can have the same value; A is a polypeptide containing 10 to 35 amino acid residues corresponding in sequence to the amino acid residue sequence of position 35 to position 95 from the amino terminus of the B subunit of the heat labile enterotoxin of Escherichia coli, wherein the position numbers include the 21 residue signal polypeptide of the B subunit; and B is a polypeptide containing up to 18 amino acid residues corresponding in sequence to at least the carboxy-terminal 14 residues of the heat-stable enterotoxin of Escherichia coli. 8. Polypeptide according to claim 7, characterized by "n" is 2; X is Light; "o" is 1; "r", "s", "p" and "m" are 0; A is a polypeptide, written from left to right and in the direction from the amino end to the carboxy end, which corresponds to the formula: and B is a polypeptide, written from left to right and in the direction from the amino end to the carboxy end corresponding to the formula: wherein amino acid residues in brackets in each polypeptide are an alternative to the immediately preceding amino acid residue in indicated preceding sequences. 9. Polymer comprising a plurality of polypeptide repeating units, characterized in that said polypeptide repeating units contain 10 to 35 amino acid residues corresponding in sequence to the amino acid residue sequence from position 35 to position 9 5 from the amino end of the B subunit of the heat-labile enterotoxin of Escherichia coli, in which the position numbers include the 21 residue signal peptide of the indicated B subunit. 10. Polymer according to claim 9, characterized in that specified repeating units are linked together by cystine-disulfide bonds formed by oxidation of Cys residues present at both the amino end and the carboxy end of specified repeating unit polypeptides before oxidation. 11. Polymer according to claim 10, characterized in that the polymer is a linear homopolymer, 12. Polymer according to claim 9, characterized in that the polymer is an arbitrary network copolymer which further comprises a plurality of other polypeptide repeating units, written from left to right and in the direction from the amino end to the carboxy end, including a polypeptide sequence corresponding to formula wherein the Asn and Tyr amino acid residues in parentheses are each an alternative to the immediately preceding amino acid residue in the sequence of the formula; a, b, c, d, e and f and g, h, i, j, k and 1 are integers each having a value of 0 or 1, provided that if the value of any of a-f or g-1 is 0, it corresponds to R?, R3, R*, R5 or R^ group or R7, R®, R?,R1:0, a b c a e xccg n i ~j R^1 or R^2 group absent, and when an R^~^ group is absent, the sulfur atom of the Cys residue with an absent Ra^ —x^ group forms a cystine disulfide bond, while if the value of which preferably of a-f or g-1 is 1, it is equivalent 1 ■* 6 7—12 Ra_f~ or Rg_-|_ group present; The Ra_, —xr groups are individually the same or different parts bonded to the sulfur atom of the Cys residue and are selected from the group consisting of hydrogen, an alkyl group containing 1 to 4 carbon atoms and a substituted alkyl group containing 2 to 4 carbon atoms; 7-12 R t are the same or different alternative amino acid-g-1 residues to each immediately preceding Cys residue shown in the formula, and is selected from the group of amino acid residues with a neutral side chain; at least 4 of a-f and 4 of g-1 are 0; and said former and other polypeptide repeating units are present in said copolymer in a molar ratio of 1:10 to 10:1; and said first and second polypeptide repeat units are linked together by cystine disulfide bonds formed by oxidation of the Cys residues present in each of said repeat units. 13. Polymer according to claim 9, characterized in that the polymer is an arbitrary network copolymer which further comprises a plurality of other polypeptide repeating units, written from left to right and in the direction from the amino end to the carboxy end, corresponding to formula wherein the Asn and Tyr amino acid residues in parentheses are each an alternative to the immediately preceding amino acid residue in the sequence of the formula; a, b, c, d, e and f and g, h, i, j, k and 1 are integers with a value of 0 or 1, provided that if the value of which rseotl\ , m lehRr a e* Rlsx ], _Rt 2 ^ -agv erulpalpe-e r f eRfrl^al-vegærr urpegn-pde 1 eee, r lol0g er , neåRr ° r 7 R deR1 a?n — —x6, ..t-Rgirl5?su, vpp rare \ ° ene , dr e r}fRr 1a1 &v, æRr^-2, end, the sulfur atom of the Cys residue with an absent R"^ ^ group forms a cystine disulfide bond, while if the value of a 1-6 any of a-f or g-1 is 1, the corresponding R is r- or 7-12 Rg_]_ group present; The R 1— 6.- groups are individually the same or different parts a—i bonded to the sulfur atom of the cys residue and are selected from the group consisting of hydrogen, an alkyl group containing 1 to 4 carbon atoms and a substituted alkyl group containing 2 to 4 carbon atoms; Rg_± are the same or different alternative amino acid residues 7-12 to each immediately preceding Cys residue shown in the formula, and is selected from the group of amino acid residues with a neutral side chain; at least 4 of a-f and 4 of g-1 are 0; said former and said second polypeptide repeating units are present in said copolymer in a :'■ molar ratio of 1:10 to 10:1; and said former and said second polypeptide repeating units are linked together by cystine disulfide bonds formed by oxidation of Cys residues present in each of said repeating units. 14. Polymer according to claim 13, characterized in that a-f and g-1 are 0. 15. Polymer according to claim 9, characterized in that said polymer is a network polymer containing a plurality of repeating units, which repeating units include said 10 to 35 amino acid residue polypeptide sequence, peptide-linked to a second polypeptide forming a composite polypeptide repeating unit containing 25 to 55 amino acid residues, which composite polypeptide, written from left to right and in the direction from the amino end to the carboxy end, corresponds to the formula in which X, Y and Z, when present, are amino acid residues which are (a) selected from the group consisting of a Lys and an Arg residue, or (b) selected from the group consisting of an Asp and a Glu residue; "n" and "m" are integers with a value of 0, 1, 2, 3, or 4, so that the respective X and Y are absent when one or both of "n" and "m" have a value of 0, while the respective X and Y residues are present when one or both of "n" and "m" has a value different from 0, where the average number of X and Y residues present per polypeptide, is equal to the values of X and Y; "r" and "s" are integers with a value of 0, 1, or 2, so that the respective Z is absent when one or both of "r" and "s" have a value equal to 0, while the respective Z is present when "r" and "s" are present, where the average number of Z residues per compound polypeptide repeating unit is equal to the square root of "r" or "s", provided that when one or the other of "r" or "s" is greater than 0, the other of "r" or "s" is equal to 0 , and the respective Z if "r" or "s" is 0 is absent; "o" and "p" are integers with a value equal to 0 or 1, so that the corresponding A is absent when one or the other of "o" and "p" has a value equal to 0, provided that "o" and "p" cannot both have the same value; A indicates the former 10 to 35 amino acid residue polypeptide sequence; and B is indicated second polypeptide comprising an amino acid sequence, written from left to right and in the direction from the amino end to the carboxy end, corresponding to the formula wherein the Asn and Tyr amino acid residues in parentheses are each an alternative to the immediately preceding amino acid residue in the sequence of the formula; a, b, c, d, e and f and g, h, i, j, k and 1 are integers each having a value of 0 or 1, provided that if the value 1 2 of any of a-f or g^ -1 is 0, the corresponding R a , R, b, R3, R^, R5 or R^ group or R7, R®, R9, R10, R?;1 or CQe _i g n i ~j jc R 1 , 2 -group absent, and when an R 1 — 6^ group is absent, a1-6 forms the sulfur atom of the Cys residue with an absent R Si x f- group a cystine disulfide bond, while if the value of any of a-f or g-1 is equal to 1, the corresponding R<1><t>~7-12 or R„ q group present; The Ra = .- x^ groups taken individually are equally or differently bonded to the sulfur atom of the Cys residue and are selected from the group consisting of hydrogen, an alkyl group containing 1 to 4 carbon atoms and a substituted alkyl group containing 2 to 4 carbon atoms; 7-12 Rg_± are the same or different alternative amino acid residues to each immediately preceding Cys residue shown in the formula, and is selected from the group consisting of amino acid residues with a neutral side chain; at least 4 of a-f and 4 of g-1 are 0; and indicated composite polypeptide repeating units are linked together by cystine disulfide bonds formed by oxidation of Cys residues present in indicated repeating units. 16. Polymer according to claim 15, characterized in that the compound polypeptide repeating unit, written from left to right and in the direction from the amino end to the carboxy end, is selected from the group of polypeptides consisting of: 17. Network polymer comprising a plurality of polypeptide repeating units, which repeating units are linked by interpolypeptide cystine bonds formed between the Cys residues of the repeating units, which repeating units, written from left to right and in the direction from the amino end to the carboxy end, individually correspond to to the formula: (X)nMetValIleIleThrPheMet (Lys)SerGlyGlu (Ala)ThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLys AsnThrPheTyrCysCysGluLeuCysCysTyr (Asn) ProAlaCysAlaGlyCysAsn (Tyr) (Y) 18. Network polymer according to claim 17, characterized by "n" is 2; X is Light; and "m" is 0. 19. Network polymer according to claim 17, characterized in that "n" and "m" are both 0. 20. Inoculum containing as active ingredient an effective amount of a synthetic polypeptide containing 10 to 35 amino acid residues corresponding in sequence to position 35 to position 95 from the amino end of the B-subunit of the heat-labile enterotoxin of Escherichia coli, in which the specified position number includes the 21-residue signal peptide of specified toxin, which polypeptide is linked to a carrier as a conjugate or as a repeating unit of a polymer and is present in a physiologically acceptable diluent, which inoculum, when introduced in a unit dose into a host animal, is capable of elicit the production of antibodies that immunoreact with the indicated B subunit. 21. Inoculum according to claim 20, characterized in that the polypeptide is present coupled to a carrier as a conjugate. 22. ' Inoculum according to claim 20, characterized in that the polypeptide is present as a polymer repeating unit. 23. Inoculum according to claim 22, characterized in that it is capable of protecting said host against the heat-labile enterotoxin of Escherichia coli. 24. Antibodies which immunoreact with the B-subunit of the heat-labile enterotoxin of Escherichia coli, which antibodies are elicited by introducing into a host mammal an effective amount of a synthetic polypeptide containing 10 to 35 amino acid residues corresponding in sequence to position 35 to position 95 from the amino terminus of the B subunit of the heat-labile enterotoxin of Escherichia coli, wherein said position numbers include the 21 residue signal peptide of said toxin, which polypeptide is present in an inoculum chained to a carrier as a conjugate or as a repeating unit of a polymer when introduced in designated host mammals. 25. Antibodies according to claim 24, characterized in that they are raised against the specified polypeptide, written from left to right and from the amino end to the carboxy end, from the group consisting of: