WO1981001101A1

WO1981001101A1 - Improvements in or relating to vaccines

Info

Publication number: WO1981001101A1
Application number: PCT/GB1980/000173
Authority: WO
Inventors: T Lehner; M Russell
Original assignee: T Lehner; M Russell
Priority date: 1979-10-18
Filing date: 1980-10-20
Publication date: 1981-04-30
Also published as: JPS56501364A

Abstract

Two antigenic substances derivable from Streptococcus mutans, and having molecular weights as determined by SDS-PAGE in the range of 146,000 to 155,000 daltons and 175,000 to 195,000 daltons, respectively, are of value for use in vaccines against dental caries. These substances are obtainable from S. mutans in purified form by procedures including anion exchange chromatography followed by gel filtration and affinity chromatography utilizing antibodies raised against the antigenic substance of higher molecular weight and against a third antigenic substance derivable from S. mutans having a molecular weight as determined by SDS-PAGE of 47,000 to 49,000 daltons.

Description

IMPROVEMENTS IN OR RELATING TO VACCINES This invention relates to vaccines and more particularly to vaccines for use against dental caries.

The association of Streptococcus mutans with the occurrence of dental caries in man has been widely accepted for many years and has led to extensive investigation of the possibility of preparing a protective vaccine from this micro-organism. Research has been pursued on many fronts and a wide variety of antigenic materials derived from S_. mutans has been reported in the litera¬ ture. However, whilst protective effects have been achieved in experimental animals with some of these materials, none of the forms of such antigenic material described in the art has been established as being suitable to form the basis of an anti-caries vaccine for use in man. The introduction of a vaccine of practi¬ cal use has been inhibited by a number of factors, the most important being the incidence of undesirable and in some cases very serious side effects accompanying the use of these antigenic materials.

We have directed our attention to the identification of an antigenic material of value as a basis for a vaccine against caries and especially to the production of this antigenic material in a purified form free from by-products of its manufacture which have deleterious side effects or which may obliterate the antigen's potential to provide immunity.

According to the present invention an antigenic material comprises one or both of that antigenic substance derivable from Streptococcus mutans having a molecular weight as determined by SDS-PAGE in the range of 146,000 to 155,000 daltons and that antigenic substance derivable from S. mutans having a molecular weight as determined by SDS-PAGE in the range of 175,000 to 195-000 daltons, said material being of a purity such that on

SDS-PAGE it exhibits a protein band or bands corresponding to one or both of said molecular weights, substantially free from similar bands corresponding to proteins or polypeptides of other molecular weights.

SDS-PAGE is an abbreviation of sodium dodecyl sulphate poly- acrylamide gel electrophoresis. The molecular weight values determined by this procedure as quoted herein are referred to the l4C-rnethylated standards described on page 24.

The two antigenic substances of different molecular weights are related, antiserum raised against either substance reacting with both substances owing to the presence in the substance of higher molecular weight of the antigenic determinant present in the substance of lower molecular weight. For simplicity of description this substance of lower molecular weight is herein¬ after referred to as antigen I. Moreover, the substance of higher molecular weight will react not only with antisera raised against antigen I and against itself but also with antiser_. raised against a further antigenic substance derivable from S_. mutans having a molecular weight as determined by SDS-PAGE in the range of 47-000 to 49.000 daltons. Such cross reaction is a result of the presence in the 175_.000 to 195_.000 daltons molecular weight substance of the antigenic determinant of the 47.000 to 49.000 daltons molecular weight substance as well as that of antigen. I. For simplicity of description this substance having a molecular weight in the range of 47.000 to 49.000 daltons is hereinafter - referred to as antigen II. Moreover, since the substance having a molecular weight in the range of 175,000 to 195,000 daltons con¬ tains at least a part (that containing the antigenic determinant) and possibly the whole of both of antigens I and II, this sub¬ stance is hereinafter referred to as antigen l/ll.

Although the present invention extends to mixtures of antigen I and antigen l/ll, it is preferred to use each of these substances in a form substantially free from the other.

The antigen I has certain characteristics, some or all of which may be used, in addition to its molecular weight, in identifying it. 1. It is immunogenic in experimental animals, forming precipi¬ tating antibodies.

2. It reacts with antisera raised against antigen l/ll as well as with antisera raised against itself, but does not react with antisera raised against antigen II.

3- It is distinct from serotype polysaccharide antigens, glycerol teichoic acid, dextran and similar glucans, and does not synthesise glucans from sucrose. 4. It is substantially, but not necessarily exclusively, of a proteinaceous nature.

5- It has an isoelectric point lying in the range of 5-0 to 5»2. 6. It has an ultraviolet absorption with a peak at less than 230nm and a peak at 270 to 28θnm; the ratio of ^E.-ό_n/^E _p(-,o is greater than 1. 7. It is eluted from DEAE-cellulose by 0.01M tris HCl of pH 7.4

(DEAE is an abbreviation of diethylaminoethyl and tris HCl is an ab_¬ breviation of 2-amino-2-hydroxymethylpropane 1,3-diol hydrochloride). 8. It does not react with concanavalin A. 9- Its immunogenic activity is destroyed by any one of the enzymes chymotrypsin, papain, pepsin, pronase and trypsin.

The combined form of antigen I, antigen l/ll, has the follow¬ ing characteristics, some or all of which may be used, in addition to its molecular weight, in identifying it.

1. It is immunogenic in experimental animals, forming precipit- ating antibodies.

2. It reacts with antisera raised against antigen I or antigen II as well as with antisera raised against itself.

3- It is distinct from serotype polysaccharide antigens, glycerol teichoic acid, dextran and similar glucans, and does not synthesise glucans from sucrose.

4. It is substantially, but not necessarily exclusively, of a proteinaceous nature.

5« It has an isoelectric point lying in the range of 4.7 to 4.9. 6. It has an ultraviolet absorption with a peak, at less than 230nm and a peak at 270 to 280nm; the ratio of ^E280^/E260 ^is greater than 1.

7. It is retained on DEAE-cellulose in the presence of 0.01M tris HGl of pH 7.4 and is eluted therefrom by O.lM NaCl/O.OlM tris HCl of pH 7.4.

8. It does not react with concanavalin A.

9. Its immunogenic activity is modified, but not destroyed, by any one of the enzymes chymotrypsin, papain, pepsin, pronase and trypsin.

Antigen II has the following characteristics, some or all of which may be used, in addition to its molecular weight, in identifying this antigen.

1. It is immunogenic in experimental animals, forming precipitating antibodies.

2. It reacts with antisera raised against antigen I/II as well as against itself (but not with antisera raised against antigen I),

3. It is distinct from serotype polysaccharide antigens, glycerol teichoic acid, dextran and similar glucans, and does not synthesise glucans from sucrose.

5. It has an isoelectric point lying in the range of 4.0 to 4.1.

6. It has an ultraviolet absorption with a peak at less than 230nm and a peak at 270 to 280nm; the ratio of E---/E-,-- is greater than 1.

7. Its immunogenic activity is not affected by any one of the enzymes chymotrypsin, papain, pepsin, pronase and trypsin.

It will be appreciated that reference above to the activity of various enzymes relates to the use of these enzymes under normal conditions (preliminary indications are that the enzymes chymotrypsin and pepsin may lead to a modification of the antigen II molecule although the immunogenic activity is unaltered) . Antigen I and antigen l/ll are both isolable from Streptococcus mutans and both are apparently originally present in the organism in vivo although the possibility cannot be entirely ruled out that antigen I as isolated is derived through degradation of antigen I/II during the isolation procedure. The two substances may con¬ veniently be obtained from a suitable culture of an appropriate strain, particularly of serotype c, for example the Guy.s and Ingbritt strains of this serotype, by processing of the culture supernatant and/or of the cells after suitable disruptive treat- ment. Other serotypes, particularly serotypes e and f, are, however, of interest as a source of these antigens or of equiva¬ lent antigens as discussed hereinafter on page 9.^' The processing may involve as an initial step the concentration of the culture supernatant and/or of a cell extract to give an antigen rich concentrate, but more conveniently involves the precipitation of protein therefrom, including these antigens, by the use of salts or other precipitants, for example ammonium sulphate.

One convenient procedure for the separation of antigen I and antigen I/II includes treatment of the initial crude product containing a mixture of antigens by anion exchange chromatography, conveniently on a column of an^" aήion exchange material which • contains dialkylarriinoalkyl groups such as DEAE groups, for example various carbohydrate based materials such as DEAE-Sephadex (Sephadex is a dextran material), DEAE-agarose and particularly DEAE-cellulose, or an equivalent anion exchange material, to which the crude product is applied in tris HCl buffer or other buffer of low ionic strength of about 0.01M and a pH of about 7.4. Elution with the same tris HCl buffer is conveniently used to remove the bulk of antigen I from the column. Increase of the ionic strength of the eluting buffer is then conveniently effected and typically removes the bulk of the antigen I/II from the column together with some contaminating antigen I and possibly also free antigen II. O.lM NaCl/O.OlM tris HCl buffer of pH 7.4 or an equivalent buffer of similar ionic strength may conveniently be used for this purpose.

Further purification of the material obtained from an anion exchange column is generally required to produce a material show- ing a single protein band on SDS-PAGE in any significant yield. Another purification step which may be carried out is the use of gel filtration, conveniently on a column of a polyacrylamide/ agarose gel such as Ultrogel AcA 22 (LKB) or an equivalent material. A buffer such as O.lM tris HC1/0.5M NaCl of pH 7.4, or one of equivalent ionic strength, may conveniently be used to prepare, to load and to elute the column. Antigen I/II is eluted from such a column before antigen I but some overlap tends to occur and purity may be enhanced at the expense of yield by taking smaller portions of eluate to work up for a particular antigen, and vice versa. It is difficult, purely by the use of anion exchange chromatography and gel filtration to produce a sample of antigen I showing a single protein band on SDS-PAGE. It is, however, poss¬ ible to achieve this for antigen I/II. hilst the anion exchange/ gel filtration technique just described maybe usedfor this pur- pose, a variant of the technique is more particularly suited there¬ to and "involves." absorption of a protein concentrate from the culture supernatant onto an anion exchange material as described above, for example DEAE-cellulose, from a urea containing buffer such as 0.01M tris HCl pH 8.O/6M urea. The anion exchange material ^~-y then be washed with the same buffer and eluted by this buffer but with an increase in ionic strength, for example through the incorporation of 0.15M sodium chloride therein, to remove antigen I/II therefrom. Following this, gel filtration of the antigen 1/ II fractions, for example on a material such as the agarose, Sepharose 6B (Pharmacia), in 1% w/v ammonium bicarbonate then con¬ veniently enables a sample of antigen l/ll showing^, one*.SDS-PAGE- protein band to be obtained.

-^DP^.

C.._ ^'"I? A purification procedure of general application in obtaining antigens showing one SDS-PAGE band is the use of affinity ^"" chromatography. This may be applied in various ways, either using an affinity column which binds the desired antigen and/or one which binds impurities, such columns containing the appro¬ priate antibody attached to a suitable support, for example an agarose material such as Sepharose 4B. The antibodies used in such procedures may conveniently be raised by conventional proce¬ dures involving injection of the relevant antigen into a suitable animal, the antigens I, I/II and II each having been found to be highly immunogenic in experiamental animals, for example in rhesus monkeys, rabbits and mice. Two specific affinity chromatography procedures are described below.

One procedure involves the use of a single affinity column containing antibody to antigen II. Antigen II, or a substance immunologically similar thereto, is detectable in the earlier purification procedures, this substance either occurring originally in S. mutans or being derived indirectly therefrom through degrada¬ tion of antigen I/II during the isolation procedures, but is difficult to isolate in any significant amount by these procedures. The antigen II characterised herein and required to effect affinity chromatography procedures is produced by the action of pronase on the antigen I/II. Antigen l/ll thus has value in the present invention not only for administration irx vivo but as a starting material of use in the preparation of antigen II and thus of antigen I. Such a procedure employing a column containing antibody to antigen II is of value in the purification of antigen l/ll when only this combination and the antigen I are present in a sample with no other antigens or protein impurities appearing on SDS-PAGE. Passage through the column will then lead to reten¬ tion of antigen l/ll whilst antigen I passes through the column, antigen l/ll then being eluted from the column, free from antigen I. This procedure is particularly applicable to the separation of

OMPI purified antigen l/ll which has already been separated from antigen II by earlier purification stages and antigen I may also be recovered from the original eluate of the column. A second procedure for the production of purified antigen I, is however preferred, and involves the use, in series, of a first column containing antibody to antigen II and a second column containing antibody to antigen l/ll. Antigen I will pass through the first column whilst antigen l/ll and any antigen II present will be retained. Antigen I will, however, be retained by the second column, although impurities will not in general be retained, and may subsequently be eluted therefrom.

In general, it is preferred to use the dual column affinity chromatography technique for the production of purified antigen I and an intermediate gel permeation step may conveniently then be omitted and anion exchange purified material used in the affinity chromatography. For the production of the purified antigen l/ll however, single column affinity chromatography is generally sufficient provided a sample which has been purified by gel per¬ meation to remove antigen II is employed in this procedure. As described above, gel permeation chromatography under appropriate condition can itself yield a purified antigen I/II, and it is also of particular value in the preparation of both antigen l/ll and also of antigen II sufficiently purified for the preparation of antisera for use in affinity chromatography (affinity chromatography purified antigen I usually being employed to produce an antiserum against this antigen).

Antigen I and antigen l/ll are both of interest for providing protection against dental caries, but antigen I is of particular interest in this respect. The presence in antigen l/ll of the antigen II determinant as well as that of antigen I, or of a proportion of antigen II in admixture with antigen I or antigen 1/ II as discussed hereinafter, is acceptable. Indeed antigen II is capable of providing allower level of protection in its own

O -^* _, VI right, but the use of antigen I substantially free from antigen II and conveniently also substantially free from antigen I/II is to be preferred. The production i__ vivo of the antibodies specific to the determinants of the antigens I and I/II is believed to be an important aspect of their action and accordingly the present invention extends to antigenic substances of equivalent immuno- logical activity to antigen I or antigen l/ll and of similar purity to that described above as being preferred for these anti¬ gens, i.e. exhibiting on SDS-PAGE a single protein band substan- tially free from similar bands corresponding to other proteins or polypeptides. Such equivalent antigenic substances may be derived from a Streptococcus microorganism and/or from these antigens, and their equivalent immunological activity is shown by their ability (a) to elicit an immune response in vivo with the production of essentially the same antibodies as either antigen I or antigen 1/ II and (b) to combine, for example in vitro, with antibodies pro¬ duced by either antigen I or antigen I/II.

Antigen I and antigen l/ll are of particular interest for providing a high level of protection both in terms of antibody production and in terms of the breadth of activity of the anti¬ bodies produced. Thus antiserum to antigen I shows an immuno- fluorescence reaction with each of S_. mutans serotypes a, c, d, e, f and g but not b, which is, however, predominantly a rat strain. Of these six serotypes, a, d and g do not show a marked reaction with antiserum to antigen II and therefore may not contain antigen l/ll or an equivalent antigen, but antiserum against antigen l/ll will nevertheless show an immunofluorescence reaction with each of serotypes a, c, d, e, f and g owing to the presence of antigen I or, particularly in the case of serotypes d and g, and especially a, possibly of an immunologically equivalent antigen therein. Both antigen I and antigen l/ll or an equivalent antigen derived from one serotype therefore provide a wide breadth of antibody activity. Vaccines containing antigen I and/or antigen I/II may be prepared by conventional methods, the antigen or antigens being incorporated in an administrable form and dosage into the vaccine. It will be appreciated that it is often desirable for the vaccine to be effective against a variety of serotypes but, as indicated above, cross occurrence or reactivity may allow this to be achieved without having to incorporate an antigen isolated from each of the serotypes in question, and some inter-strain cross reactivity may even be achieved. The vaccine may be formulated in a diluent such as isotonic saline or even a solid carrier, and may where desirable incorporate other- antigens than those described above, for example antigens derived from other serotypes than those against which the original antigens will cross-react such as serotype b, or even antigen II although this substance is of less interest than antigen I or antigen l/ll. In general, such other antigens are mixed with antigen I and/or antigen l/ll of the purity specified above, but in the particular case of antigen II, this antigenic substance may alternatively be incorporated simply by. purifying the antigen I and/or antigen l/ll to the point where on SDS-PAGE bands are present corresponding to one or both of molecular weights in ranges of 146,000 to 155_.000 daltons and 175)000 to 195.000 daltons, and additionally to a molecular weight of 47.000 to 49)000 daltons, substantially free from similar bands corresponding to proteins or polypeptides of other molecular weights weights. Such an antigenic material is included by the present invention, whether produced directly or by admixture of one or more purified components.

Although induction in a patient of the presence of the anti¬ bodies described herein is an important feature of the present invention, and antibody preparations according to the present invention may be considered for use in passive immunisation(being administered by conventional means or even possibly in milk) as well as in the purification of the antigenic materials, it is preferred to use the antigenic materials themselves in direct immunisation.

The vaccines according to the present invention may conven- iently be administered by various routes, for example subcutan- eously, intramuscularly and/or orally. Oral methods of admini¬ stration include not only those intended to produce an effect systemically but also incorporation of the antigenic material in lozenges, toothpaste and mouth washes, etc., in order to produce an effect topically. In general, however, some form of parenteral administration is favoured. The vaccines are of particular interest for prophylactic use in order to produce protection against attack by dental caries, administration at an early age therefore being preferred, although administration to a patient already having caries is also envisaged.

Dosage levels are conveniently selected with the aim of producing high levels, of protection, and the incorporation of adjuvants, for example aluminium hydroxide, into the vaccines is of interest in this context. Whilst the exact dosage levels used will therefore depend on such parameters as the nature of the adjuvant used, and the regimen of vaccination adopted, it is possible to give some general guidance. Thus, the dose of antigen for human immunisation by a subcutaneous route is conveniently of the order of 0.1 to 1 mg given with aluminium hydroxide or other suitable adjuvant. Alternatively, oral administration of the order of 1 to 5 m of antigen incorporated in a suitable adjuvant includ¬ ing, for example, the use of liposomes. (When antibodies are administered directly, topical application is preferred, for example a dose of about 1 to 5 ml of an antibody preparation of average titre incorporated in an appropriate form of composition.) The administration of the vaccine given subcutaneously to young patients is conveniently timed with the following order of fre¬ quency: 6 months, 2 years, 5 years and 10 years with the initial dose being accompanied by adjuvant and the subsequent doses being

_,O PI_ administered without adjuvant and being about one half to one quarter of the level of antigen in the initial dose. The fre¬ quency of administration of the vaccine is, however, preferably decided by monitoring the antibody levels present in the patient, for example by a radioimmunoassay carried out on serum or gingival crevicular fluid, the latter having the particular advantage with young patients that blood does not have to be withdrawn.

It will be appreciated that the invention extends to the administration of a vaccine comprising an antigenic material or substance or an antibody or antibodies as defined herein in combination with a physiologically acceptable diluent or carrier to a mammalian, particularly a human, patient in an amount effec¬ tive to provide a level of protection against dental caries in the patient, as well as to such a vaccine itself. The preparation of the antigens and antibodies, by typical methods, and the testing of the former for in vivo activity is described by way of exemplifica ion in the following Examples. EXAMPLES

Analytical grade reagents are used wherever possible and in accordance with normal practice all of the solutions used are aqueous unless stated otherwise. Example 1 : Preparation of Antigen l/ll A) CULTURE OF ORGANISM

Streptococcus mutans serotype __ (Ingbritt or Guy's strain) is cultured in 10 litres of medium (a) or medium (b) using 5 litre flasks.

Medium (a) is the tryptone-yeast dialysate medium supplemented with casamino acids, salts and glucose as described by Fukui et al, Journal of Bacteriology, 1974, _____ 796 with minor modifications in that the tryptone, yeast extract and casein hydrolysate used are Oxoid materials; bromothymol blue is omitted and no attempt is made to maintain pH by the intermittent addition of strong sodium hydroxide; and that the 0>5g/l of thioglycolate may optionally be replaced by cysteine HCl, particularly if there are

O any indications of the former being undesirably toxic in relation to the strain used.

Medium (b) is the semi-defined medium based on a casein acid hydrolysate (Acidicase Peptone 2, low salt, Baltimore Biological Laboratories) supplemented with amino acids, vitamins, salts and glucose as described by Bowden et_ ___. Journal of Dental Research,

1976, 55, A 192, with minor modi ications in that the glucose level is increased to 10g/l and that 1 ml of sterile 20% w/v

Na CO is added before inoculation for each, litre of medium. 2 3 The final composition of the medium is thus as follows: KH_P0₄ 6 g/1

K₂HPO₄ 9 g/1

MgSO, .7H 0 0.2 g/1

CaCl .2H 0 0.02 g/l

CH C00Na.2H 0 0-3 g/l 2

FeSO, .7H 0 4 mg/1

MnSO, .2H 0 0.15 mg/l

Na MoO, .2H 0 0.1 mg/l

Glucose 10 g/l Acidicase Peptone 2 (low salt; Baltimore

Biological Laboratories) 2 g/l

Cysteine hydrochloride 0.2 g/l

Glutathione (reduced) 0.05 g/l

L-Asparagine 0.1 g/l

L-Tryptophan 0.04 g/l

Glutamic acid 0.5 g/l p-Aminobenzoic acid 2 mg/l

Thiamine 2 mg/l

Riboflavin 2 mg/l

Nicotinic acid 2 mg/l

Pyridoxal hydrochloride 2 mg/l

Calcium pantothenate 2 mg/l - 14 - m-Inositol 2 mg/l

DL-Thioctic acid 0.1 mg/l

Biotin 0.1 mg/l

Haemin 0.2 mg/l

Folic acid 0.1 mg/l

(the pH of this mixture is adjusted to 7 and it is autoclaved at 10. lbs for 20 minutes; before inoculation 1 ml/l of sterile

20% w/v Na CO is added). o

The culture medium is warmed to 37 C, is inoculated with o 10 ml of an overnight culture of the cells prepared at 37 C in

Todd-Hewitt broth, and incubation at 37 C is continued until growth ceases (24 to 64 hours). The cells and supernatant are separated by centrifugation using a continuous flow rotor. B) EXTRACTION OF ANTIGENS The culture supernatant is treated with solid ammonium sulphate to 75% saturation (3M), stirred and allowed to stand overnight in the cold. The resulting precipitate is collected by centrifugation using a continuous flow rotor.

The cells initially separated from the supernatant are resuspended completely in about 3 volumes of 6M urea in 0.15M

NaCl and stirred for 1 hour at room temperature then overnight

. o at 4 C. The mixture is centrifuged at about 10,000 g for 30 minutes and the supernatant and cells are separated. The latter are re-extracted by the same procedure but using 4M urea in 0.15M NaCl and the two supernatants are combined, dialysed against 0.15M NaCl overnight in the cold and centrifuged. The supernatant is separated and treated with solid ammonium sulphate to 75% saturation, stirred and allowed to stand overnight in the cold. The resulting precipitate is collected by centrifugation. C) ANION EXCHANGE CHROMATOGRAPHY

The combined ammonium sulphate precipitates derived from cells and supernatant are redissolved in 100 ml of 0.01M tris HCl of pH 7-4 and are thoroughly dialysed overnight in the cold

(1) ^" ~^"~

In a variant of this procedure these precipitates are worked up separately. against the same buffer (at least 100 volumes). Any precipitate is removed by centrifugation. The mixture of products in solution is loaded on to a column of DEAE-cellulose (Whatman DE 52, 30 cm x 2 cm), the column being prepared using 0.01M tris HCl of pH 7.4. The column is initially eluted with 0.01M tris HCl of pH 7.4 until the first u.v. absorbing peak has been eluted (l to 2 column volumes) and is then eluted with O.lM NaCl in 0.01M tris HCl of pH 7-4 (2 column^{^}volumes), the flow rate used throughout being 25 ml/hour. The eluate is monitored at 28θnm and fractions of 5 ml are collected. The fractions are assayed for the presence of antigens by fused rocket immuno-electrophoresis against poly- specific anti-S. mutans antiserum /this is polyspecific for cell and supernatant antigens of S_. mutans of serotype c including antigens I, II and I/II and is prepared by injecting formalin- killed whole organisms, whole non-protease treated cell walls or unfractionated culture supernatant (75% saturated ammonium sulphate precipitate of culture supernatant, exhaustively dialysed and freeze-dried) in the procedure described in Footnote 1 to this Example in relation to antisera against antigen 11/ or, where possible, by single radial immunodiffusion against antisera to both antigen l/ll and antigen II (prepared using the general procedure described under Footnote l). Antigen I is eluted with starting buffer and antigen l/ll with the O.lM NaCl buffer, (antigen I/II being accompanied by some contaminating antigen I and possibly also antigen II). Antigen I and antigen I/II posi¬ tive fractions are separately pooled and the antigens are recovered by dialysis against water and freeze-drying, or by precipitation with 75% saturated ammonium sulphate in the case of the antigen 1/ II. D) GEL FILTRATION

A column of Ultrogel AcA 22 (LKB; 100 cm 2.5 cm) is prepared in O.lM tris HCI/0.5M NaCl of pH 7-4 and the void volume is determined by eluting Blue Dextran 2,000 (Pharmacia). The

O PI partially purified antigen l/ll from the DEAE-cellulose column is taken up in 5 ml of O.lM tris HC1/0.5M NaCl of pH 7.4, centrifuged to remove any precipitate, and is applied to the Ultrogel column. The column is eluted with O.lM tris HC1/0.5M NaCl of pH 7.4 at approximately 10 ml/hour, monitoring the eluate at 28θnm and collecting 5 ml fractions. The fractions are assayed for antigens by fused rocket immunoelectrophoresis against polyspecific antiserum or, where possible, by single radial immunodiffusion against antigen I/II and antigen II, antisera. Antigen l/ll typically elutes under these conditions: at an elution volume/void volume ratio in the range of 1-74 to 1.96, the general order of elution of the antigens being l/ll, I and II. Single radial immunodiffusion typically shows a symmetrical distribution of antigen I/IJC, which reacts with both antisera, in the main part of the u.v. absorbing peak, together with antigen I, which reacts with the^'antigen l/ll serum only, towards the low molecular weight tail. The fractions are selected quite broadly for high yield of antigen l/ll of lesser purity or only higher.molecular weight fractions are selected for a higher purity.but lesser yield of this antigen, and the lower molecular weight or tail fractions are selected for antigen I. The antigens are recovered by dialysis against water and freeze drying. A typical total yield of ; antigen l/ll broadly selected for high yield out low purity is 6 mg. E) IMMUNOABSORBENT CHROMATOGRAPHY

A column (5cm x 1.6cm) of Sepharose 4B linked to IgG from rabbit antiserum to antigen II (prepared as described in Footnote 1 to this Example) is prepared in O.lM phosphate/θ.5M NaCl of - pH 7.4. Three milligrams of antigen l/ll purified by gel fil- tration obtained in (D), which is contaminated with antigen I but not antigen II, is taken up in 1 to 2 ml of the same buffer and is applied to the column (such a load being acceptable to avoid overloading of a column of this size) and then eluted, again with the same buffer, at a rate of about 10 ml/hour. The eluate is monitored at 28θnm and 1 ml fractions are collected. Antigen I is eluted in the first peak and after 2 column volumes (20 ml) of the buffer have been passed through the column, elution is com- menced with 6M guanidinium chloride at a rate of approximately 10 ml/hour, collecting 1 ml. fractions. These fractions are assayed for antigens by single radial immunodiffusion against antisera to the antigen I/II and antigen II (obtained as described in footnotes to Examples 2 and 1, respectively). Appropriate fractions showing reaction to both antisera are pooled and dialysed thoroughly against 0.15M NaCl, the antigen Ϊ/II being frozen for storage.

A yield of 0.5 mg of purified (single SDS-PAGE band) antigen l/ll is typically obtained by this procedure from ^~ mg of starting material and shows the properties described hereinbefore. Thus, for instance, the figure obtained for the molecular weight as determined: by the SDS-PAGE procedure (described in detail in Footnote 2 hereto) lies in the range of 175,000 to 195,000, often in that part of the range from 175,000 to 185,000 or 190,000, for example about 185,000 daltons, whilst the figure obtained for the isoelectric point (determined essentially according to the procedure of Schmidt-Ullrich ___ a__, Proceedings of the National Academy of Sciences, 1977₁ 7_4₅ 643-647) lies in the range of 4.7 to 4.9) and the ultraviolet absorption (in 0.15M sodium chloride at substantially neutral pH) shows a peak at less than

230nm and a peak at 270 to 2δθnm; the ratio of E _ /E being

2o0 260 greater than 1 (a typical value being about 1.2). The value of the sedimentation coefficient S W for a typical sample is determined as about 8.7 S (-0.2 S) with a preparative ultra-

125 centrifuge-, and using as standards I labelled alcohol de.- hydrogenase, catalase and bovine serum albumin (assumed to be of similar partial specific volume to antigen l/ll), the deter¬ mination being effected on a 5-20% sucrose gradient in lOmM tris HCl of pH 7.4. The value for a typical sample of the ratio of Ve/Vo (elution volume/acid volume) for chromatograpy on a column (approximately 90 cm x 2.5 cm) of Ultrogel AcA22 prepared and eluted with O.lM tris HCl/0.5 NaCl of pH 7-4 is determined as about 1.79 - 0.035/(mean - standard error of the mean). Amino acid analysis of one sample of antigen I/II has indi¬ cated the substantial absence of sulphur containing amino acids, the analysis showing no significant amount of methionine or cysteine, /in contrast positive results were obtained for each of the other amino acids tested for (Trp, Gin and Asή were not deter- mined), viz: Asp, Thr, Ser, Glu, Pro, Gly, Ala, Val, lie, Leu, Phe, Lys, Tyr, His and Arg, although for the last two mentioned only at relatively low levels;/.

FOOTNOTE 1: PREPARATION OF IMMUN0ABS0RBENT COLUMN Antigen II 100 mg of antigen l/II (partially purified on DEAE-cellulose) in 5 ml of O.lM tris HCl of pH 7-4 is treated with 1 mg of pronase o and the mixture is incubated at 37 C overnight to destroy the antigen I activity, then centrifuged to remove any precipitate.

A column (100 cm x 2.5 cm) of Ultrogel AcA 34 (LKB) is prepared in 0.1M tris HC1/0.5M NaCl of pH7.4 and the void volume of the column i determined with Blue Dextran. The digest is applied to the column and eluted with 0.1M tris HC1/0.5M NaCl of pH 7.4 at 20 ml/hour, the eluate being monitored at 28θnm and 5 ml fractions being collected. The fractions are assayed for antigen using single radial immunodiffusion against antigen I/II antiserum. Antigen II is typically eluted in an isolated peak at an elution volume/void volume ratio of 1.88. Appropriate fractions are pooled and antigen II is recovered by exhaustive dialysis against water and freeze-drying. A yield of 2.5 mg of antigen II is typically obtained by this procedure and this has the properties described hereinbefore. Thus, for instance, the figure obtained for the molecular weight as determined by the SDS-PAGE procedure lies in the range of 47,000 to 49,000 daltons, for example about 48,000 daltons (since antigen II is derived by the cleavage of antigen I/II it seems likely that the whole of antigen II is incorporated (in antigen I/II), and the ultraviolet absorption (in 0.15M sodium chloride at substantially neutral pH) shows a peak at less than 230nm and a peak at 270 to 28θnm; the ratio of ^E 8o^/E26θ ^bein9 greater than 1 (a typical value being about 1.1). The value of the ratio Ve/Vo has been determined for chromatography on a column (approximately 90 cm x 2.5 cm) of Ultrogel AcA 34 prepared and eluted with 0.1M tris HC1/0.5M NaCl of pH 7-4 as being about 1.88. Amino acid analysis of one sample of antigen II gave broadly simi¬ lar qualitative results to these described herein for antigen l/II. Antiserum

1 mg of dry antigen II purified by gel filtration is dissolved in 1 ml of saline and mixed with 1 ml of Freund's complete adjuvant. The mixture is injected intramuscularly and subcutaneously at. multiple sites into a rabbit. After 3 to 4 weeks, a further simi¬ lar administration of antigen II and Freund's incomplete adjuvant is made. After a further 3 to 4 weeks, the rabbit is bled to pro- vide 25 ml of antiserum to antigen II, which is checked for mono- specificity by immunodiffusion (Ouchterlary) and immunoelectro- phoresis with unfractionated supernatant antigens, and for identity of reactions by immunodiffusion (Ouchterlary). IgG 5 ml of rabbit serum is dialysed overnight in the cold against 1 litre of O.OI75M phosphate buffer of pH 6.5, and then centrifuged to remove any precipitate. A column (10 cm x 1.6 cm) of DEAE- celliilose equilibrated with 0.0175M phosphate of pH 6.5 is pre¬ pared. The dialysed serum is applied to the column which is then eluted with 0.0175M phosphate of pH 6.5 at a rate of 20 ml/hour, monitoring the eluate at 28θnm and! collecting 5 ml fractions. The 4 or 5 main peak fractions consisting of IgG with precipitat¬ ing antibody of the same specificity as the original serum are pooled. The protein concentration is determined by optical densiτy at 28θnm, 1 mg/ml of IgG having 0D_2g0 = 1.35₅ the total yield of IgG from 5 ml of serum typically being 30 mg. IgG - Sepharose 4B 30 mg of IgG in 20-30 ml of O.lM NaHCO /0.5M NaCl of pH 8.3- is dialysed against 1 litre of this buffer at 4 C overnight. 3g of CNBr-activated Sepharose 4B (Pharmacia) is washed with 0.001M HCl as specified by the manufacture-.'. The gel is mixed with the dialysed IgG in O.lM NaHCO /0.5M NaCl and the mixture agitated gently by rotation for 1 to 4 hours at room temperature and.then overnight at 4°C. The gel is then washed with O.lM NaHCO /0.5M NaCl, treated with about 30 ml of 1M ethanolamine HCl of pH 8 for 2 hours at room temperature with agitation and finally washed again. The resultant gel is packed into a 5 x 1.6 cm column and washed with 0.-.M borate/lM NaCl of pH 8 followed by O.lM acetate/lM NaCl of pH 4. The cycle of ^• washing is then repeated, the flow rate used being about 10 ml/hour and 2 to 5 column volumes of each washing liquid being used. The column is finally washed with 2 column volumes of 6M guanidinium chloride and then with to 10 column volumes of O.lM phosphate/0.5M NaCl of pH 7.4. FOOTNOTE 2χ SDS-PAGE PROCEDURE

/The acrylamide (specially purified for electrophoresis), polyacrylamide, sodium dodecyl sulphate (SDS), tetramethyl ethylenediamine (TEMED) , ammonium persulphate, glycine and ethylamediamine tetraacetic acid disodium salt (EDTA) are obtained from British Drug Houses and the N,N'-bis-methylene acrylamide (bisacrylamide) is obtained from Sigma and is recrystallised from acetone before use/.

The main separating gel is prepared by a modification of the procedure of Laemmli, Nature, 1970, 227, 68θ, a gel which is

7-5% in acrylamide being found to show good^" results. This gel is produced by mixing the following aqueous media: A 30%: 0.8% (w/v) of acrylamide:bisacrylamide 10 ml

.(prepared at 4 C and stored at this temperature in the dark) B_ A mixture of 1.5M tris HCl of pH 8.8, 10 ml

8mM EDTA, and 0.4% w/v SDS

£ Water 13- ml

D 3% w/v polyacrylamide 6.6 ml

ImM sodium azide lmM sodium fluoride Medium D is stirred graduallly into mixed media A, B and C and this mixture is then mixed directly with

TEMED (undiluted) μ 1 ammonium persulphate (10% w/v in water, freshly made) 200 μ 1 The resulting mixture is mixed thoroughly, no degassing being necessary, and is then poured into a mould of the dimensions described by Studier, Journal of Molecular Biology, 1973, 7_9_. 37, formed from standard window glass plates with plastic spacers 0.15 cm thick and assembled with 6 paper clips, the moulds being sealed round the edges with 1.5% w/v agar in water. A gap of

2 cm is^' left below the top of the plates and 40 ml of the mixture is found to be ample for the preparation of one gel. The mixture is overlaid using a Pasteur pipette with a small volume of tris buffer made up from a mixture of 1.5M tris HCl o pH 8.8 5 ml

8mM EDTA,

0.4 w/v SDS; and water 15 ml

After setting, more overlay is added to prevent second polymeriza- tion at the top of the gel. The gel is then used as described below after an interval of several hours or up to five days after preparation.

The main gel is next treated to form a second gel on the top thereof containing sample wells. The overlay is removed from the main gel which is washed a few times with electrode buffer. The electrode buffer contains the following ingredients in water to a volume of 4 litres: tris base ^" 24 grams glycine 115-2 grams

EDTA 3-04 grams

SDS 4 grams

The following mixture is then poured over the dried main gel in order to produce the second, stacking, gel. The mixture is pro- duced by the thorough mixing of the following aqueous (except for TEMED) media:

A 30%:0.8% (w/v) of acrylamide:bisacrylamide 1.2 ml

33 0.5M tris HCl of pH 6.8 2 ml

8mM EDTA 0.4% w/v SDS

C 3% w/v polyacrylamide 1.3 ml

D_ Water 3.3 ml

E_ TEMED 10 μ 1

F 10% w/v ammonium sulphate 100 μ 1 A comb (BioRad) is then pushed into the applied mixture to pro¬ duce sample wells to take 10 or 20 samples, the comb being applied so as to leave about 1 cm below each tooth. After the stacking gel has set, the comb is removed with applications of electrode buffer from a syringe with a needle bent at 90°ι the wells being thoroughly washed out, also with electrode buffer in this way. The bottom: spacers are then removed and the plates are attached to the electrophoresis apparatus, which is a vertical slab gel apparatus based on the detailed plans described by Studier, ibid, using petroleum jelly to effect a seal and binding with two paper clips. Electrode buffer as described above is added to the tanks ensuring that no bubbles are trapped in the bottom plate.

Samples for application to the gel may contain antigen pre¬ parations in buffer solutions but should not contain guanidine hydrochloride. The antigen preparations and protein standards are made up in concentrated sample buffer containing five times the concentrations of components listed below for 1 x SDS buffer, dilution being effected with water as appropriate to give final concentrations as listed, an amount of 1 to 10 P'g of protein typically being contained in a total volume of 8θ - 100 μl of 1 x SDS buffer. The final (1 x SDS) sample buffer contains 2% w/v SDS 10% w/v glycerol 1% w/v β-mercaptoethanol 2mM EDTA

50mM tris HCl of pH 6.8 0.001% w/v bromophenol blue The final solution is boiled for ^~ minutes to dissociate the pro- teins and samples are then layered into the various wells using a fine pipette tip or syringe.

The power supply anode is connected at the bottom of the gel using a constant current and electrophoresis continued until the dye front reaches the bottom of the gel. Typically, a 0 mA current is. used for a 6 to 7 hour run and a 15 mA current with a final boost of 30 mA is used for a longer, overnight, run. On completion of the electrophoresis, the glass plates are gently prised apart with a spatula and the gel is stained with an aqueous solution of 40% w/v trichloracetic acid and 0.1% w/v Coomassie blue for 30 minutes. The stain is rinsed away and the gel - destained in 10% w/v acetic acid with a skein of white wool to absorb the dye. Protein bands typically start to become visible within half an hour and complete destaining is achieved overnight. Under the conditions indicated above antigen I/II typically runs about 1 cm into the gel, whilst antigen I runs about 2.5 cm and antigen II about f the gel length (about 8-9 cm). In order to estimate molecular weights, the l4C-methylated protein stan¬ dards supplied by the Radiochemical Centre, Amersham are used

*v Λ, IPO according to the manufacturer's instructions. These standards comprise myosin (200,000), phosphorylase B (92,500), bovine serum albumin (69,000), ovalbumin (46,000), carbonic anhydrase (30,000) and lysozyme (14,300). By plotting mobility, which is the distance moved by protein/the distance moved by dye front, against 1°9₁₀ of molecular weight, for the standards a curve is obtained which is then used to estimate the unknown molecular weights. Antigen I and antigen I/II both migrate at a rate between that of myosin and that of phosphorylase B. Even though substantially free from proteinaceous impurities as detected by Coomassie blue staining of the SDS-PAGE gel, samples of each of the three antigens may nevertheless be found to contain carbohydrate. This is probably an impurity and, indeed, on treatment of a SDS-PAGE gel with a carbohydrate- reactive reagent no bands may be detectable, the carbohydrate being located at the front and/or the base line. However, it is possible for all three antigens, for example antigen II and especially antigen I, that carbohydrate may conceivably represent a minor part of the antigen. Example 2: Preparation of Antigen I

10 mg of crude antigen I obtained from the 0.01M tris HCl (pH 7.4) eluate of the DEAE-cellulose column as described in Example 1 (C) above is applied in 1 to 2 ml of 0.1M phosphate/0.5M NaCl of pH 7-4 to a column (5 cm x 1.6 cm) of Sepharose 4B linked to IgG from rabbit antiserum to antigen II (prepared as described in Footnote 1 to Example 1) connected in series to a second column (5 cm x 1.6 cm) of Sepharose 4B linked to IgG from rabbit antiserum to antigen l/II (prepared as described in the footnote to this example), both columns being made up in 0.1M phosphate/0.5M NaCl of pH 7.4 (such a load being acceptable to avoid overloading of a column of this size). The columns are eluted with 0.1M phos- phate/0.5M NaCl of pH 7.4 at a rate of 10 ml/hour, monitoring at 28θnm and collecting 1 ml fractions. Once unabsorbed material has been eluted (after at least 2 column volumes or 40 ml of buffer) the two columns are separated and the second (I/II anti=- serum) column is eluted with 6M guanidinium chloride at a rate of 10 ml/hour, collecting 1 ml fractions. The fractions are assayed for antigens by single radial immunodiffusion against antisera to t-_e_antigen l/II and to antigen II. Those fractions which react with antiserum to antigen I/II, but not with antiserum to antigen II, are pooled, dialysed exhaustively against water and stored frozen.

A yield of 1 mg of purified (single SDS-PAGE band) antigen I is typically obtained by this procedure and shows the properties described hereinbefore. Thus, for instance, the figure obtained for the molecular weight as determined by the SDS-.PAGE procedure lies in the range of 146,000 to 155)000 daltons, for example about 150,000 daltons, and the ultraviolet absorption (in 0.15M sodium chloride at substantially neutral pH) shows a peak at less than 230nm and a peak at 270 to 2δ0nm; the ratio of E,o_n ---_)ήr- being greater than l.(a typical value being about 1.45). The value of the ratio of Ve/Vo for a typical sample for chromatography on a column (approximately 90 cm x 2.5 cm) of Ultrogel AcA 22 prepared and eluted with O.lM tris HC1/0.5M NaCl of pH 7.4 has been deter¬ mined as being in the range from about I.85 to about 2.32.

Amino acid.' analysis of one sample of antigen I has indicated the substantial absence of sulphur containing amino acids, the analysis showing no significant amounts of methionine or cysteine /JLΠ contrast positive results were found for the other amino acids tested for (Trp, Gin and were not determined) , viz: Asp, Thr, Ser, Glu, Pro, Gly, Ala, Val , lie, Leu, Tyr, Phe, Lys, His and Arg although for the last two mentioned only in trace amounts/. FOOTNOTE: PREPARATION OF ANTIGEN I/II IMMUN0ABS0RBENT COLUMN The column is produced by the general procedure of Footnote 1 to Example 1 but using antiserum to antigen I/II rather than to antigen II.

-£TJRE_Γ^

O...PI

_V^ _. ^IPO - ΛΛ lC^, Example 3: Alternative Procedure for the Preparation of Antigen I

Crude antigen I obtained from the O.OIM tris HCl (pH' 7-4) eluate of the DEAE-cellulose column as described in Example 1(C) is applied in O.lM tris HC1/0.5M NaCl of pH 7-4 to a column (85 cm x 2.6 cm) of Ultrogel AcA 22 (LKB) made up in the same buffer system. The column is eluted at a flow rate of 10 ml/hour with O.lM tris HC1/0.5M NaCl pH. 7-4, 5 ml fractions being .- collected. The fractions are assayed for antigen I as described in Exaπiple 1(C), the positive fractions being pooled and the antigen recovered by dialysis against water and freeze drying.

The antigen I obtained by this procedure typically contains minor amounts of antigens l/II and/or II, particularly the former. Example 4: Alternative Procedure for the Preparation of ' Antigen I/II Streptococcus mutans serotype c (Ingbritt or Guy's strain) is cultured substantially as described under Example 1(A) but employing 12 litres of medium (b) in which the glucose level is increased to 5% w/v. The cells and supernatant are separated as described previously ^"by centrifugation in a continuous flow rotor (MSE) at 18,000 rpm.

The culture supernatant is treated with solid ammonium sulphat to 75% saturation (3M) , stirred and allowed to stand overnight at 4oC; the msolubilized material then being recovered by continuous flow centrifugation as described above. This material is dissolved in 0.01M tris HCl pH 8.O/6M urea buffer and the solution is dialysed against water for 24 hours with frequent changes. The dialysate, and also material which precipitates therefrom, are reconstituted to 6M in urea by the addition of cry¬ stals of urea and to 0.01M tris HCl by the addition of a 1/lOth volume of O.lM tris HCl pH 8.0, the pH also being adjusted as necessary. The material is then loaded batchwise onto a 200 ml volume of DEAE-cellulose (Whatman DE 52) equilibrated with tris

^^cTUR

_ Oλ ^~ V/ HCl pH 8.O/6M urea buffer. After 15 minutes the slurry is cen¬ trifuged at 2000 rpm for 5 minutes and the clear supernatant is discarded.

The cellulose is washed twice with the tris HCl pH 8.O/6M urea buffer to remove unbound material and is then packed into a 5 cm x 10 cm column and the column eluted with the same buffer until .the absorbance at 28θnm is zero. The column is then eluted with 200 ml of tris HCl pH 8.O/6M urea buffer containing 0.15M sodium chloride to remove antigen I/II, 12 ml fractions being collected. The active fraction of the eluate is dialysed against water and lyophilized, a yield of about 17 mg of antigen I/II typically being obtained at this stage. This material is dissolved in 3 to 4 ml of 1% w/v ammonium bicarbonate and applied to a column (1.5 cm x 90 cm) of Sepharose 6B (Pharmacia) equilibrated in 1% w/v ammonium bicarbonate. The column is eluted at a rate of 20 ml/hour with 1% w/v ammonium bicarbonate, 3 ml fractions being collected and assayed for antigen l/II by fused rocket immunόelectrophoresis against, polyspecific antiserum or, where possible, by single radial immunodiffusion against rabbit anti- serum to antigen l/ll (prepared using the general procedure des¬ cribed under Footnote 1 to Example 1). The fractions containing antigen l/II are pooled and lyophilized.

It is possible to obtain from the gel filtration a material substantially free from antigens I and II, a typical yield being about 1 mg. However, where desirable, further purification may be achieved by a preparative version of the SDS-PAGE procedure described in Footnote 2 to Example 1 using 3 mm thick gels. The antigen l/II band is located by means of parallel electrophoresis of the dansylated antigen prepared using the dansylating procedure of Weiner ____ ____ Journal of Biological Chemistry, 1972, 247, 3242.

After visualization under ultraviolet light, the portion of the gel convtaining the antigen l/ll is excised using the electrophoresis run with the dansylated antigen as a guide. The gel slice is homogenised in 0.01M tris HOAc pH 8.0/0.05% w/v SDS/lmM phenyl- methysulphonyl fluoride (Sigma) , the mixture is incubated overnight at 37°C, and the eluted antigen l/II is then dialysed extensively against water and lyophilized. In a variant of the above procedure, the cells which are separated from the supernatant- are extracted with three volumes of tris HCl pH 8.0/6M urea, and the extract clarified by centri¬ fugation at 20,000 g for 30 minutes. The cell free extract is then concentrated by the addition of ammonium sulphate to 40% saturation and the precipitate processed as described above for that from the original supernatant to give a further yield of antigen l/II. The additional amount of the antigen I/II thus obtained is typically about 16.6 mg from cellulose and about 1.3 mg from the gel but minor amounts of antigen I and/or antigen II are usually present.

Example ^~ -. Immunisation Trials with Antigen I and Antigen I/II in Rhesus Monkeys

Young rhesus monkeys weighing between 1.4 and 2.2 kg were selected for the presence of all their deciduous teeth; in a few of the monkeys the first permanent molars were erupting. Twenty-four monkeys were caged, examined and within 5 days of starting the trial were kept entirely on a human type of diet as described by Lehher et al_, Archives of Oral Biology, 1975) 20, 299- The animals were divided randomly into 3 groups of 5 5 and 14 monkeys, which respectively received a subcutaneous primary immunization of antigen l/II, antigen I or saline as a control. Two samples of both antigen I and antigen I/II were used in the trial, these antigens being administered with either Freund's incomplete adjuvant (FIA) or Alhydrogel as adjuvant. The control group and some monkeys of the antigen l/II group received a secondary subcutaneous immunization at an interval of from 16 to 28 weeks after the primary immunization, but without the use of adjuvant. The full details of the immunization schedule for the twenty-four monkeys is shown in Table 1.

The monkeys were studied serially throughout the trial for the development of approximal, cervical and fissure caries by means of probe and mirror and X-rays. A standardised "caries score" was then awarded to each monkey according to the procedure described by Lehner __ al_, Archives of Oral Biology, 1977) 22, 393- Venous blood was taken to assay serum antibodies using the immunofluorescent test described by Lehner __ al_, Nature, 1976, 254, 517. The trial was continued with all of the monkeys for a period of 7 weeks after starting the trial with the primary immunization, and in the case of a proportion of the monkeys the trial was continued for a period of 92 weeks.

The..results obtained are shown in Table 2, mean caries indices and standard error being shown for decayed, missing filled surfaces (DMFS) , and also separately for the smooth surface and fissure caries types. The caries indices obtained for the differentt samples of antigens I and l/II, as identified in the footnotes to Table 1, were comparable and the results presented in Table 2 therefore refer in each case to the whole group of samples for each antigen. Statistical analysis of the 7 week results showed a significant reduction in the overall caries index found with antigen I immunization (t = 2.140 d.f. 17,p*.0.05) and with antigen l/II immunization (t = 2.181 d.f. 17, p<0.05) as compared with that found for the control group. This analysis was main¬ tained at 9 weeks with, smaller numbers of monkeys in the second and third groups. Separation- of caries into the smooth surface and fissure types showed that at 72 weeks there was a comparable, though slightly greater reduction in fissure than smooth caries for the antigen I and l/II immunized monkeys. At 92 weeks, how¬ ever, both groups showed a slightly greater reduction in smooth surface than fissure caries.

All of the antigen immunized monkeys developed an IgG class of antibody titre between 1:320 and 1:1280 as compared with the

OMPI v* *^"~wϊpδ^"~ - 30 - controls which developed a titre of less than 1:10. None of the monkeys in the trial showed any evidence of systemic abnormality. They gained weight throughout the duration of the trial and showed no evidence of red blood cell or white blood cell - abnormality. Table 1: Immunization Schedule

(1) Prepared by anion exchange chromatography and gel filtration as described in Example 3- (2)

Prepared by immunoabsorbent chromatography as described in Example 2. (3)

Prepared by anion exchange chromatography as described in Example 1. (4)

Prepared by anion exchange chromatography and gel filtration (with selection for high purity in the latter) as described in Example 1.

The antigen preparations described in footnotes and correspond to single antigens shown to be substantially free from other proteins or polypeptides by SDS-PAGE but in the case of those described in footnotes and , the preparation addi¬ tionally contains a minor amount of one—or both of the other antigen

O

^' among antigens I, I/II and II, (the preparations of higher purity being preferred for human administration) . Table 2: Mean Caries Indices (a) After 72 weeks

(b) After 92 weeks

NOTE: Strains of Streptococcus mutans for the preparation of antigens and antibodies according to the present invention are readily available, either by isolation using procedures described in the art of from culture collections, various of which contain deposits of strains of S. mutans.

- U REΛ

OMPI VvIPO

V// -

Claims

CLAIMS 1. An antigenic material comprising one or both of the antigenic substances, antigen I and antigen I/II, derivable from Streptococcus mut ns, antigen I having the following properties:-

(a) it has a molecular weight as determined by SDS-PAGE in the range of 146,000 to 155,000 daltons,

(b) it is immunogenic in experimental animals, forming precipitating antibodies,

(c) it reacts with antisera raised against antigen I/II as well as with antisera raised against itself, but does not react with antisera raised against the antigenic substance, antigen II, as defined in Claim 4,

(d) it is distinct from serotype polysaccharide antigens, glycerol teichoic acid, dextran and similar glucans, and does not synthesise glucans from sucrose, (e) it is substantially, but not necessarily exclusively, of a proteinaceous nature,

(f) it has an isoelectric point lying in the range of 5.0 to 5.2,

(g) it has an ultraviolet absorption with a peak at less than 230nm and a peak at 270 to 280nm, the ratio of *--o /- ,- being greater than 1,

(h) it is eluted from DEAE-cellulose by 0.01M tris HCl of pH 7.4, (i) it does not react with concanavalin A, and (j) its immunogenic activity is destroyed by any one of the enzymes chymotrypsin, papain, pepsin, pronase and trypsin; and antigen I/II having the following properties:-

(a) it has a molecular weight as determined by SDS-PAGE in the range of 175,000 to 195,000 daltons,

(b) it is immunogenic in experimental animals, forming precipitating antibodies, (c) it reacts with antisera raised against antigen I or antigen II as well as with antisera raised against itself,

(f) it has an isoelectric point lying in the range of 4.7 to 4.9,

(g) it has an ultraviolet absorption with a peak at less than 230nm and a peak at 270 to 280nm, the ratio of ^E28c ^E260 ^being greater than 1,

(h) it is retained on DEAE-cellulose in the presence of 0.01M tris HCl of pH 7.4, and is eluted therefrom by O.lM NaCl/O.OlM tris HCl of pH 7.4, (i) it does not react with concanavalin A, and

(j) its immunogenic activity is modified, but not destroyed, by any one of the enzymes chymotrypsin, papain, pepsin, pronase and trypsin; the antigenic material being of a purity such that on SDS-PAGE it exhibits a protein band or bands corresponding to one or both of the molecular weights attributed to antigen I and antigen I/II above, substantially free from similar bands corresponding to proteins or polypeptides of other molecular weights.

2. An antigenic material comprising the antigenic substance, antigen I, derivable from Streptococcus mutans, having the following properties:-

(c) it reacts with antisera raised against antigen I/II as defined in Claim 1; as well as with antisera raised against itself, but does not react with antisera raised against the antigenic substance, antigen II, as defined in Claim 4, (d) it is distinct from serotype polysaccharide antigens, glycerol teichoic acid, dextran and similar glucans, and does not synthesise glucans from sucrose,

(e) it is substantially, but not necessarily exclusively, of a proteinaceous nature, (f) it has an isoelectric point lying in the range of 5.0 to 5.2,

(g) it has an ultraviolet absorption with a peak at less than 230nm and a peak at 270 to 280nm, the ratio of ^E280^^E260 ^beinσ= greater than 1, (h) it is eluted from DEAE-cellulose by 0.01M tris HCl of pH 7.4, (i) it does not react with concanavalin A, and (j) its immunogenic activity is destroyed by any one of the enzymes chymotrypsin, papain, pepsin, pronase and trypsin; or an antigenic substance which is of equivalent immunological activity to antigen I; the antigenic material being of a purity such that on SDS-PAGE it exhibits a single protein band, substantially free from similar bands corresponding to other proteins or polypeptides.

3. An antigenic material comprising the antigenic substance, antigen I/II, derivable from Streptococcus mutans having the following properties:-

(c) it reacts with antisera raised against antigen I, as defined in Claim 1, or against antigen II, as defined in Claim 4, as well as with antisera raised against itself,

(d) it is distinct from serotype polysaccharide antigens, glycerol teichoic acid, dextran and similar glucans, and does not synthesise glucans from sucrose,

(e) it is substantially, but not necessarily exclusively, of a proteinaceous nature,

(f) it has an isoelectric point lying in the range of 4.7 to 4.9,

(g) it has an ultraviolet absorption with a peak at less than 230nm and a peak at 270 to 280nm; the ratio of E-_o0/E_?fin being greater than 1, (h) it is retained on DEAE-cellulose in the presence of

0.01M tris HCl of pH 7.4, and is eluted therefrom by O.lM

NaCl/O . O lM tris HCl of pH 7 .

4 ,

(i) it does not react with concanavalin A, (j) its immunogenic activity is modified, but not destroyed, by any one of the enzymes chymotrypsin, papain, pepsin, pronase and trypsin; or an antigenic substance which is of equivalent iminunological activity to antigen I/II; the antigenic material being of a purity such that on

SDS-PAGE it exhibits a single protein band, substantially free from similar bands corresponding to other proteins or polypeptides. . An antigenic material comprising one or both of the antigenic substances, antigen I and antigen I/II, derivable from

Streptococcus mutans and having the properties given for these substances in Claim 1, in admixture with the antigenic substance, antigen II, derivable from Streptococcus mutans and having the following properties:- (a) it has a molecular weight as determined by SDS-PAGE in the range of 47,000 to 49,000 daltons,

(c) it reacts with antisera raised against antigen I/II as well as with antisera raised against itself,

(f) it has an isoelectric point lying in the range of 4.0 to 4.1,

(g) it has an ultraviolet absorption with a peak at less than 230nm and a peak at 270 to 280nm, the ratio of

^bein§ greater than 1, (h) its immunogenic activity is not affected by any one of the enzymes chymotrypsin, papain, pepsin, pronase and trypsin; the antigenic material being of a purity such that on SDS-PAGE it exhibits protein bands corresponding to a molecular weight in the range of 47,000 to 49,000 daltons and to one or both of a molecular weight in the range of 146,000 to 155,000 daltons and a molecular weight in the range of 175,000 to 195,000 daltons, substantially free from similar bands corresponding to proteins or polypeptides of other molecular weights.

5. An antigenic material according to any of Claims 1 to 4, which is derived from a Streptococcus mutans strain of serotype c, e or f.

6. An antigenic material according to Claim 5, which is derived from a Streptococcus mutans strain of serotype c.

7. A process for the preparation of an antigenic material according to any of Claims 1 to 6, which comprises treating a culture supernatant and/or cell extract of Streptococcus mutans to produce a protein concentrate therefrom, and thereafter treating the protein concentrate to obtain the antigenic material.

8. A process according to Claim 7, in which the protein concentrate is produced by salt precipitation.

9. A process according to Claim 7 or 8, in which the protein concentrate is subjected to anion exchange chromatography.

10. A process according to Claim 9, in which the anion exchange chromatography is effected on an anion exchange material containing diethylaminoethyl groups.

11. A process according to Claim 9 or 10, in which the protein product purified by anion exchange chromatography is further purified by gel filtration.

12. A process according to any of Claims 7 to 11, in which the antigenic material is pruified by affinity chromatography.

13. A process according to Claim 12, in which the affinity chromatography is effected using a column comprising antibody specific for antigen I/II and/or a column comprising antibody specific for antigen II.

14. A process according to Claim 9 or 10 for the preparation of antigen I, in which material which has been purified by anion exchange chromatography is further purified by affinity chromatography using in succession firstly a column comprising antibody specific for antigen II and secondly a column comprising antibody specific for antigen I/II.

15. A process according to Claim 11 for the preparation of antigen I/II, in which material which has been purified by anion exchange chromatography and gel filtration is further purified by affinity chromatography using a column comprising antibody specific for antigen II.

16. A process for the preparation of an antigenic material derivable from Streptococcus mutans, substantially as described herein.

17. An antigenic material derivable from Streptococcus mutans, whenever prepared by a process according to any of Claims 7 to 16.

18. An antigenic material according to any of Claims 1 to 6 and 17, for use in the treatment of dental caries.

19. An antibody preparation which is specific for the determinant(s) present in an antigenic material according to any of Claims 1 to 6 and 17.

20. An antibody preparation which is monospecific for the determinant of antigen 1 as defined in Claim 1.

21. An antibody preparation according to Claims 19 or 20, in the form of a substrate for affinity chromatography.

22. A pharmaceutical composition which comprises an antigenic material according to any of Claims 1 to 6 and 17 together with a physiologically acceptable diluent or carrier.

23. A pharmaceutical composition according to Claim 22 which comprises a further added antigenic substance derivable from Streptococcus mutans■

24. A pharmaceutical composition which comprises an antigenic material which an administration _in vivo produces antibodies specific for the determinant(s) present in antigen I or antigen I/II, as defined in Claim 1, together with a physiologically acceptable diluent or carrier.

25. A pharmaceutical composition which comprises antibody against antigen I and/or antibody against antigen I/II, as defined in Claim 1, together with a physiologically acceptable diluent or carrier.

26. A pharmaceutical composition according to any of Claims 22 to 25 in a form suitable for parenteral or oral administration.

27. A pharmaceutical composition according to Claim 26, which comprises aluminium hydroxide as an adjuvant.

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