WO1981001101A1 - Improvements in or relating to vaccines - Google Patents

Improvements in or relating to vaccines Download PDF

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Publication number
WO1981001101A1
WO1981001101A1 PCT/GB1980/000173 GB8000173W WO8101101A1 WO 1981001101 A1 WO1981001101 A1 WO 1981001101A1 GB 8000173 W GB8000173 W GB 8000173W WO 8101101 A1 WO8101101 A1 WO 8101101A1
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Prior art keywords
antigen
antigenic
sds
page
antigenic material
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PCT/GB1980/000173
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English (en)
French (fr)
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T Lehner
M Russell
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T Lehner
M Russell
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Publication of WO1981001101A1 publication Critical patent/WO1981001101A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to vaccines and more particularly to vaccines for use against dental caries.
  • 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.
  • this substance of lower molecular weight is herein ⁇ after referred to as antigen I.
  • 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 .
  • antigen II the substance having a molecular weight in the range of 47.000 to 49.000 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.
  • 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.
  • antigen I 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.
  • Antigen II has the following characteristics, some or all of which may be used, in addition to its molecular weight, in identifying this antigen.
  • 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.
  • 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 " a ⁇ ion 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.
  • a ⁇ ion 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-
  • 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.
  • 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.
  • an anion exchange material as described above, for example DEAE-cellulose
  • 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.
  • a purification procedure of general application in obtaining antigens showing one SDS-PAGE band is the use of affinity "" chromatography.
  • 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.
  • 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.
  • 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.
  • antigen II is capable of providing allower level of protection in its own
  • 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 ⁇ genes, 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 ⁇ lodged 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.
  • 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.
  • 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.
  • a diluent such as isotonic saline or even a solid carrier
  • antigen II although this substance is of less interest than antigen I or antigen l/ll.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • Streptococcus mutans serotype __ (Ingbritt or Guy's strain) is cultured in 10 litres of medium (a) or medium (b) using 5 litre flasks.
  • 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
  • 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,
  • 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
  • the cells initially separated from the supernatant are resuspended completely in about 3 volumes of 6M urea in 0.15M
  • the eluate is monitored at 28 ⁇ nm 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.
  • 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).
  • 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 28 ⁇ nm 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.
  • 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/ ⁇ .5M NaCl of - pH 7.4.
  • the eluate is monitored at 28 ⁇ nm 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 ⁇ /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.
  • the figure obtained for the molecular weight as determined: by the SDS-PAGE procedure 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, 1977 1 7_4 5 643-647) 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
  • 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-
  • 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 As ⁇ 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 28 ⁇ nm 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.
  • 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 28 ⁇ nm; the ratio of E 8o /E 26 ⁇ bein9 greater than 1 (a typical value being about 1.1).
  • 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 28 ⁇ nm 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.
  • 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 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/.
  • SDS sodium dodecyl sulphate
  • TEMED tetramethyl ethylenediamine
  • EDTA ammonium persulphate
  • glycine and ethylamediamine tetraacetic acid disodium salt EDTA
  • the main separating gel is prepared by a modification of the procedure of Laemmli, Nature, 1970, 227, 68 ⁇ , a gel which is
  • This gel is produced by mixing the following aqueous media: A 30%: 0.8% (w/v) of acrylamide:bisacrylamide 10 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
  • 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
  • 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 8 ⁇ - 100 ⁇ l of 1 x SDS buffer.
  • the final (1 x SDS) sample buffer contains 2% w/v SDS 10% w/v glycerol 1% w/v ⁇ -mercaptoethanol 2mM EDTA
  • 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.
  • 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.
  • 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).
  • 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).
  • the l4C-methylated protein stan ⁇ dards supplied by the Radiochemical Centre, Amersham are used
  • 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 28 ⁇ nm and 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.
  • 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 2 ⁇ 0nm; the ratio of E,o n --- ) ⁇ r - being greater than l.(a typical value being about 1.45).
  • 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 /JL ⁇ 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.
  • 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 Exa ⁇ iple 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.
  • MSE continuous flow rotor
  • 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
  • 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 28 ⁇ nm 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 immun ⁇ electrophoresis 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.
  • 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.
  • 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.
  • 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.
  • antigen preparations described in footnotes 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

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988006455A1 (en) * 1987-02-27 1988-09-07 Council Of Governors Of The United Medical And Den Antibodies against streptococcus
US5518721A (en) * 1987-02-27 1996-05-21 Council Of Governors Of The United Medical And Dental Schools Of Guy's And St. Thomas Hospital Antibodies against Streptococcus

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6028937A (ja) * 1983-07-25 1985-02-14 Kitasato Inst:The 非う蝕性抗体および組成物
AU626049B2 (en) * 1989-05-29 1992-07-23 Lion Corporation Method for preparing vaccine for dental caries and vaccinal compositions for dental caries used as nasal drop

Citations (3)

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Publication number Priority date Publication date Assignee Title
GB1375866A (enrdf_load_stackoverflow) * 1971-06-11 1974-11-27
US3931398A (en) * 1973-06-20 1976-01-06 Colgate-Palmolive Company Method for local immunization against dental caries
EP0009872A1 (en) * 1978-09-01 1980-04-16 Secretary of State for Social Services in Her Britannic Majesty's Gov. of the U.K. of Great Britain and Northern Ireland Antigen preparation, process for producing it and pharmacological or dental preparations containing the antigen preparation or antibodies thereto

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1375866A (enrdf_load_stackoverflow) * 1971-06-11 1974-11-27
US3931398A (en) * 1973-06-20 1976-01-06 Colgate-Palmolive Company Method for local immunization against dental caries
EP0009872A1 (en) * 1978-09-01 1980-04-16 Secretary of State for Social Services in Her Britannic Majesty's Gov. of the U.K. of Great Britain and Northern Ireland Antigen preparation, process for producing it and pharmacological or dental preparations containing the antigen preparation or antibodies thereto

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Chemical Abstracts, vol. 85, no. 13, issued 1976, September 27 (Columbus, Ohio, US), S. Hamada et al: "Purification and immunochemical characterization of type e polysaccharide antigen of Streptococcus mutans", page 454, the abstract no. 92037y; & Infect. Immun. 1976, 14(1), 68-76 *
Chemical Abstracts, vol. 88, no. 17, issued 1978, April 24 (Columbus, Ohio, US), J.R. Wetherell Jr. et al: "Antigens of Streptococcus mutans: isolation of a serotype-specific and a cross-reactive antigen from walls of strain V-100 (serotype e)", page 393, the abstract no. 119292d; & Infect. Immun. 1978, 19(1), 160-9 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988006455A1 (en) * 1987-02-27 1988-09-07 Council Of Governors Of The United Medical And Den Antibodies against streptococcus
US5518721A (en) * 1987-02-27 1996-05-21 Council Of Governors Of The United Medical And Dental Schools Of Guy's And St. Thomas Hospital Antibodies against Streptococcus

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