DE2300329A1 - Anti-caries vaccine - contg antigenic material derived from buccal streptococcus ssc - Google Patents

Abstract

The antigen may be (i) a buccal Streptococcus SSC, (ii) any fraction of a buccal Streptococcus SSC, (iii) a dextransucrase type enzyme obtained from the culture liquid of a buccal Streptococcus SSC, or (iv) a distinct glucosyltransferase type enzyme isolated from the culture liquid of a buccal Steptococcus SSC. Streptococcus SSC is always found in precarious dental plaque on the smooth surfaces of teeth, and is indentical with a strain of S. mutans (NCTC 10449). Unit does of the vaccine pref. contain 1 x 106 - 2. x 1012 colony formation units of the microorganism, or 0.05-100 mg of dextransucrase or glucosyltransferase enzyme.

Description

Vaccine against tooth decay, process for their production and therewith Therapy to be Performed The present invention relates to a vaccine which to reduce the incidence of tooth decay on the smooth surfaces of the teeth, or able to prevent, it also concerns a process for the production of vaccines, also ampoules or other vessels, the unit or multiple doses of the vaccine and finally a therapy to be carried out with the vaccine.

Finding antibodies and bactericidal substances in saliva has met with great interest in the professional world, specifically in relation to their possible Relationship to human caries resistance.

Several studies have been published in which one Immunization processes have applied to the incidence and intensity of dental caries to reduce in laboratory animals.

V. Ross, F. Krasnow and J. Samet (J.D.R.

7,337) in 1927 rabbits were vaccinated with killed B.acidophilus. The sera showed agglutination to a greater or lesser extent. The spit the rabbit contained agglutinins in 7 of 14 cases, but the titer was far lower than that of the serums. They came to the conclusion: "If that is what it is the existing findings regarding the assumption that B. acidophilus eix the etiological factor in dental caries seems to substantiate it the test results presented in the present work have a certain Contribution to the experimental illustration in favor of experiments on vaccine prophylaxis and treatment.

In 1952, P. Jay et al. (J.Am.D.A.

20,2130) filtrates from 40 oral strains of B. acidophilus produced, the isolated from cases of dental caries.

A skin-reactive substance was observed in the filtrate.

In two cases there were negative skin reactions with B. acidophilus agglutinins in the blood serum after application of a polyvalent B. acidophilus-Vaceine. In 1933, P. Jay and coworkers came to us as a result of a series of attempts the following conclusion: "Bacillus acidophilus agglutinins can in general can be detected in the blood series of caries-free persons. The B. acidophilus agglutinin titer the blood series of certain susceptible Patient was given by administration a vaccine, which contained the R phase of B. acidophilus, to the titer of caries-free persons increased. Such a treatment procedure was generally accompanied by abscess formation at the vaccination site. "In 1942, C.P. Canby and J.L. Bernier (J.Am.D.A. 29,606) studies regarding the antigenic Behavior of 24 strains of Lactobacillus acidophilus from carious dentin and regarding the quantitative results of vaccination with L. acidophilus bacteria on this organism in the mouth. They reported about this: "The antigenic behavior of members of this group of organisms turns out to be a confusing complexity as determined by agglutination cross-tests. "The authors pointed out after that vaccination of human patients with vaccines obtained from strains of L. acidophilus-were manufactured to "stimulate the production of a substance appeared to have an inhibitory effect on L. acidophilus in the mouth. "They stated that the small number of cases on which they carried out their investigations made it would not allow any conclusions based of the results obtained. In 1943 V. Dietz, N.B.

Williams and W. Lawton (J.Am.D.A. 30,859) the strength of agglutinins for lactobacilli in the blood of 15 highly caries-prone and 15 against caries not vulnerable patients.

No significant differences could be demonstrated. the highest titers were accompanied by a negative Speiehel-Lactobacillus number regardless of the caries attack. The low agglutinin titers in the both Groups were not equally accompanied by high saliva counts. the authors came to the conclusion: "The high blood titers in our test results appear to be more of a sign of low levels of lactobacilli in the mouth to be regarded as a sign of caries infection.

In 1944, N. Williams (J.D.R. 23,403) vaccinated human patients both with organisms killed in the heat and with living organisms, namely different ones Lactobacillus strains. The vaccination increased the blood agglutinin titre for the in the strains used in the serum, with the maximum values around two weeks after the Vaccination have been achieved. The titers decreased after this period and were 5 Months after the last injections, the level of the unvaccinated measured values almost reached achieved.

Those measured in saliva over a period of four months Lactobacillus numbers showed that 15 out of 20 patients (i.e. 75%) were immunized in the Group an apparent decrease in average saliva counts exhibited. The statistical analysis showed that with only 5 of approximately 20 (25%) could be assumed that they had a reduction, the greater than what could be regarded as having happened by chance. The one for the lactobacilli Mean numbers measured in the controls showed changes within the limits of random variation were. The test results seemed to indicate that the immunization as carried out in this study was not even action on the number of lactobacilli, from the oral cavity could be grown.

Williams noted, "So little is there about the lactobacilli family known that the organism strains used may not have been the best.

In 1960, R.J. Fitzgerald and P.H. Keyes (Journal of the American Dental Assn. 61,9) Tooth decay in hamsters by giving them orally with a Strain of a Streptococcus infected that emerged from the plaque scrape of a carious Molaren from a hamster kept on a caries test diet had been isolated.

With the isolation of a specific microorganism that is involved in the Could be involved in triggering tooth decay has been the theoretical basis for the assumption that it should be possible to treat the disease with the help of a vaccine to fight, consolidated.

However, in Fitzgerald's early investigations, there are many organisms have been found that have been shown to be capable of Experimental animals produce tooth decay. These microorganisms include, along with many other strept. Liquefaciens (+ ND529) Strept. Faecalis (# ND547) Strept. sp. FAI and Lactobacillus Casei (# = ND465).

It appears that too few people are working on this problem Difference between the etiology of tooth decay on the smooth tooth surfaces and the Caries on the fissures, Recognized indentations and crevices in the teeth to have. The choice of laboratory animals can also give a misleading picture of tooth decay deliver; favor the contours of the hamsters' molar teeth, for example the development of carious lesions on the smooth tooth surfaces, whereas the Rat molars with their deep furrows favor the development of fissure caries.

Tooth decay or tooth decay is a disease of complex, multi-cause biology. It represents a convergent biological phenomenon in which 5 interrelated conditions must be present in order to trigger the development of tooth decay. These conditions can be described as follows: A. In the oral cavity, microorganisms with caries-producing Effect.

B. There must also be a suitable nutrient medium in the oral cavity that promotes cariogenesis and is capable of the growth of caries-causing To support organisms; and C. there must be teeth in the mouth, one Have a sub-optimal resistance to tooth decay, either directly for compositional or morphological or positional reasons or indirectly due to characteristic salivary properties, for which purpose Composition, pH, buffering capacity, saliva flow volume, viscosity and include the content of various antibacterial factors. Genetic causes can play an important role in controlling some of these factors.

Gnotobiotic studies have shown convincingly that Tooth decay does not develop in aseptic animals in the complete absence of bacteria. The complexity of the oral flora offers the possibility that more than one type of caries-producing organisms coexist in the oral cavity.

As mentioned above, a difference can be made between the tooth decay that forms on the smooth surfaces of a tooth, and the caries that has developed inside, a depression or a defect developed on the surface of a tooth. An essential prerequisite for the The development of tooth decay on the smooth surface of a tooth is the formation of a precarious dental plaque, i.e.

a circumscribed, raised stain that appears as a material deposit on the tooth surface and acts as a medium for bacterial growth. The sticky nature of the plaque keeps the caries-producing organisms contained in the plaque in intimate contact with the tooth surface. The initiation of the karien process can be summarized by the scheme -g microorganisms (MO) + suitable substrate (SS) 7 metabolized in a given time (T) with the generation of an acid as waste product, which leads to a local increase in the hydrogen ion (H) concentration the tooth enamel surface, ie

where the upward arrow indicates an increase in hydrogen ion concentration indicates.

Sufficient time for the reaction to occur can be on one can be reached by two routes. In the case of caries, the smooth surface holds the precarious Plaque the metabolizing microorganisms very close to the tooth.

In a depression or fissure, the organisms and the substrate can be protected against the expelling force of the flow of saliva by the physical contours of the depression or fissure. Therefore, the formation of precarious plaque is not necessarily an essential preliminary stage in the development of fissure caries. Numerous organisms encountered in the mouth are capable of entering into the reactions identified by the scheme be expressed. Theoretically, each of these organisms is able to trigger fissure caries. On the other hand, fewer oral microorganisms have the ability to stimulate the formation of precarious dental plaque.

These organisms can be distinguished by their ability Sucrose via certain exocellular enzymes to form complex polysaccharide polymers commonly known as dextrans. The result of this reaction is the appearance of a gummyJ sticky substance in association with the microorganisms and a large number of foreign components build up the conglomerate, known as dental plaque. The order for the formation of the precarious Dental plaque can be summarized as follows: 1. Certain types of potential Caries-producing organisms stay close to the tooth's organic cuticle at rest for a minimal amount of time.

2. The organisms begin to form certain components of the membrane metabolize.

3. Is sucrose brought into close proximity to the organisms or if it is already present in it, it becomes exocellular for processing Enzymes commonly known as dextran sucrases. These enzymes are able to produce complex polysaccharide polymers (dextrans) from the glucose and fructose units to synthesize sacoharose molecules.

4. The dextran binds the organisms, the substrate etc.

to the tooth surface, 5. The organisms can metabolize it continue within the dental plaque.

One of the objects of the present invention is now To produce a vaccine that is found to be effective in fighting tooth decay on the smooth surfaces of the teeth in humans.

Another object of the invention is to make it available a vaccine that is effective in stopping or preventing the Formation of precarious dental plaque on the smooth surfaces of the teeth.

The disclosure also belongs to the subject matter of the present invention a Vaocine therapy, thereby preventing the occurrence of tooth decay on the smooth Surface of the teeth reduced, brought to a minimum or completely prevented will.

The invention relies in part on isolation and knowledge the importance of an oral streptococcus that in the precarious Dental plaque is always present on the smooth surfaces of the teeth. This streptocoocus, referred to in this description of the invention as Streptococcus SSC, is used below more closely characterized. It should be noted that there are many tribes of Strept. SSC there.

The present invention is directed to a vaccine which can be used as Antigen or as part of the antigen at least one of the following components (a) to (d) contains: (a) Streptococcus SSC; (b) any part, especially cell walls, any oral strept. SSC; (c) a relatively crude dextran urase-type enzyme, that from the broths of any oral strept. SSC is obtained; and your or several enzymes of the particular glucosyltransferase type obtained from the culture fluids any oral strept. SSC can be isolated.

So can diaccine from the oral organism Streptococcus SSC or from a part of this organism or from enzymes such as those in the culture fluids this organism are present.

Treatment with the vaccine stimulates antibody production, so that these antibodies, if transported through saliva, are likely have a disruptive effect on the property of the Streptococcus SSC, an established component to become or remain in the oral cavity, and / or probably have a disruptive effect on the ability of the strept. SSC, certain extracellular enzymes (dextran sucrases or certain glucosyl transferases ) to create. As a result of that preventing these enzymes from synthesizing dextrans from sucrose reduces the formation of precarious dental plaque on the smooth surfaces of the teeth, inhibited or prevented. In short, the vaccine stimulates salivary antibodies either against the Streptococcus SSC or the glucosyltransferase entyme, which from Strept. SSC can be generated.

Let us now consider the insulation and the characteristic properties the oral Streptocooci, referred to in this description of the invention as Streptococous SSC will be explained in detail.

There were 246 leolates of the organism from the dental plaque material of 246 patients who were suffering from active tooth decay were obtained, which the smooth ones Surfaces of the teeth had attacked.

Cultivation characteristics Growth on Mitis Salivarius agar (Difco B298).

(a) At 370C in an atmosphere of 95% N2 + 5 ffi CO2 in 24 hours.

(b) 5 to 6 colonies are isolated from each dish and applied directly a Streptococcus broth transferred containing 0.4% (weight / volume) glucose and 0.05% (volume / volume) Tween 80 (cf. Jordan, Fitzgerald and Bowler, J.D.R. 39,116) contains.

(c) Pure cultures were obtained by alternating growth Blood agar and Mitis Salivarius agar, incubated anaerobically and then on blood agar held. Each isolate is grown as a minor culture every 4 weeks.

All isolates represent immobile, catalase-negative, gram-positive Represent cocci that occur in short chains or in chains of medium length, if they are grown in bouillon. No isolate produced a gas in glucose media.

Variations in the morphology of the colonies on anaerobic blood agar 234 isolates represented white, circular or irregular colonies that were found in their size varied between 0.5 and 1.0 mm. The elevations varied from humped to cushion or cushion-shaped, the surfaces were smooth. The colonies were hard and coherent and firmly adhered to the agar surfaces.

12 isolates (4.8%) showed a convex elevation and a circular one Shape. They were soft and sticky when touched with a platinum wire.

225 isolates retained crude colony morphology on an anaerobic Mitis Salivarius Agar. 7 developed mucous colonies and 14 smooth colonies. The 7 mucous isolates had a colony diameter of 1 to 2 mm and a "gum drop" appearance (see G.H. Chapman, J.Bact. 48,113 (1944)). The colonies were transparent in both incident and transmitted light. the 14 smooth colony isolates had a colony size of 0.5 to 1.5 mm. she were of circular shape and the elevation was convex to humped and the surface smooth and opaque.

All isolates (-246) produced tooth decay scores in albino hamsters which had been infected with the isolate. (Average Tooth decay about 240 compared to 8.9 for controls).

Fluorescent antibody testing methods were used to demonstrate that all isolates were serologically related.

The physiological properties are summarized in Table 1.

Table 1 characteristic number of the% of Properties of isolates total number Final pH after leaving for 48 hours #### in the sucrose broth PH 4.0 - 4.3 246 100% "- 4.400 in the glucose broth pH 4.0 - 4.3 246 100% "4, 4 OO Reaction in litmus milk Attenuation before curdling 0 0 Acid and coagulation 246 100% thick precipitate in sucrose after adding 1 part ethanol 246 100% Aesculin hydrolysis 220 89 9 Table 1 - continued characteristic number of the% of Properties. Isolate total number Reaction to blood agar aerobic + 10% C02 α 12 5% ß 180 73% γ 36 15% Growth at pH 9.6 0 0 Growth at 10 ° C 0 0 Growth at 45 ° C 0 0 4% growth. NaCl 231 93% 6.5% growth. NaCl O 0 Growth on blood agar (10%) 237 96% Growth to 0.04% tellurite Blood agar 0 0 Reduction of methylene blue 0 0 Acid from: D (-) - sorbitol 240 97% Lactose 246 100% Inulin 246 100% Table 1 - continued characteristic number of the% of Properties of isolates total number D (-) - Mannitol 246 100% D (-) -Mannose 246 100% Glycerin 0 0 D (+) - xylose 0 0 D (-) - arabinose O 0 Dulcit O 0 Trehalose 246 100% 10 representative isolates of Streptococcus SSC were grown in loose piles, freeze-dried and used as base material for all further investigations. These representative isolates were named Streptococcus SSC MS1 through Streptococcus SSC MS10.

The one referred to in this description of the invention as Streptococcus SSC Microorganism has characteristic properties that are identical to those of the strain known as "NCTC 10449" in the "National Collection of Type Cultured ', London, England. That tribe "NCTC 10449 "is described as" a representative strain of Streptococcus mutans, that of Dr. W. Sims isolated from the deepest layer of carious dentin in 1965 has been'. It goes without saying, therefore, that the names Streptoooccus SSC and their abbreviations Strept. SSC and S. SSC wherever they appear in this description of the invention should be replaced by the term "Streptococcus mutans (NCTC 10449)" can.

The vaccine for reducing the occurrence of tooth decay on the Smooth surfaces of the teeth can come from the living, weakened or dead organism Strept.SSC or any part of this organism, especially the cell wall will. It has also been found that the enzyme can be made from the enzyme that in the culture broths of the organism Strept. SSC is in place, either from the crude dextran sucrase type enzyme or from one or more specific ones Glucosyltransferase enzymes isolated from the culture fluid.

The Vaocine can be a simple or monovalent vaccine, or it can be a multivalent Vaocine, which is made up of two or more isolates of Strept. SSC has been established. Furthermore, the vaccine can be complex or mixed Be vaccine in which the antigen component is made up of any two or more (i) any strept. SSC organisms, from (ii) a portion of any Strept SSC organism, the end (iii) a particular enzyme of the glucosyltransferase type and consists of (iv) a crude enzyme of the dextran sucrase type, the enzymes (iii) and (iv) in a culture broth of a strept. SSC organism or isolates present are. Of course, the vaccine can consist of any mathematical permutation or a combination of the antigens (i), (ii), (iii) and (iv).

The vaecine can be an autogenous vaccine, in which case the antigen in the vaccine a strept. SSC is isolated from the recipient's own mouth or is an enzyme of the dextransucrase or glucosyltransferase type, as it is obtained from the culture fluid or fluids of such a strept. 8SC isolate have been won.

The Vaceine can contain an accompanying substance and it can additionally or alternatively contain a preservative. Of course everyone has to Accompanying substance and any preservative must be pharmaceutically acceptable and must not be corrosive to the antigen or to any other component of the Act Vaccine. Of course, the vaccine does not necessarily need an accompanying substance or to contain a preservative at all.

In addition to the antigen, the vaccine can also contain one or more others contain medicinal or therapeutic agents, provided that these agents contain non-corrosive or detrimental to the other components the Take effect of the vaccine. In particular, the vaccine can have a predetermined or measured dose of a local anesthetic such as lignocaine.

The vaccine can be in a unit dose or a multi dose form ready to use, e.g. as a liquid formulation in sealed glass or the like. Ampoules or containers are made available. Each single dose has been expedient a volume of 1 ml or 1.5 ml, but the volume of the single dose can of course be bigger or smaller.

The vaccine antigen can, whether it is in the form of the organism strept. SSC or in the form of part of such an organism or in the form of one or more Enzymes obtained from the culture fluids of the organisms are present, Freeze-dried or otherwise dried, and the vaccine can ready for injection immediately before use, e.g. by adding a suitable liquid medium such as sterile saline, i.e. 0.85% (Weight / volume) solution, or a solution of a local anesthetic.

A single or unit dose of the vaccine obtained from the living, weakened or dead organism strept. SSC has been produced, e.g. contain the equivalent of 1 x 106 to 2 x 1012 colony-forming units (c.f.u.), wherein such a binfach or unit dose is preferably in liquid form and is expediently present in a volume of 1 ml. There are samples of the vaccine that are up down to 2 x 106 c.f.u. per ml and up to 2 x 1012 c.f.u. contained per ml, has been manufactured.

Of course, however, the concentration of the antigen in the Vaccine less than 1 x 106 c.f.u. per ml and more than 2 x in12 ¢ .f.u. per ml. A preferred single or unit dose of the vaccine in liquid Form contains 2 x 109 + 10% c.f.u.

When the antigen in the vaccine comes from the relatively crude enzyme of the dextran sucrase type, which consists of the supernatant culture fluids of the Strept. SSC has been received, so are unit or single doses of the vaccine have been prepared containing 0.25 mg to 100 mg of the dextran sucrase. Such Unit or single doses should conveniently be in the form of a one volume solution from 1 to 1.5 ml. The specific amount of dextransucrase in a unit dose fluctuates with the activity of dextran sucrase. A single or unit dose suitably contains an activity (as defined below) of 500 up to 5,000 dextran sucrase units (DSU). An appropriate unit dose contains an activity of about 1,000 DSU, e.g. from 900 to 1,100 DSU.

As mentioned above, the vaccine can also be prepared are made from one or more of the purified and determined glucosyltransferases, those from the culture fluids of the strept. SSC have been isolated, and are Single or unit vaccine doses have been made that contain 0.05 mg to 100 mg each or contain more of the specific glycosyltransferases. Such doses were prepared as solutions, and the unit dose suitably has a volume of 1 ml. Again, a unit dose of the vaccine, consisting of one or more the specific glucosyltransferases prepared has been, in turn an activity of 500 to 5,000 DSU. An appropriate unit dose has an activity of about 1,000 DSU. A unit or single dose of one complex vaccine can, for example, 2 x 106 c.f.u. up to 2 x 1012 c.f.u. from the Organism strept. SSC and / or contain 0.05 mg to 100 mg of the enzyme, which the latter either from the dextransucrase enzyme or a specific glucosyltransferase enzyme, those from the culture fluids of the strept. SSC has been obtained.

Single doses of the vaccine (e.g. antigen + accompanying substance + preservative) are conveniently in sterile, sealed ampoules containing 1 to 1.5 ml of the vaccine included, made available. Ampoules have also been made that down to 0.1 ml of the vaccine and up to 100 ml (multi-dose ampoule) the Vaocine included.

The vaccine dose is preferably subcutaneous, intramuscular or intravenous injected. It is preferably injected into the mouth as it has been determined that then a faster build-up of antibodies in the saliva occurs than when one injected into a place far from your mouth. It is of particular advantage when the vaccine dose expediently in a volume of 1 ml to 1.5 ml in the buccal furrows in the oral mucosa in the four quadrants of the mouth will. The amount of vaccine injected into each of the four quadrants is preferably essentially the same, i.e., approximately 0.25 ml Case of a 1 ml dose.

A treatment procedure with the Vaooine can vary widely be performed. An average regimen can be 2 to 4 doses or Require injections, each about 2 x 109 c.f.u. or contained about 1,000 DSU. Of course, the number of doses can be larger and so can the total antigen dose to be taller.

The invention will now be described in relation to the vaccine and method for the production of the vaccine as well as using an immunization protocol in the The following examples are explained in more detail, but the invention is intended by the following Examples are in no way limited.

Example 1 (a) The strain of the organism Strept. SSC MS2 was built in in this particular case for 18 to 24 hours at 370C in a Bactobrainheart infusion broth bred.

(b) 1 ml of the strept. SSC MS2 culture was applied to the surface of a flat layer of a Bacitobrain containing an addition of 2 ffi Bacto-Agar inoculated with kyrart infusion broth, which was in a Blake Kölbehen. the Blake flasks were incubated for 24 hours at 37 ° C. and the cells in sterile saline solution [0.85% (w / v) NaCl collected.

(c) The cells were washed three times in sterile saline solution (0.85%) alternating centrifugation and re-.

suspend washed.

(d) The cells were then placed in sterile saline (0.85%) plus 0.6% formalin suspended and incubated overnight at room temperature to so kill the cells.

(e) The cells were washed again in sterile saline solution, and the bacterial strain was then tested for sterility by means of inoculation of 0.1 ml portions to 10 test tubes each with a BBL Fluid thioglycolate medium were filled and the tubes were opened at 37 ° C. for 10 days.

(f) As a preservative, merthiolate became the sterile stock suspension given up to a final concentration of 1 t 10,000.

Standardization of the antigen for immunization.

(g) The parent strain of the sterile antigen Ed.h. the product of the stage (f) 7 was aseptically sonicated for 5 minutes at lowest intensity to avoid breaking up of the chains of cells.

(h) The sonicated stock suspension was then treated with merthiolate-containing Saline solution (which is a sterile 0.85% saline solution that merthiolate is in a concentration of 1: 10,000) up to an equivalent of 4 x 109 c.f.u. (colony-forming units) per ml, based on a pre-standardized McFarland nephelometer curve recorded in a Klett-Summerson colorimeter with a 420 millimicron filter was read.

(i) The final antigen suspension for injection was prepared by diluting in a ratio of 1: 1 with a merthiolate saline solution for the purpose of Preparation of a finished suspension which is an equivalent of 2 x 109 a.f.u. Per ml.

Immunization Protocol (One Method).

The first injection of a total of 0.5 ml (1 x 109 c.f.u.) in the form the merthiolate saline suspension of stage (i) was absorbed into the oral mucosa injected into several places. The same doses were given on the 7th and 14th day, and on day 21, a total of 1 ml (2 x 109 c.f.u.) of antigen was introduced into the mucous membrane of the cheek injected. A repeat injection (1 ml = 2 x 109 c.f.u.) was given on 120. Day given. Three days after this repetitive injection was a significant one An increase in the level of antibody production in the saliva can be detected.

Example 2 (a) The cells of that Streptococcus SSC strain, which further has been designated as MSI above, were as in steps (a) and (b) of the example 1 grown and collected in sterile distilled water. The cells were three times in distilled water by alternating centrifugation and resuspension washed.

(b) The cells were suspended in distilled water + 0.6 ffi formalin and incubated overnight to kill the cells.

(o) The cells were again washed three times in distilled water, and sterility tests were carried out as in Example 1, step (e).

(d) Merthiolate was now added to the suspension as a preservative up to the setting of a final concentration of 1: 10,000.

(e) The stem mass suspension was exposed to a sound source for 10 minutes Sonicated at the lowest intensity.

The sonicated suspension was further washed with distilled water, the merthiolate contained in a concentration of 1 t 10,000 ,. up to an equivalent of 2 x 109 c.f.u. per ml diluted based on a pre-standardized McFarland Nephelometer curve recorded in a Klett-Summerson colorimeter with a 420 millimicron filter was read.

(g) The suspension was further sonicated for 10 minutes.

(h) One liter of the stock suspension was placed on a water bath and heated to 450e. 0.75% (weight / volume) Carbopol was added to the suspension 934 (a carboxyvinyl polymer) or Carbopol 940 or 941 added. After an hour With stirring, 0.5 ffi (w / v) sodium carbonate was added to the suspension and stirring was continued for 2 hours.

(i) The suspension was placed in stoppered glass containers using no more than 50 ml capacity poured and saturated steam in for 30 minutes exposed to an autoclave at 115 to 11600.

(j) Each serving was tested for sterility as in Example 1, step (e) tested.

(k) Under strictly aseptic conditions, the vaccine was administered in single-dose and multi-dose vials filled.

Immunization Protocol The first injection of a total of 1 ml (2 x 109 c.f.u.) was placed in several places on the oral mucosa. One second injection of the same dose was given on day 14. One repeat injection (the same dose) was given on the 60th day.

Days after this repeat injection could be significant Increases in the level of antibody production in saliva may be noted.

Example 3 (an autogenous vaccine) In this example the organism a strain of Streptococcus SSC, isolated from a particular patient, and the Vaccine was made from that strain of the organism.

The organism was isolated in pure culture, grown in bulk, collected and washed thoroughly.

The organisms were suspended in sterile saline. It was then estimate the approximate number of organisms per ml of suspension. For the production of the autogenous vaccines, living, attenuated, or dead organisms can be used will. Any dose or series of doses consisting of a certain number of Organisms (which are calculated in millions) can exist in a volume amount of 1 ml to be prepared in such a way that one suitable from the original suspension Dilutions in either sterile saline (0.85 ig) or carbolic saline (0.85% NaCl + 0.5% phenol).

A portion of the sterile saline suspension was added with more Quantities of sterile saline solution (0.85%) diluted to obtain a vaccine, the approximately 2 x 109 o.f.u. per ml. The other serving was made with the carbolic saline solution diluted to produce an attenuated autogenous vaccine that is approximately 2 x lO9 c.f.u. per ml dose. - Multivalent vaccines can be prepared by using more than 1 isolate of Streptococcus SSC. In this case, pure cultures of the organism must be preserved and standardized separately Suspensions are produced. Appropriate concentrations of each are then combined in the final preparation.

Example 4 Two or more organisms producing different isolates of the Streptococcus SSC, were isolated in pure culture, grown in bulk, collected and washed thoroughly. Individual standardized suspensions of the Organisms were prepared in sterile saline (0.85%). The individual Suspensions were then combined in substantially equal amounts to a total of 1 x 106 to 2 x 1012 c.f.u. per ml, preferably from about 1 x 1Q9 to about 2 x 109 c.f.u.

per mole, to be delivered.

The example was repeated, but using carbolic saline solution (0.85% NaCl + 0.5% phenol) instead of the sterile saline solution (0.85% NaCl).

Example 5 Following the procedure of Example 1 up to step (i) worked with the modification that the strain of Streptococcus SSC, known as MS3 is designated instead of the strain MS2 and thiomersal as a preservative were used in place of merthiolate. The antigen suspension was then placed in a Added suspension of aluminum hydroxide in saline solution. (NB: every finished dose must not contain more than 15 mg aluminum hydroxide).

Example 6 Example 5 was repeated with the modification that Aluminum phosphate was used instead of aluminum hydroxide.

Example 7 Example 2 was reworked up to stage (g), and then the following work steps were carried out: (h) It was Potassium alum solution added to the stock suspension so as to cause precipitation obtain.

(i) The precipitate was separated and thoroughly distilled three times Washed water, after which the precipitate was then resuspended in distilled water became.

(j) 1 liter of the suspension was as in step (h) of Example 2 treated.

(k) The sterilization and filling of the vaccines into the vials was carried out as in steps (i), (j) and (k) of Example 2.

As will be explained below, the vaccine can use enzymes of the dextran sucrase type contained as the sole antigen component or as part of the antigen component, the enzymes used for the production of the vaccines of the dextran sucrase type from the supernatant culture fluids of one or more strains of Streptococcus SSC have been obtained.

Dextransucrase activity is determined by measuring the release reducing sugar according to the method of M, Somogyi, J. Biol. Chem 160.61 (1945). Dextransucrase activity is expressed as dextransucrase units (DSU) expressed as the amount of enzyme that dissolves 1 mg of sucrose in dextran in 1 hour converts with the release of 0.52 mg fructose under standard conditions (cf. H. Koepsell and Tsuchiya, J. Bact. 63, 293 (1952)). Those for use in the Anticaries vaccines, certain dextran suorase, can be extracted from the supernatant culture fluids of Streptococcus SSC can be obtained in several ways. The following is a Description of five suitable methods for obtaining the enzymes of the dextransucrase type given.

Method 1 An aqueous medium of 4 liters, the yeast extract (1), Contained Na2NH4P04 (0.5%), KH2P04 (0.1%), MgS04.7 H2Q (O, T%) and sucrose (10%), was adjusted to pH 7 with sodium hydroxide and then at 1.05 for 30 minutes Steam sterilized kg / cm² (15 lb / sq. in.). After inoculation with an actively-growing one Culture of the selected organism Streptococcus SSC M52 was there for 17 hours 250C incubated. The culture (PH 6.6) was cooled to OOC and it became gradual Ethanol (2160 ml; final concentration 35% by volume) was added with stirring. It was done carefully careful attention to the temperature during this procedure and all subsequent procedures to hold at 0 to 1 0C. After 1 hour a dark, gummy precipitate became collected by centrifugation (2,000 rpm), in 0.1 molar citrate buffer (500 ccm; PH 6.0) and 30 minutes at 5,000 rpm. centrifuged so as to be cell-free Obtain enzyme solution. Then 0.1 molar citrate buffer (2,500 ccm; pH 6.0) added to obtain a solution containing approximately .1.5 units of activity per ccm contained. The dextran sucrase was isolated from this solution, by removing the precipitate, which is obtained by adding ethanol (1 liter; final concentration 25% by volume) overnight, dissolved in water (200 ccm). After the clarification by centrifugation at 5,000 rpm. the supernatant solution was freeze-dried and stored at OOC as a brown powder (4.06 g).

Method 2 Cultures of the organism Streptococcus SSC MS2 were in 2 liter flask grown on a sucrose-peptone-salt medium similar to that corresponded to that of H.J. Koepsell and H.M. Tsuchiya (J.Bact. 63 (1952) 293) and H.M. Tsuchiya and others (1952 J.Bact. 64,521) had been used, but in which Peptone (Bactopeptone from Difco Labs. Inc., Detroit, Mich.) The corn steep liquor replaced. Using a 5% inoculum from a previous approach good growth was achieved in about 16 hours at 2500. The PH fell from one Initial value from about 7.2 to a final value of about 5.5 s with no attempt being made in order to keep the pH constant, for example by adding alkali. The final values of the Dextransucrase levels were usually 80 to 100 DSU per ml.

When the enzyme level reached a peak, fermentation stopped terminated by adding a small amount of toluene, the + was with aqueous sodium hydroxide adjusted to about 7.0 to precipitate foreign material and the bacteria were removed by centrifugation in a Servall Angle centrifuge, Type SS-1A, or for larger batches by filtering through a sparkler filter. After removal the bacteria became the enzyme solution with aqueous sodium hydroxide adjusted to a pH of about 5.0 and the enzyme was purified by adding 400 g Ammonium sulfate precipitated per liter. The precipitation was carried out in a cold room 5 0C carried out. Precipitation with ethanol, as described by H.M. Tsuchiya in 1955 in J.Am.Chem.Soc. 77,2412, it was found that more dextran precipitated than it did Ammonium sulfate did, and consequently made a less pure enzyme preparation. The enzyme precipitates were centrifuged off, with cold ammonium sulfate (40%, calculated as weight / volume) Washed and cold ethanol and then in an acetate buffer of pH 5.2 again solved. Analyzed on the basis of their dry weight, they pointed to this Enzyme preparations made in this way usually have about 200 DSU per mg.

Method 3 Using the Streptococcus SSC MS5, the enzyme according to the method of J. Cybuloka and R. Pakula (1963, Exp. Med. Microbiol. 15, 187) and partially purified by means of elution from hydroxyapatite (R. Dedonder et al., 1965, Bull. Soc.

Chim. Biol. 45,477).

Method 4 An aqueous medium (140 cc), the yeast extract (1%), Na2NH4P04 (0.5%), KH2P04 (0.1%), Mg04 7 H20 (0.5%), sucrose (2%) and maltose (10%) was adjusted to pH 7.0 with sodium hydroxide for 50 minutes at Steam sterilized and re-sterilized under sterile conditions adjusted to pH 7.0.

After inoculation with the selected organism Streptococcus SSC MS4 it was worked at 250C for 30 to 36 hours until the pH of the culture was 5.9. The bacterial cells were removed by centrifugation and it became ethanol (47 ccm; final concentration 25 vol.-k) added to the culture liquids, whereby the temperature was maintained at 0 ° C during this and subsequent operations became. After 1 hour the suspension was centrifuged, and the precipitate, the 6% of the dextran sucrase originally present in the culture, as after the standard method was determined in the presence of 100 mg of maltose discarded. Another precipitation representing 25% of the original dextransucrase activity was also discarded after centrifuging the suspension that contained by further addition of ethanol (1) cc; Final concentration 50% by volume) had been. The supernatant liquid was cooled to -200C and it became gradually a further amount of ethanol (80 ccm; final concentrate 50% by volume) was added with stirring. The suspension was kept at -20 ° C. for 1 hour and then at -18 ° C. for 20 minutes centrifuged (2,000 rpm). The heavy precipitate was dissolved in 0.05 molar citrate buffer (PH 5.0; 40 ccm) and 48 hours against 0.05 molar citrate buffer (PH 5.0; 2 x 2 liters) dialyzed at 0 ° in a dialysis tube that is thoroughly distilled with Water and 0.05 molar citrate buffer (pH 5.0). The solution was then freeze-dried to a powder (0.702 g).

Method 5 Designated an isolate of Streptococcus SSC, namely MS7 Strain, was in a five-liter fermenter in a Bactobrainbeart infusion broth 18 hours at 5700 grown. The pH was constant 6.0 and the atmosphere consisted of 5% 002 and 95% N2.

At the end of the 18 hour period, the culture was at 10,000 x g Centrifuged for 5 minutes at 40C. The cells were then discarded, and the supernatant Liquid was washed with cold (NH4) 2 SO4 g53 Xig; (Weight / volume) J treated. To 8 hours, the liquid and the precipitation through a glass suction filter with a sintered glass filter part (D5: D4) filtered and the filtrate discarded.

The precipitate was dissolved in 0.005 molar phosphate buffer (pH 6.0) and dialyzed against the same buffer. The supernatant liquid was discarded, and the dialyzed dextran sucrases were treated with 87 volume percent acetone (at -200C), The solution was centrifuged at 20,000 x g for 10 minutes at 400C. The sediment was then dissolved in 0.001 molar phosphate buffer (pH 6.0) and lyophilized.

In the following, different methods for the production of Anti-caries vaccines are described, both as the antigen component of the vaccines the dextran sucrase-type enzyme obtained from the culture supernatants was used any isolated strain of Streptococcus SSC was obtained.

Example 8 Using Method 4 for the Production of Dextran Sucrase it was found that 1 liter of the liquid medium containing Streptococcus SSC MS6 had been inoculated, ultimately delivered 7.5 g of dextran sucrase, the activity of the Enzyme was determined to be 200 DSU per mg.

(a) 0.5 g of the dextran sucrase was added to each of 15 containers, each 100 ml sterile saline solution (0.85 Xig) with merthiolate as a preservative, which had been added to a final concentration of 1: 10,000.

(b) The contents of the containers were placed on a water bath for 6 hours stirred at 40 ° C under strictly aseptic conditions.

(c) The sterility tests as in Example 1 were then carried out.

(d) The vaccine was supplied in either 1 ml or filled in multi-dose containers under aseptic conditions.

Each 1 ml dose of the vaccine contained 5 mg dextran sucrase with a DSU activity in this example of about 1,000 units per dose.

Immunization protocol.

A 1 ml dose was injected on the first day, and the injection of the Dose was repeated on day 14. Another dose was on day 21 and one Repeat dose given on day 120. All injections were into the buccal lining given. 3 days after the repeat injection there was a sharp increase in magnitude antibody production in saliva, i.e. on the 123rd day.

Example 9 An approximately equal amount of the enzyme was used in that in Example 8 but manufactured using the strain of Strept. SSC, referred to as MS8 instead of MS6.

(a) 0.5 g of the dextran sucrase was added to each of 15 containers filled, each containing 100 ml of sterile distilled water, which a Addition of merthiolate as a preservative in a final concentration of 1: 10,000.

(b) The containers were placed on a Dei 400C water bath for 1 hour intensely stirred. Then 0.5 ffi (weight / volume) of Carbopol was added to each container 940 or Carbopol 941 or Carbopol 934 added. The stirring became another Continued for hour.

(c) 0.3% sodium carbonate was added to each container, and that Stirring was continued for an additional two hours.

(d) The contents of each container were then subjected to sterility tests carried out as described in Example 1.

(e) The vaccine was then under strict aeseptic conditions in Filled single-dose or multiple-dose containers.

Each finished 1 ml dose of the vaccine contained 5 mg of dextran sucrase with a DSU activity of approximately 1,000 units per dose.

Example 10 Example 8 was reworked up to step (b) with the modification that thiomersal as a preservative instead of merthiolate was used. Then the procedure was as follows: (c) In each with the vaccine filled container was a suspension of aluminum hydroxide in sterile saline solution admitted (NB: Each ready-to-use dose of 1 ml of the vaccine must not exceed 15 mg Contain aluminum hydroxide).

(d) The sterility tests were carried out as in Example 1, and then the vaccine was packaged.

Example 11 The procedure of Example 10 was repeated but with the modification that aluminum phosphate instead of aluminum hydroxide was used.

Example 12 A mixed multivalent vaccine was made.

Using the isolate Strept. SSC MS2 were the stages (a) reworked to (g) of Example 1, and a sonicated one was obtained Stammassen suspension.

In addition, dextran sucrase, which has an activity of 200 DSU per mg exhibited, from the strept. SSC MS6 as described in Example 8, prepared, and 0.5 g of the dextran sucrase was added to each of a number of containers, each containing 100 ml of the sonicated strain suspension, followed by merthiolate was added up to a concentration of 1: 10,000. After that were the stages (b), (c) and (d) of Example 8 were carried out.

The formation and significant importance of extra -cellular bacterial Polysaccharides in the aetiology of tooth decay is in. Full details by Fitzgerald and Jordan 1968 under the title "The art and science of caries research't, publisher R.S. Harris, Academy Press., New York, and von Newbrun, 1967 in Odontol. Rev. 18,373 has been published.

The ability to synthesize insoluble glucans is a fixed property of the streptococci that cause tooth decay on the smooth surface of the tooth and are referred to in this description of the invention as Strept. SSC are designated. The enzymes responsible for this synthesis are classified by the "International Commission on Enzymes" as "Glucosyltransferases" and belong to the subgroup of "Hexosyltransferases" EC 2.4.1.x. For the final identification of each glucosyltransferase, further knowledge is required regarding the exact binding that occurs during the synthesis. However, the general reaction they catalyze can be represented as follows: whereby a glucosyl unit is transferred to a growing glucan polymer (glucan) n to form the larger, growing polymer molecule (glucan) ni, and 1 molecule of fructose is set free for each sucrose molecule used. No glucan initiator is required to start the transfer, since the sucrose itself can apparently serve as both an acceptor and a glucosyl donor.

Polysaccharides are important components of precarious dental plaque. Numerous authors including Wood, 1967, Arch.

Orally. Biol. 12, 1959; Gibbons and Keyes, 1969, Arch. Oral Biol. 14,721; and Gibbons and Fitzgerald, 1969, J. Bact.

98,341, have referred to dextran sucrase, which is obtained from oral streptococci and concluded that an enzyme is responsible for dextran synthesis was.

However, the polysaccharides produced by these organisms are as well as chemically and morphologically heterogeneous. From the inventor of the present invention however, it has been found that several (at least 4) certain glucosyltransferases can be isolated from the supernatant culture fluids of the Streptococcus SSC can. The glucosyltransferases differ in their isoelectric points, the pH optima, the Km value and the like. What their characterization as in certain Wise differentiated enzymes justify. Any or all of these particular glucosyl transferases can be used as the antigen for the production of the anti-caries vaccines according to the invention should be made available.

Certain glucosyl transferases were obtained from the culture supernatants of Streptococcus SSC isolates are obtained in the following manner.

Approaches of the selected organisms were made in five-liter laboratory fermenters with automatic pH control adjusted to the value 6.0 under a constant flowing gas mixture (0.5 liters / minute) of N2 (95%) and C02 (5%) allowed to grow. During the intervals between the addition of alkali (2-n NaOH), stirring took place (400 Rpm). When acid production stopped, the surviving culture fluid became collected after centrifugation for 15 minutes at 25,000 x g and 40 ° C. All Cleaning stages were carried out at a temperature between 40 and 60C.

2 liters of distilled water became the culture supernatant given to decrease the ion concentration to facilitate absorption. there has been a Slurry of hydroxyapatite (250 ml to 275 ml) was added, and clumping of the hydroxyapatite was observed immediately thereafter. The mixture was run continuously for several hours, leaving the slurry Sediment overnight and the supernatant was then removed. That Sediment was washed three times with 0.01 molar phosphate buffer, pH 6.0, to remove the remove unabsorbed material. The hydroxyapatite was then put into a column filled (2.5 cm diameter). When the column thus formed was eluted stepwise using 0.2 molar and 0.5 molar phosphate buffer, PH 6, o so entered the enzymatic activity as 2 peaks, namely after the 0.2 molar Stage using phosphate buffer and as a further peak after the stage with the 0.5 molar Buffer.

The amount of activity (DSU) that emerges from the stumps (pools) of each Stage recovered was about 15% in the stage with the 0.2 molar buffer and about 18, at the same level as the 0.5 molar buffer.

In another example in which an elution with a linear gradient (0.05 molar phosphate buffer, PH 6, o to o, 4 molar; phosphate buffer, PH 6.8) 3 specific enzyme peaks were detected. The first and low peak occurred at the 0.08 molar phosphate concentration, and it coincided with the bulk of the amount of the protein. The second enzyme peak appeared at the 0.12 molar phosphate concentration. The third and major activity peak was at 0.17 molar phosphate concentration eluted; it gave the best purification and it corresponded to a yield of about 15.% of the initial activity.

A representative cleaning operation is shown in Table 2. Hydroxyapatite chromatography with step-by-step elution (0.2 molar and 0.5 molar Phosphate buffer) resulted in two pools which, taken together, were ten times smaller of the volume of the culture filtrate and a recovery of the activity of about 43% corresponded.

The subsequent isoelectric focusing of the two swamps had result in the occurrence of up to 7 peaks of glucosyltransferase activity.

Table 2 Purification of Glucosyltransferases from Streptococcus SSC. Volume total pro spec. The end- men enzyme units in active Procedure ml DSU / ml th mg / ml would take place Culture filtrate 5100 3.0 15 180 18 0.17 100% absorbed on Hydroxyapatite 13050 86% Hydroxyapatite Chromatography 0.2 mol. Eluate 385 8.5 3273 1.2 7.08 21.6% (Fr. 33 to 100) 0.5 mol. Eluate 223 14.75 3290 0.8 18.44 21.7% (Fr. 101 to 140) isoelectric Focus 0.2 mol. Eluate Sump 1 Ip 5.65 58 3.1 180 0.02 155 1.2% Sump 2 Ip 4.2 70 4.25 298 0.14 30.36 2.0% Bottom 3 rest 132 2.6 343 0.15 17.33 2.3% 0.5 mol eluate Sump 1 Ip 6.03, 5.85 and 5.65 67 6.25 419 0.02 312.5 2.8% (Fr. 25 to 34) Sump 2 Ip 5.4 6 7.25 44 0.10 72.5 Q3% (Fr. 36) Sump 3 Ip 5.0 68 9.75 663 0.12 81.25 4.0% (Fr. 39 to 47) NB. Fr. means "fraction", each fraction having a volume of approximately 6 ml.

Ip means "isoelectric point".

Glucosyltransferases with isoelectric points of 4.2, 4.6 and 5.6 predominate in the 0.2 molar sump. The more important glucosyl transferases in the 0.5 molar eluate have an isoelectric point of 5.0 and 5.6.

The relative amounts of the glucosyltransferases are shown in Table 3.

Table 3 Ip estimated% - values of the total activity according to LEF +) of Bottom 0.2 mol eluate 0.5 mol eluate combined eluate 4.2 40 + 16.9 2 t 2.5 21 t 22.4 4.58 17 + 6.7 1 t 2, 0 9 t 10.3 5.00 7 5 .3.0 60 + 20.7 34 + 32.1 5.4 ------ 2 t 5.0 1 + 3.8 5.65 26 + 15.6 25 # 20.5 25 1 17.1 5.8 4 # 1.7 3 # 6, o 4 + 4.4 6.03 4 1 3.2 6 + 9.0 5 t 6.7 +) IEF means isoelectric focusing. Some key figures of the main glucosyltransferases from strept. SSC MS2 are summarized in Table 4.

Table 4 isoelectri- optimal Km +) enzyme denotes shear point PH (mM) net as 4.24 6.0 7.23 X1 5.00 7.0 0.98 X2 5.65 6.0 3.37 x3 6.03 5.0 4.43 x4 +) Km is the Michaelis constant. The products formed by the specific glucosyltransferases were examined more closely in order to further characterize the glucosyltransferases. For this purpose, aliquots from each peak were inoculated with 0.125 molar sucrose in phosphate buffer, pH 6.8. Each fraction formed water-insoluble polysaccharides, but differences in the appearance of the polysaccharides could be found. After washing, the polysaccharides were analyzed for their carbohydrate composition.

Glucose was the only sugar that could be detected. the further properties of the glucosyltransferases, denoted by X1 to X4 and from Strept. SSC MS2 that have been isolated in this way are shown in Table 5 compiled.

Table 5 Appearance of the charcoal number of bands that Ip insoluble hydrate in acrylamide gel Enzyme of the Polysaccha- composite electrophercgram Swamp ride settlement occur Enzyme protein Gangs gangs X1 4.24 flaky glucose 2 7 X2 5.00 cloudy glucose 2 2 - 6 X; 5.65 gel-like glucose 1 - 2 1 - 4 X4 6, oRi flaky glucose 2 3 The particular glucosyl transferase enzymes thus polished from the relatively crude "dextran sucrase" enzyme extract can be used to produce the vaccines.

Example 13 A vaccine was produced, with the particular glucosyltransferase mentioned above and referred to as X3 has been designated.

This glucosyltransferase X3 has an analyzed DSU activity of 20 units per mg.

100 doses of the vaccine were prepared as follows: (a) 0.5 g des Enzyme X3 was added to 100 cc of sterile saline (0.85 kig).

(b) The suspension was left on a water bath at 40 ° C for 30 minutes stirred, and meanwhile, merthiolate was used as a preservative up to one Concentration added which was equivalent to a ratio of 1: 10,000.

(c) The solution was tested for sterility as in Example 1.

(d) The vaccine was in single-dose or multiple-dose containers bottled.

Example 14 One of the strains of Streptococcus SSC underwent treatment as in steps (a) and (b) of Example 1, and the cells were subjected collected in sterile 0.85% (w / v) NaCl solution. The cells were washed three times in sterile saline by centrifugation and resuspension. The cells, washed three times, were resuspended in sterile saline (0.85%) and then sonicated at a pitch sufficient to tear the cell walls apart. The sonicated suspension was then centrifuged to settle the cell walls or to be separated. Cans of the Vaccines were prepared by making suitable dilutions in the manner described in the preceding examples of the cell wall fraction in sterile, 0.85 pointer saline solution and for Suspension merthiolate added up to a concentration of 1: 10,000.

There could of course be numerous other individual examples for the vaccine, referring to the combinations and permutations of bacterial antigen, cell wall antigen, 1'-dextransucrase "type enzyme as antigen and think of each and every one of the four particular glucosyltransferase enzymes as an antigen got to.

Any pharmaceutically acceptable preservative can also be used be incorporated into preparations or vaccines, provided that they are not corrosive Has an effect on any other component. Anyone who has been tested can do it and safe accompanying substances are incorporated into the preparations. To the appropriate Preservatives include phenol, esters of p-hydroxybenzoic acid, formaldehyde, Propylene glycol, benzoic acid and its sodium salt, hexachlorophene, quaternary germicides, Thiomersal, merthiolate and glycerin. Suitable accompanying substances are NaCl solution, potassium alum, Protamine, aluminum phosphate, aluminum hydroxide, calcium phosphate, a variety of non-volatile oils and isolated and synthetic esters of higher molecular weight Fatty acids.

Of course, the preparations of the vaccine can contain other components except dax contain antigen with or without preservatives or accompanying substances. In particular, the preparations can be a local anesthetic, e.g. lignocaine, Amethocaine (Tetracaine hydrochloride), amylocaine and benzocaine. That's how preparations are containing the vaccines of each of Examples 1 to 13 and Lignooain included as a local anesthetic.

All stages of the production of the vaccine should be strictly aseptic Conditions and applying the regular tests to be carried out to determine the Ensure the sterility of the vaccine at all relevant manufacturing stages. For a detailed overview of the applicable sterility tests, see referred to World Health Org. Tech. Report Serv. 1960, 200 and Appendix 44 Speo, for quality control of pharmaceutical preps. "" International Pharmacopoeia " from W.H.O., Geneva, 1967.

The vaccine doses must be in glass or other containers Material is filled that does not react with the components of the vaccine occurs.

The containers are preferably clear, colorless or light amber in color. The containers should preferably be closed by melting or by Application of other suitable closures. Multi-dose containers should be preferred the removal of the content without removal.

Allow removal or destruction of the lock.

The closure of the multi-dose container 5011 preferably involves piercing with a sharp needle without leaving any fragments of the closure at the Content extraction are torn off. When removing, the closure should immediately close the container against external contamination. Provided the single-dose container are provided with rubber caps, the caps should be made of a suitable synthetic material Be made of rubber or plastic. The caps are said to be oil resistant, in case the vaccine is provided in an oil base.

The antibodies are specialized serum or saliva proteins, the immunoglobulins. The immunoglobulin IgA is preferentially secreted in saliva. Secreted IgA differs from serum IgA in that it has an extra protein component owns. The immunoglobulins are produced in the body in the lymphatic tissues. The Mechanism by which the antibodies are formed is still unclear. Depending on nature of the antigen, the antibody can be found in the serum or in the saliva several days after the First injections of a minimal dose of the antigen can be detected. The antibody titer then gradually increases to a low peak, slowly decreases and disappears in the end. A second injection of the antigen is given while the antibodies are being raised from the first stimulus or incentive are still present, there is only a slight decrease of the titer, followed by a rapid increase to one much higher peak than can be achieved by an injection alone. In connection at the second stimulus the antibodies disappear much more slowly than after the first stimulus. The rapid rise in antibody titer following a such a 'repetition' shock or

such a repeat injection presumably indicates that the cells producing the antibodies are stimulated by the first contact with the antigen or have been primed and therefore much more effective and more quickly respond when they encounter the antigen for the second time.

One of the circumstances that can stimulate the effectiveness of an antigen The stimulus to determine antibody production is the rate at which the antigen is absorbed and eliminated at the site of its administration. In general the more slowly the antibody is released, the stronger and more pronounced it is Antigen is absorbed at the injection site. For this reason, in immunization preparations Concomitant substances used to delay this absorption. In the case of the anti-caries vaccine, as they are prepared according to the invention, clinical trials have shown that the use of a solution of a local anesthetic as a vehicle for the antigen The absorption of the antigen is delayed if it is in one place in the oral mucosa is injected.

A number of experiments have been carried out with the vaccine and one of these attempts will be briefly described below.

Application Trial This trial consisted of one for three years conducted clinical trials of the anti-caries vaccine.

The group of subjects selected for this study consisted of children who were 5 1/2 to 6 years old at the start of the experiment. 120 children total made up the total number of test subjects, of whom 60 were the control group and 60 received the vaccines.

From the outset, 72 children were selected for each group to avoid failures of all kinds, and Table 24 shows the number of children which were examined every year in the two groups.

Table 24 Number of children examined each year became Initial number for all un- on the trial second third examinations at the beginning of year year were not available Test- group 72 64 60 60 Control group 72 68 60 60 At the beginning of the experiment, all test subjects were given precise instructions on oral hygiene. These were supported by regular, repeated reminders of the importance of correctly practiced oral hygiene.

The clinical and radiographic examinations were approximately 6 monthly intervals. The standardized conditions for the Investigations were maintained during the entire duration of the experiment.

The dental caries was assessed according to the standard guidelines, such as by D. Jackson (1950, British Dental Journal, 88.207) and G. Slack et al (1958, British Dental Journal, 104,399). Oral cleanliness became classified as good, mediocre or bad, depending on the amount of leftover or Deposits on the front teeth.

Bite plate X-ray images (bitewing radiographs) of the first molars immediately following the clinical examination. The radiographs were taken under standard conditions by evaluated by the examiner - regardless of the clinical entries.

Defects in the first molars that almost represent holes, such as they were determined on the basis of the x-rays were classified as follows: Y1 = a hole confined to the tooth enamel; Y2 = a hole that enamel and Dentin detected; Y3 = a hole that engages tooth enamel, dentin and pulp.

For the purpose of summarizing the results, all The degree of hole formation determined radiographically was analyzed together.

Each member of the test group received a first injection on April 13. May 1969, a second repeat (booster) injection on June 13, 1969, one third injection on June 22, 1970, a fourth injection on July 14, 1971, and one fifth injection on July 7, 1972. A clinical and radiographic examination was on the same five days and also on the following, intervening days Days, namely on November 13, 1969, January 12, 1971 and January 3, 1972. Each dose was 1 ml, and it was taken in approximately equal aliquots of 0.25 ml injected four places of the oral mucosa as it continues is described above in experiment E.

The formulation of the vaccines used was put together in such a way that that each ml dose contained: dextran sucrase antigen 0.025 g lignocaine HCl BP. 0.01 g Epinephrine U.S.P. 0.000005 g sodium metabisulfite B.P. 0.001 g sodium chloride BP. o, oo6 g of methyl p-hydroxybenzoate B.P. 0.001 g sodium hydroxide B.P. and until for a disodium phosphate position of the to 6.9 water for injection up to Make up to 1.0 ml.

The dextran sucrase used in the vaccine was prepared in this way as described in Example 8 (i.e. according to Method 4). the Dextransucrase was such that the DSU activity of each ml dose of the vaccine, i.e., each injection, was approximately 1,000 units.

It was the increase in tooth decay in each subject who was responsible for the three-year test duration was available. It became all of those Teeth and tooth surfaces that were available for the examinations and included proved to be free of caries during the first examination, and subsequently by the tooth decay was affected, filled with a filling or extracted, noted down.

Teeth that were present at the start of the experiment and teeth that were present during had passed the ongoing investigation, were separately analyzed. The test group and the control group at the start of the experiment were compared in relation to: (a) age; (b) kaiies-free teeth; (c) available permanent teeth; (d) -lnvolten; teeth with filling; (e) rotten, missing, with filling provided teeth; (f) rotten, filled tooth surfaces; (g) rotten, missing tooth surfaces with filling.

Address the results compiled in this experimental work only with the increasing attack on individual tooth types.

It was an analysis of the increasing attack on the four smooth Areas of the first permanent molar teeth and also the appearance of an increasing one Attack on the depressions and fissures of the first permanent molars. Finally, there is also the increasing attack on the mesial, distal and buccal surfaces of the upper permanent central teeth.

A. Four smooth surfaces of the first permanent molars, these are the ones Mesial, distal, lingual and buccal surfaces of the first molars.

For the duration of the trial, the first four were permanent molars present in or penetrated into the mouth of each test subject; at 6o people in the test group and 60 people in the control group.

Total areas recorded in the control group = 60 x 4 x 4 = 960 total areas recorded in the test group = 60 x 4 x 4 = 960 B. Buccal, distal and mesial surfaces of the upper central inclined teeth The two upper permanent central teeth were present for the duration of the experiment in or penetrated into the mouth of each subject; at 60 people in the test group and 60 people in the control group.

Therefore, the total areas in the control group were = 60 x 2 x 3 = 360 total areas in the test group = 60 x 2 x 3 = 360.

C. Depressions and fissures on each first permanent molar Each Subject had four permanent molars, and one area of this, the "occlusal" area, was taken into account in this investigation. Accordingly, the total areas were in the control group = 60 x 4 x 1 = 240 total areas in the test group = 60 x 4 x 1 = 240.

Study A In the control group, 960 smooth surfaces were at risk and at the end of the three-year test period, 130 areas were rotten or of attacked by tooth decay. So it was (130 x 100) / 960 or 13.54% of the total tooth surface; which had been available for the examination, infected by the tooth decay.

In the test group, 960 surfaces were at risk, and at the end of the During the three-year test, 8 surfaces were rotten or affected by caries. So it was (8 x 100) / 960 or 0.38% of the total surfaces that were used for the investigation had been available, infected by the tooth decay. From a comparison of the such values can be found in the test group and in the control group Calculate a reduction in the incidence of caries by approximately 93%.

Study B In the control group, 360 surfaces were at risk, and at the end of the three-year test period, 30 surfaces were rotten or from the Caries affected. So it was (30 x 100) / 360 or 8.3, 4 of the total areas, which had been available for investigation have been destroyed.

In the test group, 360 areas were at risk, and at the end of the three-year-old During the test, two surfaces were rotten or affected by caries. It were therefore (2 x 100) / 360 or 0.55% of the total areas that were used for the investigation had been available had been destroyed. This corresponded to a reduction of the areas affected by the tooth decay by almost 93% if you compare the values with compared to the values determined in the control group.

Study C In the control group, 240 tooth surfaces were at risk, and at the end of the three-year test period, 87 areas were rotten or destroyed. So it was (87 x 100) / 240 or 36.2% of all surfaces that were for the examination to the Had been available had been destroyed.

In the test group, 240 tooth surfaces were at risk; at the end of the three year old During the test, 16 tooth surfaces were affected by the caries. So there were (16 x 100) / 240 or 6.6 ffi of the surfaces that were available for the investigation had been rotten or destroyed (decayed), which is a reduction of approximately 82% corresponds if these values are compared with the values measured in the control group can be compared.

So it was in the specified teeth of the 60 test subjects the control group formed 247 cavities during the three-year test period, 130 of which are holes in one or the other of the four smooth surfaces of the first permanent molars, 30 holes in the mesial, distal or Buccal surfaces of the permanent upper central teeth were located and 87 holes were in the pits and fissures of the first permanent molars. This matches with a number of 4.11 holes per subject.

With the corresponding teeth of the 60 members who made up the test group or belonged to the vaccinated group, formed in the three-year trial period a total of only 26 holes, an average of 0.43 holes per subject is equivalent to.

In the control group there were a total of 1560 tooth surfaces during the three-year duration of the experiment at risk.

Of these, 247 surfaces were attacked, rotten or destroyed, and this corresponds to a value of 15.8 g.

In the test group there were a total of 1560 tooth surfaces during the three-year period The duration of the experiment was at risk, and 26 holes were formed in this time. the 26 surfaces that were destroyed or rotten made up 1.6% of the total of the available 1560 surfaces. The overall reduction in the number of holes developed in the test group was - compared to the number of the control group - 89.8%.

To prevent the risk of anaphylactic shock, the Antigens are only administered when it is ensured that the patient is opposite is not hypersensitive to the antigen being used. There were therefore Tests for hypersensitivity performed before any human patient a clinical dose of the vaccine was given. A total of around 200 people both in terms of immediate and delayed hypersensitivity tested.

No reaction was found in any of the cases. In any case, were a skin test and a conjunctival test were carried out for this purpose.

Claims (21)

"Vaccine 0 * Claims 1. Vaccine against tooth decay, characterized in that that they have an antigen from at least one representative of the following groups of substances, (i) a oral Streptococcus SSC, (ii) part of an oral Streptococcus SSC, (iii) an enzyme of the dextran sucrase type, which is obtained from a culture fluid of an oral Streptococcus SSC and (iv) a specific enzyme of the glucosyltransferase type, that has been isolated from a culture liquid of any oral Streptococcus SSC as well as possibly accompanying substances and preservatives. 2. Vaccine according to claim 1, characterized in that the vaccine an antigen from at least two representatives of the substance groups (i), (ii), (iii) and (iv) contains. Vaccine according to claim 1, characterized in that the antigen from a representative of substance group (ii), namely from the cell walls of the organism, consists. 4. Vaccine according to claim 1, characterized in that the antigen consists of at least one representative of substance group (iv). 5. Vaccine according to claim 1, characterized in that the antigen consists of the dextran sucrase enzyme (iii). 6. Vaccine according to claim 1, characterized in that the vaccine contains a medicinal or therapeutic agent in addition to the antigen. 7. Vaccine according to claim 1, characterized in that it is a Contains local anesthetic. 8. Vaccine according to claim 7, characterized in that the local anesthetic is one that is used in dentistry. 9. Vaccine according to claim 1, characterized in that it is in the form a multivalent vaccine is present and contains antigens from at least two Streptococcus SSC isolates. 10. Vaccine according to claim 1, characterized in that the antigen consists of a live, attenuated or decayed Streptococcus SSC and a unit dose of the vaccine is the equivalent of about 1 x 10 6 to 2 x 10 12 colony-forming Contains units. 11. Vaccine according to claim 10, characterized in that the unit dose the vaccine contains about 2 x 109 + 10 (colony-forming units, 12. Vaccine according to claim 11, characterized in that the unit dose of the vaccine is a Has a volume of about 0.75 ml to about 1.5 ml. 13. Vaccine according to claim 1, characterized in that the antigen consists of at least one representative of the substance groups (iii) and (iv) and one Unit dose of the vaccine has a dextran sucrase activity of about 500 to about 5 000 dextran sucrase units. 14. Vaccine according to claim 13, characterized in that the unit dose the vaccine has a dextran sucrase activity of about 900 to about 1100 dextran sucrase units having. 15. Vaccine according to Claim 14, characterized in that the unit dose the vaccine has a volume of about 0.75 ml to about 1.5 ml. 16. Vaccine according to one of claims 1 to 15, characterized in that that they need at least one representative of the following groups of substances as the effective one (i) the Streptococcus mutans (NCTC 10449), (ii) the cell walls of Streptococcus mutans (NCTC 104 ;; 9) and (iii) an enzyme that is produced from the culture liquid Streptococcus mutans (NCTC 10449) has been preserved and production capable of effecting a polysaccharide from sucrose. 17. Antigen according to one of claims 1 to 16, characterized in that that it is in freeze-dried form. 10. Method of making a vaccine, which will remove the teeth able to effectively prevent on the smooth tooth surfaces, according to one of the claims 1 to 17, characterized in that an oral Streptococcus SSC is grown, the enzyme dextransucrase or a specific glucosyltransfcrase enzyme from the culture fluids separated and the enzyme diluted in such an ice that the unit dose of Vaccine has a dextran sucrase activity of about 500 to about 5000 dextran sucrase units having. 19. The method according to claim 18, characterized in that the dilution is made such that the dextran sucrase activity of the unit dose of the Vaccine amounts to about 900 to about 1100 dextran sucrase units. 20. The method according to claim 1S, characterized in that at least a representative from the group of accompanying substances and ion preservatives in vaccines is incorporated. 21. Method of making a vaccine which removes tooth decay can effectively prevent on the smooth tooth surfaces according to one of the claims 1 to 17, characterized in that one is living, weakened or killed Streptococcus SSC diluted with a liquid medium to such an extent that that he was a unit dose of the vaccine containing the equivalent of about Provides 2 x 10 to about 2 x 1012 colony-forming units. 22o method according to claim 21, characterized in that the dilution is made such that a unit dose of the vaccine is approximately 2 x 10 9 + 10 having colony-forming units. 2Do method according to claim 22, characterized in that at least a representative from the group of accompanying substances and preservatives in vaccines is incorporated.