DE2212739A1 - Vaccine for immunization against dental caries - Google Patents

Description

Colgate Palmolive Company

300 Park Avenue

New York N.Y./V.St.A.

(prio 22.371 US 126 933-9078)

Hamburg, March 14, 1972

Vaccine for immunization against dental caries

The invention relates to vaccines for immunization against dental caries.

For about 10 years it has been known that the formation of dental caries is due to an interaction of carbohydrates, in particular Sucrose and specific bacteria are based on the tooth surfaces. Quite a number of germs have emerged prove to be the cause of this infectious disease,

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and in particular streptococci. Numerous streptococci have been isolated from the oral cavity and have been shown to have caused dental caries in animal experiments.

Even in the course of this research, attempts were made to prevent the formation of dental caries by immunological means; however, no suitable method has yet been found. For example, M. Wagner, PhD thesis 1966, Purdue University, the immunization of aseptically raised Rats were carried out against dental caries with a vaccine obtained from a strain of Streptococcus faecalis. W.H. Bowen reported in J. Br. Dent. Res., Vol. 129, pp. 159-160 on the use of dextran-forming caries causing ^ viable germs for the immunization of monkeys against dental caries. The germs used came from a strain of Streptococcus mutans; this is one of two types of bacteria which are often regarded as the most important germs in the development of dental caries. The other germ is a strain of streptococci referred to as Streptococcus, strain SS2 and isolated by Gibbons and Banghart and described in Archives of Oral Biology, 1967, Volume 12, pp. 11-21 became. This strain is from numerous laboratories in the United States, and most notably from Forsyth Dental Center, Boston, Massachusetts. This

However, the trunk can also be removed from the mouth without difficulty.

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Cave, in particular of teeth affected by caries, to be isolated in a manner known per se.

Differentiate between Streptococcus mutans and Streptococcus, strain SS2 clearly from each other. One of these differences is in the physiological behavior of these germs in the presence of sucrose clearly. Both Streptococcus mutans and Streptococcus strain SS2 produce when grown in the presence of sucrose extracellular, sticky, rubber-like polysaccharides. The polysaccharides formed differ however, with the two germs, apart from the fact that they are thereby involved in the formation of Plague, with which the development of caries begins. Streptococcus mutans predominantly forms a dextran polysaccharide, while Streptococcus, Strain SS2, predominantly forming a fructosan polysaccharide.

Surprisingly, it has now been found that the Streptococci, and in particular Streptococcus, strain SS2, polyfructosans produced excellent immunization properties against dental caries.

According to the invention, vaccines are used for immunization against Dental caries which are characterized in that they are present in a liquid carrier during anaerobic culture of a polyfructosane-forming strain of the genus Strep-

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tococcus contain polyfructosan formed.

When using the vaccines produced according to the invention, immunizing antibodies are formed against the causally active streptococci, which is associated with a decrease in the formation of caries. The exact mechanism of action between antibodies and the harmful germs is not known. In any case, a significant reduction in caries formation can be determined.

According to the invention, a polyfructosane-forming strain of Streptococci, in particular Streptococcus, strain SS2, cultivated for the formation of the extracellular polyfructosans. The germs become anaerobic in a nutrient medium with content Cultivated on sucrose until a sufficient amount of extracellular polyfructosans can be formed.

As a nutrient medium for Streptococcus, strain SS2, a nutrient medium containing sucrose as a carbon source is used, other carbon sources preferably being reduced to a minimum. The sucrose content is between 2 and 16 and preferably between 5 and 8% by weight , based on the total medium. In addition, the culture medium contains a suitable nitrogen source, such as peptones, meat extract, yeast extract or soy extract. Ammonduin-

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sulfate, ammonium chloride or ammonium nitrate can be used. In addition, the nutrient medium contains inorganic salts such as phosphates, magnesium salts such as magnesium sulfate, metal ions such as Iron, sodium, potassium, manganese, zinc and calcium and anions such as chloride and nitrate. Others if necessary Nutrients necessary for the growth of streptococci are used as usual.

The culture is carried out in the usual manner under anaerobic conditions. The incubation temperature is 35 to 38 ⁰ C and preferably 37 ° C, the pH of the medium is 6.8 to 7.4, and preferably 7 "0 to 7". 2 The incubation is usually 12 to 72 and preferably 2 ¹ J to 48 hours; During this period, sufficient amounts of extracellular polyfructosans are formed in the culture medium.

The polyfructosans can be used to produce the vaccines according to the invention in the form of the culture medium, preferably after removal any germs that have been killed can be used. In preferred embodiments, however, the polyfructosans are isolated cleaned and used in the vaccines in a relatively pure form. The polyfructosans can in itself can be obtained from the culture medium in a known manner. The precipitation of the polyfructosans has proven to be particularly favorable from the cell-free mother liquors of the culture medium by adding

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Alcanols, such as ethanol, proved. In general, 1.5 to 2 volumes of ethanol are used. Afterward are preferably, although not necessarily, the low molecular weight fraction of the polyfructosans, by dialysis or in another known manner. The polyfructosans generally have a molecular weight of about 16 to 23 million, only about 0.25 to 0.5 55 have one low molecular weight of about 2000 to 5000, and this fraction is preferably removed by dialysis.

Subsequent to dialysis, any protein is preferably removed from the polyfructosans. The separation of proteins can be carried out by various methods known per se, the method of Sevag et al, Journal of Biological Chemistry, Volume 12 * 1, pp. 425 bis ^ 35, (1938) proved. According to this rule, chloroform and butanol or another alcanol, such as isopropanol or hexanol, is used to dissolve the protein. The cleaned Polyfructosan can then be precipitated with ethanol, for example, washed and dried.

The polyfructosans pretreated in this way, i.e. the low molecular weight ones Fractions and proteinaceous substances present have been separated off are particularly useful for production suitable for vaccines. The structural formula of these polyfructosans

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could not yet be determined, elemental analyzes and IR spectra were used for characterization. The characteristic peaks in the IR spectrum are at 920 cm, 875 cm ¹ and 805 cm ¹ .

The acid-hydrolyzed polyfruetosans leave in the chromatographic Analysis only recognize fructose as a building block. The polyfruetosans obtained by the process described can be characterized by the following properties:

Intrinsic viscosity at 25 ° C - 0.15 to 0.16

20th
specific rotation Γβ * «~] _η - 43 to 54

Total hexose content - 98.4 %

Protein nitrogen - less than 0.5 %

The vaccines according to the invention are preferably in the form of aqueous solutions or emulsions containing 0.1 to 1.0 and preferably 0.4 to 0.6 g polyfructosan / l, these amounts being based on the purified compound. The liquid carriers used are preferably sterile saline solutions which, if necessary, can be processed into emulsions ^{by means of auxiliaries such as, for example, Freund 1 see auxiliaries or similar additives.} The above-mentioned specific rotation was measured in a 1% aqueous solution of Lävan. The Freund'sehe aids containing 1, '5 ml mannide monooleate and 8.5 ml paraffin

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oil and 8 mg killed and dried mycobacterium cutyricum. Freund's auxiliary is preferably added in amounts of 1: 1. The salts usually used for the preparation Salts used by saline solutions, such as sodium or potassium chloride, can be used.

Instead of the preferably purified polyfructosans, killed germs of Streptococcus, strain SS2, can also be used will. Germs killed with formalin are particularly suitable; Even in these cases, Freund'shee can use auxiliaries can be used. When using killed germs, a sufficient amount must be used to ensure that the described Amounts of dextran are present. The number of germs thus corresponds

7 7 7

about 0.5 χ 10 'to Ί χ 10 and preferably 1 χ 10 to 2 χ

10 'germs per milliliter of solution. Lower bacterial counts should only be used if booster vaccinations after the first vaccination administered. Even if non-purified polyfructosans are used, these must be sufficient Quantities are used to achieve the levels described.

Vaccination with the vaccines according to the invention can be carried out in a known manner, for example subcutaneously, intramuscularly or intravenously. The single doses contain 1 to 20 µg and preferably 5 to 10 µg of polyfructosans per kg of body weight. Booster vaccinations can be given from time to time.

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The following examples are intended to explain the invention in more detail.

example 1

Number of diets previous streptococcal immunity

Antibiotic inoculation treatment with ab

killed germs

group I. 10 Mitchell group II 10 Mitchell group III 10 Mitchell

Thirty newborn golden hamsters were obtained from the same litter and bred by the National Institutes of Health divided into three balanced groups. All animals were fed the Mitchell diet and deionized water provided. Group I received only the Mitchell diet as a control group.

The normally gram-positive flora of golden hamsters in groups II and III was suppressed after birth by administering 100 units of penicillin per gram of the Mitchell diet on four consecutive days. On the fifth day the antibiotic treatment was discontinued and the animals in groups II and III were inoculated with 0.1 ml of an 18-hour-old culture of a streptornycin-resistant mutant of the polyfructosane-forming Streptocuccus, strain SS2 _{3, in the} hamster pockets. The usage

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a streptomycin-resistant mutant was carried out from purely experimental ones Reasons, namely starting the inoculation by further breeding the germs on a Mitus Salivarius Streptomycin agar could be verified.

The Streptococcus used in these experiments, strain SS2, was founded by Dr. R. J. Gibbons at the Porsyth Dental Center, Boston, Massachusetts. The trunk is in the American type

Culture Collection, Rockville, Maryland, deposited and carries the ATCC accession number 27006.

The germs were kept on a trypticase soy extract medium without grape sugar (BBL) but with the addition of 0.5 % sucrose at a temperature of 37 ° C. and a pH of 7.2. The sucrose was previously sterilized in the autoclave and added to the medium under antiseptic conditions. The germs were kept anaerobically in a BBL anaerobic jar at 37 ° C. in an atmosphere of 90 % hydrogen and 10 % carbon dioxide.

On the day of inoculation, the animals in Group III were injected intraperitoneally Injection of 0.5 ml of a vaccine with a content of formalin killed germs in a ratio of 1: 1, complete friend's view aids immunized. the Vaccine required for immunization was prepared as follows. After 18 hours of anaerobic growth of the germs on a tryptic

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case soya extract medium with a content of 0.5 / 5 sucrose and 100 μg streptomycin per ml, the cell suspension washed with saline was treated with ultrasound to break the chains. The ultrasound treatment was carried out for 5 minutes with a Raytheon ultrasound device with O _{3 65 amps before the germs were killed.} The suspension was then adjusted with saline solution to an optical density of 0.22, measured at 610 μm, in a Bausch and Lomb colorimeter. The germs were then suspended in a 0.6 # formalin solution overnight in order to ensure complete destruction.

The test animals received four at an interval of about two weeks additional injections, each with a decreasing concentration

6 ¹ J of the germs in the preparation, namely from 1 χ 10 to 2 χ 10. After 12 weeks, the test animals were sacrificed and the antibody titer of saliva and serum in each group was examined. The antibody titers from saliva and serum were compared against an extract prepared with saline from Streptococcus, strain SS2, and polyfructosans prepared therefrom, and it was found that a corresponding antibody reaction had occurred in saliva and serum in the immunized animals.

The animal skulls exposed according to the well-known method of Russell were prepared with the help of a modified Keyes method examined for dental caries. Dae examination for caries was done with

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BATH ORIGINAL

second and third maxillary molars performed. One animal from group I, the control group, and two animals from group II died during the attempt for other reasons.

The experiment can be seen in the table on page 9,

The examination for dental caries in the three groups showed the following Results:

Frequency of
Caries infection % Change Group I. 26.89- Group II 37.66 + $ 40.05 Group III 12.18 - 67.662

These results show that the vaccines according to the invention lead to an outstanding reduction in the formation of dental caries. The animals of group II vaccinated with caries-producing germs showed an increase in caries formation over HO # compared to the animals of control group I not vaccinated with caries-producing germs. All three groups were constantly kept on a caries-promoting diet.

The immunized group III showed a 67 / S reduction of caries infestation compared to group II, so that the effective

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immunization becomes very clear. The results show a statistical certainty of about 99 % .

Example 2 Representation of the polyfructosans

Streptococcus, strain SS2, became anaerobic in a trypticase soy extract medium without glucose (Baltimore Biological Laboratories - BBL) with the addition of 8 % sucrose at a temperature of 37 ° C. and a pH of 7.2 in an atmosphere of 90 % Hydrogen and 10 % carbon dioxide -48 hours incubated. The germs were then centrifuged off in a Sorvall freezing centrifuge for 10 minutes with an SS ^^ head at 10,000 rpm. The polyfructosans were precipitated from the clear supernatant liquid by adding 2.5 volumes of 95 # ethanol. The precipitated gummy mass was redissolved in water and then repeatedly treated by adding 0.25 volumes of chloroform and 0.1 volumes of amyl alcohol to separate the proteins. The mixture was then shaken for 15 minutes and the aqueous layer containing the polyfructosans was then dialyzed against distilled water for 2 * 1 hours to remove the low molecular weight fractions.

The polyfructosans were then reprecipitated from the aqueous layer by adding ethanol, forming a suspension of a fine powder. The purified polyfructosans were

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collected on an acid washed filter paper (Whatman 52) in a Buchner funnel, washed with absolute ethanol, Washed acetone and petroleum ether and immediately transferred to an executor.

Example 3

The procedure described in Example 1 was repeated, except that in this case Group III animals were used instead of immunization with germs of Streptococcus, strain SS2 killed with formal, an immunization with 0.5 ml of a solution with a Content of 500 µg polyfructosans per ml in a salt solution with a content of Freund's Schein aids in a ratio of 1: 1. The polyfructosans used had been prepared according to the method described in Example 2. After first vaccination with the vaccine were taking four more injections at an interval of two weeks after the first injection Use of the initially used polyfructosan solution given. The frequency of caries infestation was determined with two other groups, as in Example 1, determined and evaluated according to the method described there. The following results were obtained for the animals of group III:

Frequency of % change

Caries infection

Group III 11.2 - 70 %

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The animals immunized with the purified polyfructosans show a reduction of 70 % with regard to the caries infestation compared to the animals of group II in Example 1. This shows that the immunization with the vaccines according to the invention is actually very effective. From a statistical point of view, these results have a probability of 99 %

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Claims (9)

- 16 claims 1. Vaccine for 'immunization against dental caries, characterized in that that in a liquid carrier a strain of the genus which forms by anaerobic culture of a polyfructosane Contains polyfructosan formed by streptocuccus. 2. Vaccine according to claim 1, characterized in that the carrier is an aqueous salt solution. 3. Vaccine according to claim 1 or 2, characterized in that it contains the polyfructosans from Streptococcus, strain SS2. 4. Vaccine according to claim 3, characterized in that it the Contains polyfructosans in the killed cells of the Streptococcus, strain SS2. 5. Vaccine according to claim 3 and 4, characterized in that the polyfructosan has a molecular weight of 16 to 23 million. 6. Vaccine according to claim 5, characterized in that it contains 0.1 to 1 g of polyfructosans per liter of solution contains. 2098-1 / 1126 7, vaccine according to claim 5, characterized in that it contains 0.4 to 0.6 g of polyfructosans per liter of solution. 8. Vaccine according to claim 3 and 7 _9, characterized in that the polyfructosan contained characteristic IR absorption peaks at 920 cm, 875 and 805 cm, an intrinsic viscosity at 25 ° C of 0.15 to 0.16 and a specific rotation fr η ^{20 has} from 43 ° to 54 °. 9. Vaccine according to claim 3 to 8, characterized in that a by incubation of Streptococcus, strain SS2, under anaerobic conditions in the presence of sucrose at temperatures of 35 to 38 ⁰ C and a pH of 6.8 to 7.4 formed polyfructosan is included. 2 0 9841/1126