RU2129436C1 - Adjuvants - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: invention relates to the use of amino acid and dipeptide derivatives of fullerene as immune response stimulating agents - adjuvants and can be used in biological industry for development and production of vaccines. Invention relates to the use of the early described fullerene derivatives of amino acid and dipeptides of the general formula (I) as adjuvants: C₆₀-X where C₆₀ is fullerene ring; X is NH-CHR-COOH, NH-(CH₂)_h-COOH, NH-CHR-CO-NH- -CHR-COOH where n = from 2 to 6; R - side radical of natural amino acid. In experiments with human erythrocytes an invention provides a capability to use high doses (400 mcM) but even they do not exhibit hemolytic properties. During incubation cells are not agglutinated and their morphology is constant. Administration of these compounds to mice at high dose 80 mg/kg does not change their viability and behavior for 6 months. EFFECT: improved quality and properties of adjuvants. 2 tbl, 9 ex

Description

 The invention relates to biotechnology, in particular to the use of amino acid and dipeptide derivatives of fullerene as stimulants of the immune response - adjuvants, and can be used in research practice, as well as in the biological industry in the development and production of vaccines.

 It is known that microorganisms are used as adjuvants, their metabolic products, their suspensions in mineral oils (Freund's adjuvant), aluminum hydroxide, insoluble inorganic salts, saponins, and plant extracts of hydrophobic nature.

 However, it is also known that immunization with adjuvants, which are water-in-oil emulsions and suspensions, often leads to undesirable complications, such as local infiltrations, ulcerations, and allergic reactions [1–3].

 The use of a low molecular weight soluble adjuvant, a synthetic fragment of peptidoglycan from the wall of mycobacteria, N-acetyl-muramyl-L-alanyl-D-iso-glutamine (MDP), has been described [4].

 The use of a water-soluble adjuvant, MDP, eliminates the undesirable consequences that are manifested during immunization with adjuvants based on water-in-oil emulsions and suspensions, however, it has a number of other disadvantages, namely pyrogenicity [5] and neuropharmacological effect [3], stimulating activity is manifested only at relatively high doses, with some antigens requiring covalent attachment of MDP to the antigen molecule. In addition, this adjuvant only replaces the bacterial component in Freund's adjuvant, but does not cancel the use of the oil component, and its use is ineffective for a number of antigens [6].

The present invention relates to the use of the compounds described above, fullerene derivatives of amino acids and dipeptides of the general formula I as adjuvants
C ₆₀ -X,
where C ₆₀ is the fullerene core;
X = NH-CHR-COOH, NH- (CH ₂ ) _n -COOH,
NH-CHR-CO-NH-CHR-COOH,
where n = from 2 to 6, R is the side radical of a natural amino acid.

 Derivatives of fullerene of the general formula I are a relatively new class of organic compounds. They are synthesized directly by the addition of residues of amino acids or dipeptides to the fullerene nucleus [7]. They are solid powdery substances of a dark brown color, soluble in water. Their biological properties are currently being widely studied.

 Assessment of the adjuvant properties of compounds I is carried out in two stages. At the 1st stage, their antigenic properties are evaluated. For this, hyperimmune serum is obtained by immunizing rabbits and mice with preparations of various fullerene derivatives together with Freund's strong adjuvant. Analysis of sera is carried out according to standard methods by ELISA.

 At the 2nd stage, the adjuvant properties of compounds I are determined. For this, one group of mice is immunized with a model antigen - egg albumin (Ova), the other several groups - with a mixture of Ova with various compounds I (Ova-I).

 To detect IgG antibodies, peroxidase-labeled mouse anti-IgG (Sigma) is used. The titer is the reciprocal of the dilution of a sample whose optical density (OD) is equal to the OD of a normal serum sample (before immunization) diluted 50 times (OD of normal serum ≤ 0.2, which is achieved by selection of ELISA conditions). Adjuvant efficacy is defined as the ratio of anti-Ova-I serum titer to anti-Ova serum titer.

 The invention is illustrated by the following examples.

 I. Determination of immunogenicity.

 Getting serum.

 Example 1

To prepare hyperimmune serum, 100 μg of C ₆₀ -Ser preparation is dissolved in 0.5 ml of 0.05 M phosphate buffer with a pH of 7.4. The first immunization is carried out in complete Freund's adjuvant (Gibco, 1: 1 v / v), the next three immunizations are carried out in Freund's incomplete adjuvant. Chinchilla rabbits are injected subcutaneously with a two-week interval. Blood sampling is carried out at the same intervals, and serum samples obtained from blood are analyzed for antibodies to C ₆₀ -Ser enzyme-linked immunosorbent assay.

 Example 2

The method is carried out as described in example 1, but C ₆₀ -Ala is used as antigen.

 Example 3

The method is carried out as described in examples 1 and 2, but the dipeptide derivative of C ₆₀ -Ala-Ala is used as the antigen to obtain antiserum.

 II. Enzyme-linked immunosorbent assay.

For ELISA, the fullerene derivatives described in Examples 1-3 are dissolved in 60 mM carbonate-bicarbonate buffer (pH 9.6) and applied to GREINER plates at a concentration of 20 μg / ml. After incubation for 1.5 hours at 37 ^° C, the plates are washed from unbound components with phosphate-buffered saline (PBS) pH 7.4 with 0.05% tween-20. The test sera obtained respectively in examples 1 to 3, are diluted 50 times with a solution of 0.5% bovine serum albumin (Sigma) with 0.05% tween-20 in PBS and incubated with preparations adsorbed on the tablet for 30 min at 37 ^o C. After incubation the tablets are washed 4 times with a solution of FSB / 0.05% tween-20. A solution of rabbit anti-IgG conjugate with peroxidase (Sigma) in working dilution was incubated in the wells of the plate for 30 minutes at 37 ^° C and a similar washing was carried out. To detect immune complexes, a developing mixture is used: 4 mg of o-phenylenediamine / 10 ml of 0.1 M citrate phosphate buffer / 80 μl of 3% H ₂ O ₂ . The reaction was stopped with 2N H ₂ SO ₄ and the absorbance in the wells was measured at 492 nm using a Multiscan Plus automated scanner (Flow Laboratories, Switzerland). The value of the background absorption (Cut off) is estimated as the average value of the optical density for normal rabbit sera (n = 5) + 3SD. Serum titer was evaluated by binary dilution of sera, primary dilution was 1:50. The data are shown in table 1.

 As the 4 series of immunizations shown in Table 1 show, the amino acid and dipeptide derivatives of fullerene do not possess antigenic properties, which is probably due to its special foreignness, i.e. a sharp difference between their structures and the structures of natural antigens and the absence of suitable lymphocyte clones in the natural repertoire.

 III. Determination of adjuvant properties.

 1. Getting serum.

 Example 1

 One group of mice (7 animals) of Fl breed (CBA / BALB / c) was immunized intraperitoneally 3 times at 3-week intervals, injecting 30 μg of Ova into each mouse in 0.5 ml of 0.05 M phosphate buffer with pH 7.4.

Another group of mice (7 animals) was immunized in a similar way, however, using 10 μg Ova with 330 μg C ₆₀ -Ser / ratio of 3: 100 (w / w) / for each mouse.

 Blood sampling is carried out at the same 3-week intervals, and serum samples obtained from blood are analyzed in ELISA, as described in paragraph II. To detect IgG antibodies, peroxylase-labeled mouse anti-IgG (Sigma) is used. The results are shown in table 2.

 Example 2

The method is carried out as described in example III.1, but a mixture of 10 μg Ova with 30 μg C ₆₀ -Ala / weight ratio 1: 3 / is used as antigen.

 Example 3

The method is carried out as described in example III.1, but a mixture of 15 μg Ova with 105 μg C ₆₀ -Ala-Ala / weight ratio 1: 7 / is used as antigen.

 Example 4

The method is carried out as described in example III.1, but a mixture of 10 μg Ova with 200 μg C ₆₀ - ε-aminocaproic acid (C ₆₀ -ε-Acp) / weight ratio 1: 20 / is used as antigen.

 Example 5

The method is carried out as described in example III.1, 2, but a mixture of 20 μg Ova with 260 μg C ₆₀ -Phe-Lys / ratio 1: 13 / is used as antigen.

 Example 6

The method is carried out as described in example III.1, 2, but a mixture of 20 μg Ova with 60 μg C ₆₀ -Lys-Tyr / ratio 1: 3 / is used as antigen.

 Example 7

The method is carried out as described in example III.1, 2, but a mixture of 45 μg Ova with 300 μg C ₆₀ -Ala-Arg / ratio 3: 20 / is used as antigen.

 Example 8

The method is carried out as described in example III.1, 2, but a mixture of 20 μg Ova with 100 μg C ₆₀ -Lys-Ala / ratio 1: 5 / is used as antigen.

 Example 9

The method is carried out as described in examples III.1, 2, but a mixture of 20 μg Ova with 120 μg C ₆₀ -Ala-Thr is used as antigen in a ratio of 1: 6.

 Determination of toxicity.

 Experiments with human erythrocytes showed that even at high doses - 400 μM - they do not have hemolytic properties, the cells do not agglutinate during the incubation process, and their morphology does not change. The administration to mice of a high dose of these compounds (80 mg / kg) does not affect the viability and behavior of mice for 6 months.

 Thus, the fullerene derivatives of amino acids and dipeptides claimed as adjuvants, as can be seen from Table 2, well stimulate the immune response to a protein antigen. As shown by testing, these derivatives do not have their own immunogenicity, hemolytic properties, they are non-toxic when administered in high doses.

References
1. Immunological adjuvants. Report of the WHO scientific team. M., 1978.

 2. Murhay R., Cohen P. and Hardegree M.C.//Ann. Allergy, 1972, 30, 146.

 3. Chedid L. // Ann. Inst. Pasteur / Lmmunol., 1985, 136 D, 283 - 291.

 4. Merser C., Sinay P. and Adam A. // Biochem. Biophys. Res. Commun., 1975, 66, 1316 - 1322.

 5. Kotani S., Watanabe Y., Shimono T., Harada K., Shiba T., Kusumoto S., Yokogawa K, and Taniguchi M. // Biken J., 1976, 19, 9-13.

 6. Ivanov B. B. Creation of immunopotentiating systems for experimental peptide vaccines. Cand. dissertation, 1992.

 7. V.S. Romanova, V.A. Tsyryapkin, Yu.A. Lyakhovetsky, Z.N. Parnes, M.E. Volpin // Izv. RAS, ser. Chem., 1994, 1151.

Claims (1)

The use of fullerene derivatives of amino acids and dipeptides of the General formula
C ₆₀ -X,
where C ₆₀ is a fullerene core,
X = NH - CHR - COOH, NH - (CH ₂ ) _n COOH, NH - CHR - CO - NH - CHR - COOH;
n = 2 to 6;
R is a side radical of a natural amino acid,
as adjuvants.

Images