RU2697527C1 - Immunoadjuvant composition for vaccines for infectious agents of viral and bacterial nature - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, namely immunology, and can be used to produce an adjuvant composition consisting of chitosan and oligodeoxynucleotide. Adjuvant composition contains chitosan with molecular weight in range of 200 to 1000 kDa, degree of deacetylation from 75 to 98 %, polydispersity index of not more than 2.0, level of endotoxins and extraneous protein of less than 100 EU/ml and 0.05 %, respectively, A oligodeoxynucleotide is a synthetic oligodeoxynucleotide, which consists of 5 repeating segments of CG nucleotides with thiophosphoryl bonds in internucleotide phosphates; at ratio of components of chitosan: synthetic oligodeoxynucleotide (20–50):1.EFFECT: use of the given invention provides high immunogenicity and protective effect when adding an adjuvant composition to vaccines against infectious agents of bacterial and viral origin.3 cl, 5 ex, 3 tbl

Description

The invention relates to medicine, relates to vaccine prophylaxis of infectious diseases and describes an adjuvant composition consisting of chitosan and an oligodeoxynucleotide (oligodeoxynucleotide or ODN). Primary area of application: adjuvant composition provides increased immunogenicity and protective effect when included in the composition of various types and methods of administration of vaccines against topical infectious agents of humans and animals of bacterial and viral nature.

Adding immunoadjuvants (adjuvants) to vaccines allows us to solve a number of problems in the prevention of relevant infectious diseases in humans and animals [WHO. State of the world's vaccines and immunization, 3rd ed. 2009, p. 118-122]: increasing efficiency in all population groups without compromising safety, inducing a broadly cross-immune response, reducing the dose and switching to simple (single) immunization regimens. However, there are currently no adjuvants that optimally combine efficacy, safety, and economic feasibility, especially with mass immunization.

A known method of increasing the immunogenicity of an inactivated influenza vaccine by adding an adjuvant containing a mixture of high and low molecular weight chitosan with a molecular weight (MM) of 100-500 and 0.5-20 kDa, respectively, the degree of deacetylation (DM) in the range of 60-90% [ RU 2323742]. However, the method of producing low molecular weight chitosan using nitrous acid leads to the fact that the terminal reducing anhydromannose residue of low molecular weight chitosan formed during the reaction is unstable, the aldehyde group of the mannose residue reacts with the amino group in chitosan oligomers to form the Schiff base, which promotes the cleavage of the next glycosidic residue with the removal of water and the formation of 5-hydroxymethylfurfural [Mourya VK, Inamdar NN, Choudhari YM Chitooligosaccharides: synthesis, characterization and applications. Polymer Science. 2011, 53, N.7, p. 583-612]. From literature data it is known that oxymethylfurfural and its metabolic products have toxic and mutagenic effects. Studies in rats confirm the toxicity of oxymethylfurfural with subcutaneous administration (200-500 mg / kg) [Abraham K., Gurtler R., Berg K. et al. Toxicology and risk assessment of 5-Hydroxymethylfurfural in food. Mol Nutr Food Res. 2011, 55, N. 5, p. 667-678]. In addition, the patent does not indicate the results of tests for bacterial endotoxins for parenteral administration of the vaccine with adjuvant.

A known method of increasing the immunogenicity of an inactivated polio vaccine; as adjuvant use a suspension of micro / nanoparticles of chitosonium sulfate (100-1300 nm) at a concentration of 0.05-0.5%. The patent application [RU 2013154748] does not specify the main physicochemical characteristics of an adjuvant based on chitosan: MM and DM.

The closest to the essence and purpose of the present invention is an immunogenic composition [US 2004258702] containing a non-ionic block copolymer of Pluronic F127 and / or oligodeoxynucleotide (ODN) - CpG or chitosan, which is added to tetanus, diphtheria and anthrax antigens. Chitosan medium MM (Sigma-Aldrich, St. Louis, Missouri) or Protasan® (Chitosan chloride, UP CL 213; ProNova Biomedical, Oslo, Norway), prepared at a concentration of 3% (wt.) In 1% (vol. ) acetic acid, in 0.9% (w / v) sodium chloride. However, the patent does not discuss the sharing of a mixture of CpG and chitosan.

A method for preparing chitosan with ODN is described in CN patent 104689325. A mixture of chitosan with ODN is oligochitosan nanoparticles with MM 80-160 kDa, SD 90-98%, with ODN unmethylated DNA sequence (5'-GGCGTT-3 ', 5-ATCGAT -3, two 5-GTCGTC-3 and two 5'-AACGTT-3 'sequences). Filtering is used as a sterilizing method, while the patent does not discuss the stability of the complex of chitosan nanoparticles with ODN during storage and methods of using the complex to increase the immunogenicity of vaccines.

The problem solved by the present invention is to create an adjuvant composition, which, when included in the vaccines, will increase the immunogenicity and protective effect of immunobiological preparations, regardless of the type of antigen, route of administration, or the origin of the antigen.

The technical result of the invention is to increase the immunogenicity and protective effect of various vaccines against topical infectious agents in humans and animals. The technical result is achieved in that the adjuvant composition contains chitosan and a synthetic oligodeoxynucleotide. Chitosan in the adjuvant composition provides depot formation followed by delayed release of antigen, and oligodeoxynucleotide provides direct immunomodulating activity by the TLR9 agonist mechanism. As chitosan, the composition contains a cationic polysaccharide obtained from crab chitin, with a molecular weight in the range of 200 to 1000 kDa, a degree of deacetylation of 75 to 98%, a polydispersity index of not more than 2.0, an endotoxin and extraneous protein level of less than 100 U / ml and 0.05%, respectively; as an oligodeoxynucleotide, a synthetic oligodeoxynucleotide, which is 5 repeating ODN-Cg motifs with thiophosphoryl bonds in internucleotide phosphates in the following ratio (mass.) of components, chitosanchynthetic ODN as (20-50): 1, dissolved in 1% glutamic acid. The technical result is achieved in the entire range of the indicated chitosan characteristics: MM from 200 to 1000 kDa, DM from 75 to 98%, endotoxins and foreign protein less than 100 U / ml and 0.05%, respectively, and with a different ratio of chitosan: ODN as -50): 1.

It is known that chitosan can be used as the determining adjuvant component in the vaccine, or it can have an additive effect when combined with direct immunomodulators [Heffernan M.J., Zaharoff D.A., Fallon J.K. et al. In vivo efficacy of a chitosanAL-12 adjuvant system for protein-based vaccines. Biomaterials. 2011, 32, N.3, p. 926-932]. The relationship of the characteristics of the biopolymer and its adjuvant properties in the composition of various vaccines is not traced.

It is known that one of the mechanisms of the immunoadjuvant action of high molecular weight chitosan is the formation of a depot at the injection site, followed by a delayed release of antigen [Bertram U., Bernard M., Haensler J. et al. In situ gelling nasal inserts for influenza vaccine delivery. Drug Dev. Ind. Pharm. 2010, 36, N.5, p. 581-585]. In order to enhance the immunogenic properties of vaccines as adjuvants, immunostimulants of innate immunity factors, in particular, TLR9 agonists - CpG-ODN [Krieg A.M. Therapeutic potential of Toll-like receptor 9 activation. Nat Rev Drug Discov. 2006, 5, N. 6, p. 471-484].

We have developed an adjuvant composition containing chitosan and synthetic ODN to increase the immunogenicity and protective effect of vaccines against bacterial and viral nature due to the potentiating effect of the minimum doses of individual substances.

The invention is illustrated by the following graphic materials:

FIG. Chemical structure of chitosan (n> m):

m is the residue of acetylglucosamine;

n is the glucosamine residue.

Examples

Example 1. Obtaining an adjuvant composition

The adjuvant composition is a mixture of a solution of chitosan in acid, which is a natural cationic polysaccharide (Fig. 1), obtained from crab chitin, with MM 200-1000 kDa, SD 75-95%, polydispersity index (PI) 1.8-2, 3, the level of endotoxins and foreign protein is less than 100 U / ml and 0.05%, respectively, and synthetic ODN, which is 5 repeating segments of Cg with thiophosphoryl bonds in internucleotide phosphates.

Chitosan (manufactured by Bioprogress LLC, Schelkovo, Moscow Region) with a viscosity average molecular weight of 200 and 1000 kDa, SD 95 and 85%, respectively, was subjected to additional purification. For this, chitosan with MM 200 and 1000 kDa, DM 85% was dissolved in 1M acetic acid at pH 3.8 for 4 hours. Filtration from coarse impurities was carried out using a filter with a pore diameter of 100-160 μm (ISO 4793-80). The filtrate was titrated with a 12% NH4OH solution to pH 8.0-8.5. The precipitated chitosan precipitate was dialyzed with repeated water changes. At the end of dialysis, chitosan was lyophilized. Chitosans (Sigma-Aldrich, USA) with MM from 200 to 600 kDa, DM from 75 to 85% were used without preliminary purification. Various methods for chitosan were used to determine the main characteristics - MM and DM. Using magnetic resonance spectrometry, conductometric titration, and UV spectrometry, SD was determined. Using HPLC and capillary viscometry, MM was determined. Using HPLC, IP was also determined. The purity characterization of the starting substances and chitosan preparations was carried out according to the level of specific impurity - endotoxins using the chromogenic LAL test, residual protein - by amino acid analysis. ODN, representing 5 repeating segments of Cg with thiophosphoryl bonds in internucleotide phosphates, is commercial. To prepare the adjuvant composition, the components were dissolved in 1% glutamic acid in the following ratio of chitosan: ODN as (20-50): 1. The adjuvant composition was autoclaved at 121 ° C for 15 minutes.

Example 2. Potentiating effect

Adjuvants were added to the split H1N1 vaccine: chitosan, or ODN, or the adjuvant composition in the form of a solution so as to obtain a final adjuvant concentration of 0.5% and 3 μg of the vaccine in a total volume of 0.2 ml. Mice were immunized intramuscularly, twice, injecting 0.2 ml of the vaccine with or without adjuvant (phosphate-saline solution (FSF)). Chitosan and ODN individually are quite immunogenic, however, they have a potentiating effect in the composition: they significantly increase serum antibody titers against all 3 components of the split seasonal influenza vaccine. The immunogenicity of the split vaccine against H1N1 virus is presented in Table 1. The adjuvant composition containing chitosan and ODN increases the average antibody titer by 32 times compared to the vaccine without adjuvant, whereas the adjuvants based on chitosan or ODN were 8 and 2 times, respectively.

Example 3. Immunogenicity and protective effect

Adjuvants: chitosan, or ODN, or adjuvant composition, or aluminum hydroxide, or Freund's adjuvant (CFA) were added to the inactivated whole-virion influenza vaccine based on strain A / California / 07/2009 X-179A (H1N1) so as to obtain a final concentration adjuvants,%: 0.5 or 0.002 or 0.5 or 0.01 or 0.5, respectively, and vaccines in a ratio of 1: 1 (by volume). Mice were immunized as described in Example 2. Immunogenicity was assessed by the RHA method by the presence of antibodies in the sera against the vaccine virus. The results are presented in Table 2. The adjuvant composition containing chitosan and ODN significantly increases the immunogenicity of the inactivated whole-virus swine influenza vaccine based on the H1N1 strain in comparison with adjuvants of other groups by nature and mechanism of action (Table 2).

Compared to a vaccine without adjuvant, the average antibody titers increase by 20 times when immunized with the adjuvant composition consisting of chitosan and ODN; chitosan - 10 times; ODN or aluminum hydroxide - do not increase; Freund's adjuvant (CFA) - 3 times. With the intranasal administration of live attenuated vaccines based on cold-adapted influenza A and B strains, the adjuvant composition increases immunogenicity (local and systemic virus-specific immunoglobulins of class G), as well as a protective effect up to sterile immunity.

Example 4. Immunogenicity of polio vaccines

Adjuvants: chitosan or ODN or adjuvant composition or aluminum hydroxide or Freund's adjuvant (CFA) were added to the inactivated whole-virion type I, II, and III polio vaccine based on live attenuated (Sabin) and wild strains to obtain a final concentration adjuvants, as described in example 3. The presence of antibodies in blood serum was determined in the neutralization reaction (PH). The immunogenicity of the type III Sabin virus vaccine is presented in Table 3.

Adding to the vaccine an adjuvant composition consisting of chitosan and ODN resulted in a 128-fold increase in antibodies that neutralize a productive infection compared to a vaccine without adjuvant; chitosan - 32 times, ODN - 4 times, aluminum hydroxide - 8 times, Freund's adjuvant (CFA) - 16 times.

An adjuvant composition containing chitosan and ODN significantly increases the immunogenicity of inactivated whole-virion vaccines against poliomyelitis type I, II and III based on live attenuated (Sabin) and wild strains in comparison with adjuvants of other groups by nature and mechanism of action.

Example 5. The protective efficacy of vaccines against Pseudomonas aeruginosa (recombinant antigens)

Adjuvants: chitosan, or adjuvant composition, or aluminum hydroxide were added to the recombinant Pseudomonas aeruginosa vaccine, so as to obtain the final concentration of adjuvants as described in Example 3. Adding the adjuvant composition containing chitosan and ODN to the recombinant vaccine led to a significant increase in protective efficacy vaccines on a model of a lethal Pseudomonas aeruginosa infection. The efficiency index when using the composition exceeded 6.0, chitosan - 3.0, aluminum hydroxide - 2.0. After a double intramuscular immunization of mice with a vaccine supplemented with an adjuvant composition, complete protection against infection was provided up to LD50 80 mln.m.

Claims (3)

1. Adjuvant composition containing chitosan and a synthetic oligodeoxynucleotide, characterized in that chitosan has a molecular weight in the range from 200 to 1000 kDa, a degree of deacetylation from 75 to 98%, a polydispersity index of not more than 2.0, the level of endotoxins and foreign protein is less than 100 EU / ml and 0.05%, respectively, and the oligodeoxynucleotide is a synthetic oligodeoxynucleotide, which consists of 5 repeating segments of CG nucleotides with thiophosphoryl bonds at internucleotide phosphates; when the ratio (mass.) of the components of chitosan: synthetic oligodeoxynucleotide (20-50): 1. 2. The composition according to p. 1, characterized in that as chitosan it contains a cationic polysaccharide obtained from crab chitin. 3. The composition according to p. 1, characterized in that chitosan and a synthetic oligodeoxynucleotide are dissolved in 1% glutamic acid.

Images