RU2217162C2 - Vaccine against viral infections - Google Patents

Abstract

FIELD: biotechnology, medicine, biochemistry, immunology. SUBSTANCE: invention relates to vaccine that represents particles 25-30 nm by diameter containing polynucleotide complex (recombinant plasmid DNA comprising genes of infectious agent or double-stranded RNA-stimulator of nonspecific resistance of organism) in center and fused protein on surface comprising epitopes (polypeptides) of infectious agent and enzyme (for example, glutathione S- transferase or β- galactosidae). Linkage between polynucleotide complex and fused proteins is carried out conjugate : spermidine (for linkage of conjugate with polynucleotide complex) polyglucine- substrate for enzyme (for example, glutathione or galactopyranoside) for affinity sorption of fused proteins for construction. Vaccine provides the broader titer of antibodies and more prolonged immune response in immunization. EFFECT: enhanced effectiveness and valuable medicinal properties of vaccine. 6 dwg, 4 ex

Description

 The invention relates to the field of biotechnology, medicine, immunology; it can be used in the pharmaceutical industry and molecular pharmacology and is a method of delivery and presentation of immunogens to the body’s immune system.

 Advances in genetics, molecular biology and biotechnology have identified a modern vaccine development strategy. To date, one of the most promising and most developed areas is the creation of genetically engineered vaccines by producing antigens by microbiological synthesis and their subsequent purification to a homogeneous state. It is preferable to the improvement of vaccines based on live attenuated or inactivated pathogens. This is due to the fact that the presence of a portion of the proteins of the infectious agent in the vaccine is undesirable [1, 2]. For example, it is known that among the proteins of the HIV virus there are B-cell epitopes that stimulate the production of antibodies that increase the infectivity, and T-cell epitopes that stimulate suppressive cells, and regions that cross-react with normal cell proteins, and immunosuppressive regions. Therefore, to create effective and safe vaccines of a new generation, it is promising to create synthetic peptide vaccines, which are expected to be free from many of the disadvantages inherent in live and inactivated vaccines [3, 4]. The most promising may be vaccines containing several T and B-cell epitopes of an infectious agent. The disadvantages of genetic engineering vaccines include the relatively small molecular weight of the synthesized immunogen polypeptides, from a thousand to several tens of thousands of daltons. Therefore, various adjuvants are used for vaccination, which is not always desirable and causes side effects. The most powerful of the adjuvants is Freund's adjuvant, which is forbidden for people to use.

 Known methods for exposing the epitopes of an infectious agent on the surface of HBcAg capsid structures synthesized in E. coli [5-9] and S. cerevisiae yeast [10] and obtaining an immune response to them. HBcAg is a hepatitis B virus nucleocapsid and consists of identical 21 kD protein subunits, has a diameter of about 25-27 nm [5, 6] or 30-34 nm [9] according to different authors and is an immunogen. To realize the immune response to the built-in epitopes, the following three basic conditions must be met [5, 6]: the built-in sequence a) does not violate the capsid-forming properties of the chimera, b) is exposed on its outer surface, c) it retains its native conformation. The most promising resistance to the introduction of foreign sequences is the region of the 144th amino acid of the gene. The introduction of alien sequences of 40–50 amino acids in length in this region does not disrupt the self-assembly of capsids, which preserve the morphology of non-mutant HBcAg and expose the introduced sequences on their surface while preserving their antigenic and immunogenic activity [5, 6].

 The disadvantages of this design include the limited size of embedded foreign epitopes (about 40-50, up to 100 amino acids [5, 6]). In addition, not all of the built-in epitopes showed their antigenicity in the composition of hybrid particles [9, 11]. For example, it was found that the monkey immunodeficiency virus epitope inserted at the C-terminus after the 183rd amino acid is inaccessible to antibodies and immunologically inactive [11].

 An object of the invention is the creation of a highly effective molecular design of a virus-like species (vaccine) as a means of delivery of immunogens. For vaccination of people, it seems promising to create such a vaccine design that would have both antigenic properties and an immune response stimulator, and at the same time have a molecular weight comparable to that of viruses, i.e. would be a complete antigen.

 As stimulants of the immune response, polysaccharides, lipid (including liposomes) and polynucleotide complexes (double-stranded RNA, fragmented DNA), etc. are used.

 The problem is solved by creating a new generation vaccine - a molecular construct, which is a virus-like particles in the center of which is a polynucleotide complex (recombinant plasmid DNA or double-stranded RNA), and on the surface are hybrid proteins containing epitopes of an infectious agent (Fig. 1). Recombinant plasmid DNA may contain various genes of the infectious agent, thereby transforming the construct also into a “DNA vaccine”.

 The invention consists in the following.

 A vaccine (molecular design of a virus-like species) is a particle (Fig. 1) 25-30 nm in diameter, containing in the center a polynucleotide complex (recombinant plasmid DNA containing the genes of an infectious agent or double-stranded RNA - a stimulator of nonspecific resistance of the body), and on the surface - fusion proteins containing epitopes (polypeptides) of an infectious agent and an enzyme (e.g. glutathione S-transferase or β-galactosidase). The connection between the polynucleotide complex and fusion proteins is carried out by means of a conjugate: spermidine (for linking the conjugate to the polynucleotide complex) - polyglucin - substrate for the enzyme (for example, glutathione or galactopyranoside) for affinity sorption of hybrid proteins on the construct. When immunized with the created construct, the antibody titer is higher and lasts longer than that when immunized with the original immunogen protein.

 To assemble the molecular construct, a recombinant plasmid DNA is obtained, isolated by the alkaline method [12], and containing the genes of the infectious agent or double-stranded RNA, isolated from yeast, for example, from the Saccharomyces cerevisiae strain M437 Y116 [13], which is used as the main component of the drug to stimulate non-specific resistance organism "Ridostin" [14, 15].

The polynucleotide complex is coated with a conjugate layer (FIG. 1):
- polyglucin with a molecular weight of about 60,000 D is activated with sodium periodate, which is then removed by gel filtration on a Sephadex G-50 column in potassium phosphate buffer;
- spermidine and a substrate for the hybrid protein enzyme, for example glutathione or aminophenylgalactopyranoside, are added to activated polyglucin;
- after incubation, unbound components are removed by gel filtration on Sephadex G-50 in buffer: 0.1 M Tris-HCl, pH 8.3;
- the resulting conjugate is added in excess to the polynucleotide complex and, after incubation, the conjugate is removed from the excess by gel filtration on a CL-6B Sepharose column in physiological saline.

 Next, hybrid proteins containing epitopes of an infectious agent and an enzyme, for example, glutathione S-transferase or β-galactosidase, are added to the obtained complex, and after incubation, the resulting molecular structure is separated by gel filtration on CL-6B Sepharose using physiological saline for elution.

 Hybrid proteins are obtained according to the standard procedure for gene cloning in the same reading frame with marker enzymes (glutathione S-transferase, β-galactosidase, etc.) in developed or commercial vectors [16, 17]. Isolation and purification of proteins is carried out according to the developed method or according to the recommendations of firms.

 The process of obtaining the design shown in figure 2. The resulting design is used for immunization. The titer of produced antibodies, the strength and duration of the immune response is determined by standard methods.

 New features, compared with known immunogenic constructs, are: the use of a virus-like species for immunization of a molecular construct containing a polynucleotide complex (recombinant plasmid DNA containing the genes of an infectious agent or double-stranded RNA - a stimulator of nonspecific resistance of the body), and hybrid proteins on the surface containing epitopes (polypeptides) of the infectious agent and glutathione S-transferase (or β-galactosidase). The connection between the polynucleotide complex and hybrid proteins is carried out by means of a polyanion - conjugate: spermidine (for linking the conjugate to the polynucleotide complex) - polyglucin - glutathione (or galactopyranoside) (for affinity sorption of hybrid proteins on the construct). It is this combination of features that provides a higher antibody titer and a longer immune response during immunization with the created molecular construct as compared to analogous immunizations with the initial protein antigen. The molecular construction allows exposing on the surface of the particles the epitopes of any infectious agents lasting from tens to thousands of amino acids (in analogs - up to 50-100 amino acids) and creating multivalent vaccines.

The list of graphic materials:
Figure 1. Molecular construction of a virus-like species:
1 - plasmid DNA, 2 - conjugate: spermidine-polyglucin-glutathione, 3 - hybrid protein glutathione-S-transferase - TBI (GST-TBI).

Figure 2. Gel filtration on a column with sepharose CL-6B:
1 - plasmid DNA,
2 - the same after incubation with the conjugate: spermidine-polyglucin-glutathione,
3 - after incubation with the glutathione-S-transferase fusion protein - TBI.

FIG. 3. Scheme for constructing a recombinant plasmid: bla — β-lactamase gene encoding ampicillin resistance (Ap ^r ); gst is a gene encoding glutathione-S-transferase; tbi is a gene encoding the TBI protein.

 Figure 4. Electron diffraction pattern of a negatively stained vaccine microparticle preparation. An increase of 70,000.

 FIG. 5. The dynamics of changes in the titer of antibodies to TBI protein in animal sera immunized with TBI-GST protein (black bars); virus-like particles with TBI-GST protein (white columns). Control animals (gray columns). In each experiment, serum from five animals was used.

 FIG. 6. Electrophoregram of vaccine samples in 10% polyacrylamide gel: a - Coomassie stain R-250: 1 - construct with GST protein - TBI, 2 - protein gp41-β-galactosidase, 3 - construct with GST proteins - TBI and gp41- β-galactosidase; b - immunoblotting with antibodies positive for HIV-I human serum. M - molar mass markers.

 The invention is illustrated by the following examples of specific performance.

 Example 1. The creation of plasmids carrying the genes of hybrid proteins.

 The goal of the pGEX-2T-TBI plasmid was to produce the glutathione-S-transferase-TBI (GST-TBI) fusion protein, capable of binding affinity to the glutathione-polysaccharide conjugate with one half of the molecule and exposing HIV-I epitopes to the other half (TBI). on the surface of the structure [18]. When creating the plasmid pGEX-2T-TBI, the original plasmid pUC19 / TBI [18] is digested with restriction enzymes Pst I and EcoR I (Fig. 3), a 445 bp fragment is isolated, which is then ligated with the commercial Pharmacia pGEX- vector 2T [16] treated with restriction enzymes EcoR I and Sma I. The correctness of the insertion is determined by DNA sequencing in the region of the junction of genes encoding the components of the hybrid protein. The reading frame of the gene is not violated. In the created plasmid, recognition sites for restriction enzymes Pst I and Sma I are absent.

Competent cells of the recipient strain of E. coli SG 200-50 are transformed with the obtained plasmid, and the suspension is plated on agar medium containing ampicillin (100 μg / ml). To produce a plasmid, a cell culture is grown in L-broth to an optical density of D ₅₅₀ ≅1.5-2.0 pu, chloramphenicol is added and incubated for 3-5 hours at 32 ^o C. Plasmid DNA is isolated by alkaline method followed by purification by hydroxylapatite chromatography.

 Plasmid pGP41-16 [19], containing the gene encoding the fusion protein pGP41-16 - β-galactosidase, was created according to traditional technology and was kindly provided by the authors.

 Example 2. Isolation and purification of fusion proteins.

To produce biomass of recombinant cells, the clones grown on a selective (Ap) medium are added to 100 ml of L-broth; they are grown to an optical density of D ₅₅₀ ≅1.0 pu, ampicillin is added to a final concentration of 500 μg / ml and incubated for 2 h for lysis of plasmid-free cells. After that, 1.6 L of L-broth is inoculated (8 flasks of 200 ml each). The induction of the biosynthesis of the target protein is carried out by adding isopropylthiogalactopyranoside to a final concentration of 0.1 mM. The GST-TBI fusion protein accumulates in cells in an insoluble form in inclusion bodies.

 Purification of GST-TBI protein is carried out according to the method developed for the artificial TBI protein [20]. For this, the cell suspension is sonicated and the inclusion bodies are washed. The hybrid protein is dissolved in a 6 M urea solution for 2 h, the precipitate is removed by centrifugation, and then the protein is renatured by incubating the solution successively at urea concentrations of 3 M and 1.5 M, after which dialysis is performed against a buffer that does not contain a denaturing agent. With this mode of renaturation, the precipitation of the target protein is not observed. For purification using affinity chromatography on glutathione-sepharose.

 Purification of the fusion protein gp 41 (607-620) -β-galactosidase is carried out according to the recommendations of the authors [19].

 Example 3. The creation of a virus-like construct (vaccine).

 A) In viruses, DNA and RNA molecules are not bound to proteins by covalent bonds. A similar structure was created by us. At the center of the vaccine design is an intact polynucleotide complex, and on the surface is a hybrid protein containing HIV-I antigens. For communication, a polyglucin conjugate with spermidine and glutathione is used. A positively charged spermidine provides binding of the conjugate to the polynucleotide complex, glutathione affinity sorbes the TBI-GST fusion protein. Schematic view of the structure shown in figure 1.

 The plasmid pGEX-2T-TBI (molecular weight about 3250000 D) containing the gene encoding the HIV-I epitopes is used as the “central nucleus” of the construct.

 To obtain a polysaccharide insert, 50 mg (0.8 μM) of polyglucin (60,000 D) are treated with a 0.5 M sodium periodate solution for 20-30 minutes and then gel filtration is carried out on a Sephadex G-50 column in 20 mm phosphate buffer ( pH 7.6). After that, 5 mg of glutathione (16 μM) and 2.5 mg of spermidine (16 μM) are added to the activated polyglucin solution. The mixture was incubated overnight, then sodium borohydride was added, stirred for another 2 hours, and unreacted components were removed by gel filtration on Sephadex G-50 using 100 mM Tris-Hcl (pH 8.3). Thus obtained drug contains 3.3 mg of protein. Since the initial components (glutathione and spermidine) are introduced in an equimolar ratio, it can be assumed that the resulting conjugate contains the same amounts, i.e. 0.8 μM polyglucin 7.2 μM glutathione and spermidine (ratio of about 1: 9: 9).

To assemble the structure, a polysaccharide insert (0.1 mg protein) is added to a solution containing 400 μg of nucleotide material (about 0.12 nM) in physiological solution, the mixture is incubated for 2 hours at 4 ^° C, and the resulting complex is separated by gel filtration on Sepharose CL-6B. After that add the hybrid protein (1.5 mg) in physical. solution and also incubated for 2 hours. The resulting structure was separated from unbound components by gel filtration on Sepharose CL-6B (figure 2).

 Investigation of the physical properties of molecular construction.

 When studying the complex [nucleotide material - polysaccharide insert], it was found that 0.12 nM plasmid DNA contained 0.025 mg of protein, i.e. for 1 molecule - 540 molecules of glutathione and spermidine, or 60 molecules of the polysaccharide insert, respectively.

 The final construct contained 0.96 mg of protein (about 20 nM fusion protein with a molecular weight of about 45,000 D). Thus, the final design had the ratio: for 1 molecule of the nucleotide material - 60 molecules of the polysaccharide insert - 170 molecules of the hybrid protein. Her pier. weight about: 3250000 + 60 • 60000 + 170 • 45000 = 14500000 D and when gel chromatography on a column with CL-6B Sepharose, it elutes in free volume (figure 2).

 From theoretical calculations it follows that a spherical nucleic acid molecule with a molecular mass of about 3250 kDa can adsorb about 55 polyglucin molecules. The complex [nucleotide material - polysaccharide insert] at a molecular weight of about 3250 kDa + 55 • 60 kDa = 6850 kDa can absorb about 120 spherical molecules of the hybrid protein (mol. Weight 45 kDa). Since in the experiment this ratio was 1: 60: 170, it can probably be explained by some non-compactness of the polysaccharide conjugate and the dipole spatial structure of the hybrid protein. With a dipole spatial structure, only TBI protein (antigen) is located on the surface of the structure.

 To check the completeness of packaging of the nucleotide material in the assembled construct, it was processed with a mixture of DNase and RNase. Experience has shown that while for the initial plasmid DNA complete degradation was observed after 30 min of incubation, the nucleotide material remained intact for 24 hours in the assembled construct.

 According to electron microscopy, the molecular construction preparation contained spherical particles having a generally diameter of 10 to 25 im (Fig. 4). There were also larger particles, the diameter of which reached 50-100 nm. Perhaps this is due to the fact that the polysaccharide insert contains not one spermidine molecule on the surface, but nine, and a number of particles are combined. Nevertheless, the size of the resulting construct is comparable to the size of poliovirus and poliomyelitis virus, and approaches the size of HIV.

 B) Similar results were obtained when creating a molecular construct containing double-stranded RNA from the yeast strain Saccharomyces cerevisiae M437 Y116 as the central nucleus.

 C) When creating a vaccine design containing two hybrid proteins: GST-TBI and gp 41 (607-620) -β-galactosidase, spermidine is added to the activated polyglucin at the polysaccharide insertion stage, and aminophenylgalactopyranoside is added instead of glutathione. And then, during assembly, one part of the conjugate [polyglucin-spermidine-spermidine-galactopyranoside] is introduced into the solution with plasmid DNA into nine parts of the conjugate [polyglucin-spermidine-glutathione]. Then, hybrid proteins are added in appropriate ratios. The design exhibits total antigenic properties. It should be noted that the artificial TBI protein does not include the sequence gp 41 (607-620) and the combination of both proteins in the vaccine design should increase its immunogenic properties.

 Example 4. Biological tests of molecular construction.

 BALB / c mice weighing 18-20 grams were used for immunization. Animals were kept on a standard diet. All manipulations with animals in the experiment were performed using sedatives in accordance with veterinary legislation.

 Immunization. In order to study the immune response to artificial virus-like particles exhibiting TBI protein, BALB / c mice were immunized. Animals were injected intramuscularly three times with an interval of two weeks at 45 μg (protein) of the assembled construct. As a positive control, purified TBI-GST protein (80 μg per mouse) was used. Every two weeks, blood was taken and serum was prepared to determine the titer of antibodies to the TBI protein.

 An enzyme-linked immunosorbent assay (ELISA) was used to determine the titer of antibodies to HIV-1 epitopes (TBI protein). The antigen at a final concentration of 5 μg / ml in a volume of 100 μl was sorbed overnight in polystyrene plates. Serial triple dilutions of sera were used for titration, starting at 1:15. Bound specific antibodies were detected using a rabbit anti-mouse IgG conjugated antibody with horseradish peroxidase ("Sigma").

The accumulation of antibodies in the serum of mice immunized with pure TBI protein and particles is shown in FIG. 5. As can be seen from the graph, virus-like particles induce a higher antibody titer compared to the titer induced by the pure TBI-GST protein, despite the fact that the amount of TBI-GST protein introduced into the animal in the particles was two times less. Two groups of mice also differ in the dynamics of changes in serum titers. Groups of mice immunized with TBI-GST and virus-like particles also differ in the dynamics of changes in serum titers. One week after the last immunization, the serum titer of animals immunized with TBI-GST reached 1.8 • 10 ⁷ , but after three weeks it sharply decreased to 1 • 10 ⁵ (Fig. 5). In mice immunized with virus-like particles, a week after the last immunization, the reverse titer reaches 0.9 • 10 ⁷ and practically does not change after three weeks. Thus, virus-like particles cause a prolongation of the immune response, acting as a kind of depot of antigens of the infectious agent. Apparently, the TBI protein in the composition of virus-like particles in the animal is more stable and less susceptible to proteases.

 When the enzyme immunoassay was performed, proteins were transferred from the gel to a nitrocellulose filter. The immunoblot was conducted with human serum containing antibodies to HIV-1 proteins. Immune complexes were detected using a protein A conjugate with peroxidase. As can be seen from Fig.6, the first vaccine design contains GST-TBI protein, the second - both proteins. Both constructs exhibit antigenic properties.

Thus, the use of the proposed molecular structure in comparison with analogues allows you to:
- expose on the surface of the molecular structure any epitopes of infectious agents lasting from tens to thousands of amino acids;
- use the genes of infectious agents in recombinant plasmid DNA (the "central core" of the particles) as a DNA vaccine;
- use double-stranded RNA in the molecular structure (“central core” of particles) as a nonspecific stimulator of the immune response;
- polyglucin used to produce the conjugate is also a stimulant of the immune response;
- the relatively large particle sizes of the molecular structure (about 25 nm) allow it to be used for immunization without adjuvants as complete antigens.

Literature
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Claims (1)

A viral infection vaccine, characterized in that it is a molecular construct in the center of which is a polynucleotide complex representing a recombinant plasmid DNA containing the genes of an infectious agent or double-stranded RNA isolated from yeast, while hybrid proteins are exposed on the surface of the polynucleotide complex including the epitopes of the infectious agent and the enzyme glutathione S-transferase or β-galactosidase, the polynucleotide complex being linked to hybrid proteins dstvom conjugate containing spermidine-polyglukin glutathione or spermidine polyglukin-galactopyranoside.

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