RU2442604C1 - The method of immune response enhancement - Google Patents

Abstract

FIELD: medicine, immunology. ^ SUBSTANCE: group of inventions relates to medicine in particular to immunology and can be applied to enhance the immune response of animals and human to antigen consisting of one or a number of epitopes; for the above purpose the parenteral immunization of the patient is performed either with the mixture of the above antigen and adjuvant in the shape of spherical particles or with the above antigen fixed on the surface of the spherical particles; at that the spherical particles consist of thermo-denaturated coat protein of spiral plant viruses, predominantly tobacco mosaic virus. ^ EFFECT: invention secures the efficient stimulation of the immune response to the antigen upon pareneteral immunization of the patient with small doses of the antigen. ^ 2 cl, 9 ex, 9 dwg

Description

The invention relates to the field of immunology, namely to methods of enhancing the immune response in animals and humans to various antigens, and can be used in medicine, veterinary medicine, virus-free crop production and biotechnology to produce antibodies.

Vaccination i.e. the deliberate introduction into the body of an animal or person of weakened or killed pathogens of infections is the most effective means of preventing infectious diseases. As a result of vaccination, an immune response (AI) develops to the pathogen antigens, aimed at their removal from the body. With repeated contact of the vaccinated person or animal with this antigen, AI is much faster and more effective, so that the disease does not develop or proceeds much easier.

Vaccination with whole viral particles or bacterial cells, even killed or weakened, is often accompanied by undesirable side effects. In addition, of the many pathogen antigens, only a fraction are effective immunogens (i.e., antigens that cause strong AI). With the current state of the art, it is possible to accurately identify those pathogen fragments that have maximum immunogenicity, and to obtain them in sufficient quantities for vaccination by isolation from the pathogen or artificial synthesis. In most cases, such fragments are short oligopeptides with the length of several amino acid residues (antigenic determinants, epitopes). Due to their small size, they have very low immunogenicity and cannot be used for vaccination on their own.

One approach to enhancing AI on weak antigens is to use the so-called immunological adjuvants. Adjuvants are any substances or their compositions that activate the immune system, as a result of which a specific AI for a particular antigen is significantly enhanced. The most famous and most commonly used is Freund's complete adjuvant, consisting of a water-in-oil emulsion and killed mycobacterial cells. Of all the known, this adjuvant is the most effective, however it is toxic, it can not be used for humans, domestic and farm animals and used intravenously.

Another approach to enhancing IO for weak antigens is to increase (or restore) their immunogenicity by assembling on the surface of the carrier (platform), the dimensions of which are usually many times larger than the dimensions of an individual epitope. The basis of this method is the innate property of the immune system of warm-blooded animals with high efficiency to respond to macromolecular complexes, the surface of which is a system of regularly repeated antigenic determinants. Viral particles are natural resistant macromolecular complexes of this kind, therefore, in recent years they have been actively used as a platform for creating immunogenic compositions.

The methods for creating immunogenic compositions based on viruses (plants), which determine the current state of the art, are based on the construction of chimeric viral particles. For such particles, a fragment of the target protein (CB), for example, one of its epitopes, is inserted in the amino acid sequence of the membrane protein (BO). Such compositions are viral or RNA-free virus-like particles (HPV) that carry foreign epitopes on their surface. They are obtained by the polymerization of recombinant BO molecules constructed by fusion of a viral BO with a foreign epitope by genetic engineering methods. During immunization, viral particles act as a carrier (platform), and the epitope exposed on the particle surface acts as an antigen. Vaccines derived from the practical implementation of this approach stimulate long-term cellular and humoral protective immunity (Turpen et al. Production of peptides in plants as viral coat protein fusions. US Patent 5977438. Nov. 1999; Werner et al. International Patent Application PCT / EP 2006/009029 (WO / 2007/031339); Wemer et al. Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A. Proc. Natl. Acad. Sci. USA, 2006, 103, 17678-17683; McCormick & Palmer. Genetically engineered Tobacco mosaic virus as nanoparticle vaccines. Expert Rev Vaccines. 2008, 7, 33-41; Steinmetz et al., Structure-based engineering of an icosahedral vims for nanomedicine and nanotechnology. Curr. Top. Microbiol. Immunol. 2009, 327, 23 -58; Denis et al., Immunogenicity of papaya mosaic virus-like particles fused to a hepatitis C virus epitope: evidence fo r the critical function of multimerization. Virology 2007, 363, 59-68; Acosta-Ramirez et al., Translating innate response into long-lasting antibody response by the intrinsic antigen-adjuvant properties of papaya mosaic virus. Immunology, 2007, 124, 186-197; Lacasse et al., Novel plant virus-based vaccine induces protective cytotoxic T-lymphocyte-mediated antiviral immunity through dendritic cell maturation. J. Virology, 2008, 82, 785-794; Leclerc et al., Adjuvant viral particle. US Patent 7641896, Jan. 5, 2010).

The disadvantage of these methods is the difficulty of obtaining an immunogenic composition. A simplified scheme of such work is as follows. In a reverse transcription reaction on a genomic viral RNA template, a cDNA copy of the BO gene is obtained. The resulting cDNA is propagated by polymerase chain reaction (PCR). The PCR product (BO gene) is cloned into a bacterial plasmid. Similarly receive cDNA encoding a fragment of the target protein (CB). Then both DNAs are combined in vitro (“fused”), while at the same time adding to the construct a number of elements that ensure efficient expression of the obtained gene in bacterial cells and the possibility of purification of the recombinant protein from the lysate. The resulting plasmid infects Escherichia coli cells. As a result of expression of the recombinant gene, a BO of the virus is formed, fused with CB (“fusion protein”), which is assembled into virus-like particles (HPV) directly in bacterial cells due to the BO's ability to self-assemble. HPV is isolated from the cell lysate (usually using expensive affinity chromatography) and is used as intended. Despite the fact that many important details are omitted in this description, it can be seen that obtaining each type of recombinant virus carrying a chimeric BO is a complex and time-consuming process. Nevertheless, this approach is used in the case of native viruses, since it is impossible to fix the CB in other ways on intact viral particles (TMV, or the papaya mosaic virus, or any other viruses).

Another important drawback of immunogenic compositions based on chimeric viruses is their lack of universality. For each new Central Bank or epitope, you need to get your platform and do the newly described genetic engineering manipulations. The conditions of these operations for different CBs can vary greatly, and preliminary creation of a new immunogenic composition often requires preliminary long-term optimization of these conditions.

A known method of enhancing AI, combining both of the above technical solutions, which is advisable to take as a prototype (Leclerc D., Immunogenic affinity-conjugated antigen systems based on papaya mosaic virus and uses this. US Patent applications 2010/0047264).

According to one embodiment of the prototype, a purified preparation of papaya mosaic virus (VMP) is used as an adjuvant. Animals are immunized with a mixture of antigen and VMP. It has been shown that native particles of HMW have adjuvant activity. This activity was demonstrated on two model antigens - lysozyme and ovalbumin, and is expressed in the fact that the titer of mouse antisera obtained by combined immunization with these antigens and VMP is higher than when immunized with these antigens without VMP.

According to another embodiment of the prototype, HPV-based HPV is used as a carrier (platform) to create immunogenic compositions. HPV is obtained by polymerization of the chimeric BMP BM, combined (fused) with the target peptide as a result of genetic modification of the BM gene. Said target peptide has a high affinity for Salmonella typhi porins. To create an immunogenic composition, chimeric HPVs are mixed with antigens (porins), which are affinity adsorbed on carrier particles. It has been shown that parenteral or oral administration of such a composition induces protective immunity in the animal to the causative agent of salmonellosis.

The disadvantages of the prototype are the complexity of obtaining the immunogenic composition and the weak adjuvant activity of VMP, expressed in a low titer of antisera obtained with its help.

The aim of the invention is to simplify the method of obtaining adjuvant and immunogenic composition and increase their effectiveness in stimulation of AI. The problem is solved in that as an adjuvant and as a carrier (platform) to create an immunogenic composition using spherical particles (MF) obtained by thermal restructuring of the structure of spiral plant viruses, in particular the tobacco mosaic virus (TMV). The proposed solution is based on the fact that the aforementioned MFs have significantly greater immunogenicity than the original particles of the native virus.

The invention is carried out in two ways.

One way of implementing the present invention is as follows. A suspension of TMV particles at a concentration of 0.1-10 mg / ml is subjected to short-term heat treatment at temperatures above 94 ° C. MF resulting from this treatment is mixed with the antigen in a mass ratio of 1: 1-1: 25. The mixture is prepared immediately before injection or some time before it. A mixture of antigen and MF is parenterally administered to the animal. As a result of the introduction of such a mixture, the animal develops AI on the introduced antigen, which is expressed in the appearance in the blood serum of antibodies specific for this antigen.

Another way of implementing the present invention is as follows. A suspension of TMV particles at a concentration of 0.1-10 mg / ml is subjected to short-term heat treatment at temperatures above 94 ° C. MF resulting from this treatment is used as a carrier to create an immunogenic composition. MF is mixed with the antigen in a ratio that ensures the formation of a monolayer of antigen molecules on the surface of the carrier. The antigen is covalently fixed to the surface of the carrier by treating the mixture with formaldehyde for 10 minutes. The resulting composition is administered to the animal parenterally. As a result of the introduction of the composition, the animal develops AI on the introduced antigen, which is expressed in the appearance in the blood serum of antibodies specific for this antigen.

Brief Description of Drawings

Figure 1 illustrates the appearance of the MF obtained by heating a suspension of native TMV particles to 98 ° C and the dependence of the size of the MF on the initial concentration of TMV: (a) 0.1 mg / ml; (b) 1.0 mg / ml; (c) 10 mg / ml. Scanning electron microscopy.

Figure 2 is a graph illustrating the results of determining the titer of mouse antisera to TMV and to MF and demonstrating a higher immunogenicity of MF compared to native TMV particles. Enzyme-linked immunosorbent assay (ELISA).

Figure 3 shows that the surface of all SCs is completely covered by BVC BO molecules labeled with fluorescein isothiocyanate (FITC). (a) Fluorescence microscopy; Label size 3 microns. (b) phase contrast.

Figure 4 illustrates that recombinant P2 protein expressed in bacteria binds to the surface of MF and is detected using rabbit antibodies to plum pox virus and secondary donkey anti-rabbit antibodies conjugated with Alexa 555 red fluorophore. Label size 5 microns. Fluorescence microscopy.

Figure 5 is a graph illustrating the results of determining the titer of murine antisera to BO X-potato virus (CVC) obtained by immunizing animals with a mixture of antigen and MF of different sizes. IFA.

Figure 6 is a graph illustrating the results of determining the titer of mouse antisera to BVCV obtained by immunizing animals with an antigen fixed on the surface of MFs of various sizes. IFA.

Figure 7 is a graph illustrating the adjuvant effect of MF and the immunogenicity of a composition prepared on their basis, when stimulating AI in recombinant protein N1. IFA.

Figure 8 is a graph illustrating the results of determining the titer of mouse virus antisera to the virus obtained by immunization of animals with CPI BV mixed with MF or fixed on their surface. IFA.

Figure 9 is a graph illustrating the effect of a dose of MF on the intensity of stimulation of the immune response to an antigen. IFA.

EXAMPLES

Example 1

Obtaining spherical particles (MF)

Obtaining a purified preparation of TMV. TMVs were accumulated in tobacco plants Nicotiana tabacum s. Samsun. For preparative virus isolation, infected leaves were homogenized in 0.02 M sodium phosphate buffer, pH 7.5, containing 0.2% Na ₂ SO ₃ , 0.01 M EDTA and 0.2 M sucrose, in a 1: 5 ratio ( g / ml). The extract was clarified by centrifugation in a JA-20 rotor of 15,000 rpm for 20 min at 4 ° C. An aqueous solution of Triton X-100 was added to the clarified extract to a final concentration of 5% and stirred for 30 min at 4 ° C. The extract was layered on a 20% (g / g) sucrose pad prepared on 0.02 M sodium phosphate buffer, pH 7.5 with 0.01 M EDTA, and centrifuged in a 50.2 Ti rotor on an L5-50 ultracentrifuge (Beckman) 29000 rpm for 210 min at 4 ° C. Precipitation was resuspended in 0.02 M sodium phosphate buffer, pH 7.5, containing 0.01 M EDTA, and left overnight at 4 ° C. The viral preparation was clarified on an Eppendorf 5414 benchtop centrifuge for 10 min, diluted in 0.02 M sodium phosphate buffer, pH 7.5 with 0.01 M EDTA, and centrifuged in a 50.2 Ti rotor on an L5-50 30,000 rpm ultracentrifuge for 90 minutes at 4 ° C. The pellet was resuspended in 0.02 M sodium phosphate buffer, pH 7.5, and clarified on an Eppendorf benchtop centrifuge for 10 minutes. The concentration of TMV in the purified preparation was determined using an extinction coefficient of A260 = 2.7. The virus yield was about 500 mg, calculated on 100 g of leaf mass. Analysis of TMV by electrophoresis in a denaturing polyacrylamide gel did not reveal impurities in the purified viral preparation. Virus isolation according to the described method took 2 working days.

Obtaining spherical particles (MF). Into 0.5 ml thin-walled polypropylene tubes, 50 μl of purified TMV preparation was added and 30 μl of mineral oil was layered. The tubes were placed in the cells of the Tertsik thermal cycler (DNA-Technology, Russia) and a program was launched that regulates the temperature in the cell: 1) heating from 10 to 98 ° С, 90 sec; 2) incubation at a temperature of 98 ° C, 10 sec; 3) cooling to 10 ° C, 90 sec. At the end of the program, the tubes were removed, the oil removed and the contents of the tubes 1000 g were centrifuged for 5 min. The supernatant was collected and the pellet was resuspended in 50 μl of deionized water. All operations were carried out at room temperature.

The process of converting native TMV to MF was studied using scanning electron microscopy. The dried MF samples were placed in an IB-3 Ion Coater (Eico, Japan) ion-deposition apparatus and the surface was ionized by sputtering gold with palladium. The analysis was performed using a JSM-6380LA scanning electron microscope (JEOL, Japan) at 80 kV. A digital image was obtained using a Gatan Eriangshen ES500W camera using Gatan Digital Micrograph ™ software. Under these conditions, rod-shaped particles of TMV turn into MFs, the size of which depends on the initial concentration of TMV. The average diameter of the midrange at a TMV concentration of 0.1, 1.0, and 5 mg / ml is 50, 100, and 500 nm, respectively (Fig. 1).

This example shows that our proposed method can significantly simplify, in comparison with the prototype, the procedure for obtaining media (platforms). Carrier (MF) is obtained by short-term (10 sec) heating a TMV suspension at a temperature of 98 ° C, as a result of which rod-shaped TMV particles are transformed into MFs of a certain size. Thus, the result of the invention is a significant simplification of the method of obtaining media (platform).

Example 2

Comparison of the immunogenicity of MF and native TMV.

BALB / c mice aged 6-8 weeks were immunized intraperitoneally with MF or TMV virions at a dose of 100 μg antigen per injection. Conducted 3 injections with a two-week interval between them. At the first injection, the antigen was administered in a mixture with an equal volume of Freund's complete adjuvant, and with the next 2, with an equal volume of Freund's incomplete adjuvant. Blood, individually from each mouse, was taken from the tail vein a week after the last immunization. Blood was kept for 2 hours at room temperature until a clot formed, placed in a refrigerator overnight, and the next day the clot was separated by centrifugation at 10,000 g for 10 min. The obtained serum was used to determine the titer of antibodies to MF and to TMV. The titer was determined in a pool obtained by mixing equal volumes of antisera from each animal using an indirect enzyme-linked immunosorbent assay (ELISA). On the MaxiSorp (Nunc) plates, MF or VTM (6 μg / ml in PBS: 0.05 M K ₂ NRA ₄ , 0.1 M NaCl, pH 7.4) were sorbed in a volume of 100 μd per well. The plates were allowed to stand overnight in the refrigerator, washed with PBST (PBS, 0.1% Tween 20, pH 7.4) 5 times in 200 μl per well and the test antisera were added in double serial dilutions prepared on PBST starting from 1/1000 dilution. As a negative control, pre-immune mouse serum was used. Incubated for 1 h at 37 ° C on a thermostatic shaker, washed as described, and peroxidase conjugate (Promega) was added at a working dilution of 1/20000 prepared in PBS containing 0.5% bovine serum albumin. Incubated for 1 h at 37 ° C on a shaker, washed as described and the substrate - ABTS with hydrogen peroxide in 0.05 M citrate-phosphate buffer pH 4.4 was added. The optical density in the wells was determined on a Titerteck Multiscan reader at a wavelength of 405 nm 20 minutes after adding the substrate. The antiserum titer was considered its last dilution, in which the optical density was three times higher than the corresponding value of preimmune mouse serum. The data presented in Fig. 2 show that the titer of antibodies to MF was 20 times higher than the titer of antibodies to TMV.

This example demonstrates the non-obvious fact that MFs have a much higher immunogenicity than the original particles of native TMV. This circumstance is crucial to achieve the objectives of the present invention. It is known that the induction of a humoral immune response (antibody synthesis) requires the cooperation of B and T-lymphocytes, in which B-lymphocyte receptors recognize the antigen, and T-lymphocytes interact with the carrier. It is also known that the antibody response to an antigen attached to a carrier directly depends on the immunogenicity of the carrier. Thus, the effectiveness of immunization (vaccination) with a particular composition is determined not only by the immunogenicity of the CB attached to the carrier, but also mainly by the immunogenicity of the carrier itself.

Example 3

Obtaining the protein coat of the virus of potato X

For preparative isolation of potato virus X (CVC), the leaves of the infected Datura stramonium dope plants were homogenized using a blender in 200 ml of 0.05 M sodium phosphate buffer, pH 8.0, containing 0.15% 2-mercaptoethanol and 0.5% DIECA ) One volume of chloroform was added to two volumes of the extract filtered through a double gauze layer, the mixture was vigorously shaken for 10 min and clarified by centrifugation in a JA-20 rotor (Beckman) 10,000 g for 15 min at 4 ° C. The clarified supernatant was acidified with 0.5 M citric acid to pH 4.8, stirring the extract on a magnetic stirrer for 20 min at 4 ° C. The resulting precipitate of cellular components was removed by centrifugation at 10,000 g for 10 minutes. While stirring on a magnetic stirrer, 1 N NaOH was added to the clarified extract, adjusting the pH to neutral values, dry NaCl to a final concentration of 0.1%, and polyethylene glycol (mol.m. 6 kDa) to a final concentration of 8%. After complete dissolution of the added components, the contents were left at 4 ° C for 30 min to form a virus-containing precipitate. The precipitate was collected by centrifugation at 10,000 g for 15 min in a JA-20 rotor and poured overnight with 0.05 M sodium phosphate buffer, pH 7.2. The final purification of the virus was carried out by ultracentrifugation of the drug through a 30% sucrose cushion in a 50.2 Ti rotor for 180 min at 36,000 rpm. The virus was resuspended in 0.05 M sodium phosphate buffer, pH 7.2, and clarified by low speed centrifugation on an Eppendorf 10,000 g benchtop centrifuge for 10 minutes. The concentration of PVA in the purified preparation was determined using an extinction coefficient A 260 = 2.9. Shell protein (BO) PVI was isolated by treating the viral suspension with a neutral solution of 2M LiCl. The suspension was incubated overnight at -20 ° C and centrifuged at 10,000 g. The supernatant containing PVX BO was dialyzed against tri-distilled water. The protein after dialysis was ultracentrifuged at 100,000 g to remove impurities of BO oligomers. The concentration of BO was determined using an extinction coefficient A280 = 1.2.

Example 4

Obtaining recombinant CVC BO containing epitopes of plum pox virus

Epitopes of BO of plum pox virus (BOC) VNTNRDRDVDAG of 12 amino acid residues and SMLNPIFTPA of 10 amino acid residues (Femandez-Femandez, et al. 2002. Identification of immunogenic hot spots within plum pox potyvirus capsid protein for efficient antigen presentation. J . Virol. 76, 12646-12653) were connected, respectively, with the N-terminus of the full-sized BVC PV (recombinant protein P2) or the N-terminus of the BV CVC with a deletion of the first 20 amino acids (recombinant protein N1). Recombinant constructs expressing recombinant proteins fused to the (His) ₆ - peptide were obtained by cloning the corresponding PCR fragments into the pQE plasmid vector. Restriction PCR fragments were ligated at the corresponding sites into an expression vector as previously described (Ivanov, KI, et al. 1994. The immobilized movement proteins of two tobamoviruses form stable ribonucleoprotein complexes with full-length viral genomic RNA. FEBS Lett. 346 , 217-220). For cloning of the created constructs and their superexpression, E. coli DH5ot and M15 strains [pREP4] were used. E. coli cells transformed with the recombinant vector were grown in culture fluid at 37 ° C to an OD ₆₀₀ of 0.8-0.9. All recombinant DNA manipulations were performed according to standard procedures (Sambrook, J., Ftitsch, EFand Maniatis T. (1989) Molecular cloning: A laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The expression of recombinant proteins was induced by adding 1 mm IPTG, the cell culture was grown for an additional 3 hours at 37 ° C. Chromatographic purification of (His) ₆ recombinant proteins on Ni-HTA agarose was performed according to the manufacturer's protocol (QIAGEN).

Example 5

The binding of target proteins to the surface of the MF

Adsorption of CVC BO. CPV BO was obtained as described in Example 3. To monitor the binding of BO to the surface of the MF (obtained as described in Example 1), the protein was preliminarily labeled with a fluorescent label, 18 μg of fluorescein isothiocyanate (FITC) was added to 1 mg of BVC, and incubated for 24 hours at 4 ° C and dialyzed against water to remove unbound fluorescence tags. 50 μg of MF were incubated with 1 μg of protein in water. BO adsorption on the surface of the MF was observed using a LeicaDRMB fluorescence microscope equipped with an integrated ORCAII-ERG2 camera (Hamamatsu, Japan). The data presented in Figure 3 show that BO PVC covers the entire surface of the midrange.

Adsorption of recombinant protein P2. Recombinant protein P2 was obtained as described in example 4. 50 μg of MF were incubated with 1 μg of protein in water. P2 binding to the MF surface was observed by confocal fluorescence microscopy using immunochemical staining based on the use of rabbit antibodies to BOC and secondary donkey anti-rabbit antibodies conjugated with Alexa 555 red fluorophore. The data presented in Figure 4 show that the P2 protein covers the entire surface of the midrange.

This example demonstrates the non-obvious circumstance that, unlike native viral particles, the surface of the midrange has a high sorption capacity with respect to proteins of various origins. Due to this feature of the midrange, the method for preparing a composition based on them consists in simply mixing the midrange and central bank in a ratio that ensures the formation of a monolayer of central bank on the surface of the midrange. In this regard, when creating an immunogenic composition, the need for genetic engineering manipulations described above disappears. In addition, unlike the prototype, MFs have versatility: once received, they can be used to adsorb any CB.

Thus, another result of the invention is to obtain universal carriers (platforms) for creating various types of complexes and immunogenic compositions.

Example 6

Adjuvant activity of midrange

Outbred white mice were immunized intraperitoneally with a mixture of MF with a diameter of 100 or 500 nm and the antigen - BV CVC, in a mass ratio of 25: 1. The dose of MF was 500 μg, and BO - 20 μg per 1 injection. The control group of mice was immunized according to the same scheme with one BO. A mixture for injection was prepared immediately before immunization. The volume of the injected mixture was 0.5 ml per animal. Each group consisted of 5 animals. Conducted 3 immunizations with a two-week interval between them. Blood was individually taken from each mouse from the tail vein one week after the last immunization. Blood was kept for 2 hours at room temperature until a clot formed, placed in a refrigerator overnight, and the next day the clot was separated by centrifugation at 10,000 g for 10 min. The obtained serum was used to determine the titer of antibodies to BO. To this end, MaxiSorp (Nwic) plates adsorbed PVX BO (6 μg / ml in deionized water). ELISA was performed as described in example 2.

The results of determining the titers of antisera show that immunization of animals with a mixture of MF and BO leads to a ten-fold increase in the concentration of antibodies to BV PVA in the blood of an immunized animal compared to immunization with one BO. The antibody titer on the 7th day after the last immunization with a mixture of MF and BO was 1/64000 (Fig. 5).

In the prototype, the titers of antisera to lysozyme and to ovalbumin on the 10th day after injection (2 mg of protein and 30 mg of VMP) were, respectively, 1/640 and 1/2560 and practically did not differ from the titers obtained by immunization without VMP. On the 30th day after the injection, the titers increased and amounted to 1/1280 and 1/10240, respectively. The titer was determined by indirect ELISA, adsorbing lysozyme and ovalbumin on a tablet at a concentration of 100 and 150 μg / ml, respectively.

This example shows that an MF-based adjuvant is significantly more effective than VMP. The titers of antisera obtained using MF in a comparable time interval were 10-100 times higher than the titers obtained using VMP. This effect was achieved by using a 100-fold lower dose of antigen, a 60-fold lower dose of adjuvant, and 16–25-fold lower concentration of antigen adsorbed on an ELISA plate.

Thus, another result of the invention is the creation of a new effective adjuvant based on the midrange.

Example 7

The study of the immunogenicity of compositions based on midrange

Immunogenicity of a composition from MF and BO CVC. Outbred white mice aged 8-10 weeks were immunized intraperitoneally with an MF with a diameter of 100 or 500 nm, the surface of which was coated with PVX BO, as described in Example 5. The protein complex with MF was stabilized by adding formaldehyde to a concentration of 0.05% (10 min, 20 ° FROM). The dose of MF for 1 injection was 500 mcg. The volume of the injected mixture was 0.5 ml per animal. The control group of mice was immunized according to the same scheme with one BO. Each group consisted of 5 animals. Conducted 3 immunizations with a two-week interval between them. The processes of blood collection, its processing and determination of the titer of antibodies to BV PVA were carried out as described in example 6.

The results of determining the titers of antisera show that the immunization of animals with MF coated with BVCV leads to a fifteen-fold increase in the number of antibodies to BVCVC in the blood of an immunized animal compared to immunization with one BV. The antibody titer on the 7th day after the last immunization was 1/128000 (Fig. 6).

Immunogenicity of a composition from MF and recombinant BOC N1 protein. BALB / c mice were immunized with recombinant protein N1, the same protein mixed with MF, and the same protein adsorbed on the surface of MF. The method for producing the recombinant protein N1 is described in Example 4. Protein N1 was adsorbed on the surface of the SC, as described in Example 5, and the complex was stabilized by adding formaldehyde to a concentration of 0.05% (10 min, 20 ° C). The amount of recombinant protein N1 in all cases was 28 μg per 1 injection. Three intraperitoneal immunizations were carried out with a two-week interval between them. One week after the last immunization, the resulting antiserum was used to determine the titer of antibodies to the BOC epitope. Determination of titer by ELISA was performed as described in example 6. The results presented in Figure 7 show that MFs enhance the antibody response to chimeric protein N1, i.e. possess adjuvant activity. When immunized with a mixture of MF and protein N1, the antiserum titer increased 10-fold compared with immunization with a single N1 protein. When immunized with protein N1, fixed on the surface of the midrange, the titer increased by 80 times and amounted to 1/600000.

The prototype studied the humoral immune response to an immunogenic composition, which is a chimeric HPV VMP, on which model antigens are affinity adsorbed: OmpC and OmpF Salmonella typhi porins (Example 4, Figure 6) or hepatitis C core antigen (Example 8, Figure 13 A) . The presented results show that the titer of IgG antibodies to OmpC on the 15th day after the injection was 1/20000, and on the 15th day after the booster injection, it reached a maximum and averaged 1/160000. The titer of antibodies to hepatitis C virus antigen was 1/4500.

This example shows that the compositions based on the midrange superior to the compositions described in the prototype, in terms of immunogenicity and rate of formation of antibodies with approximately the same amount of antigen used for immunization (10 μg in the prototype, 20 μg in our invention). However, the method for producing immunogenic compositions in our invention differs from the prototype in simplicity.

Thus, another result of the invention is a simple way to create highly active immunogenic compositions based on the use of MF as a carrier (platform).

Example 8

The formation of antibodies to the intact virus during the joint immunization of BO PVA and MF

The antisera to BO PVA obtained as described in examples 6 and 7 were titrated by ELISA on plates coated with intact PVA. Plates of MaxiSorp (Nunc) adsorbed PVX (6 μg / ml in PBS) in a volume of 100 μl per well. The remaining procedures were carried out as described in Example 6.

The results of determining the titers of antisera are shown in Figure 8. They show that when animals are co-immunized with a mixture of BO and MF or BO fixed on MF, antibodies to the intact virus appear in the blood of the immunized animals. The combined immunization of animals with PVA BO and MF leads to a fifteen-fold increase in the concentration of antibodies to PVA in the blood of an immunized animal compared with immunization with BO alone. The antibody titer on day 7 after the last immunization was 1/128000.

This example shows that the joint immunization of MF and a fragment of the virus, namely its BO, mixed with MF or fixed on the surface of the MF, induces the formation of antibodies to the intact virus.

Example 9

The effect of the dose of MF on the formation of antibodies to BO and to the intact virus when co-immunized with an antigen and MF.

Outbred white mice were immunized intraperitoneally with a mixture of midrange with a diameter of 500 nm and BO PVC in a mass ratio of 1: 1. The dose of MF was 30 μg, BO - 30 μg per 1 injection. For the enzyme-linked immunosorbent assay, the titer of antibodies to BO and to the virus on MaxiSorp (Nunc) plates sorbed BV PVA (6 μg / ml in deionized water) or PVI (6 μg / ml in PBS) in a volume of 100 μl per well. The remaining procedures were carried out as described in Example 6.

The results of determining the titers of mouse antisera shown in Figure 9 showed that when animals are combined with a mixture of BO and MF in a mass ratio of 1: 1, antibodies to BO and to the intact virus appear in the blood of immunized animals. The antibody titer on day 7 after the last immunization was 1/128000.

This example shows that MFs used as an adjuvant or as a carrier for creating an immunogenic composition have high immunogenicity in a wide range of concentrations.

Thus, the proposed method of enhancing the immune response has the following advantages compared to the prototype: 1) can significantly simplify the procedure for obtaining a carrier (platform); 2) allows to obtain a universal and more immunogenic carrier for creating various types of complexes and immunogenic compositions; 3) allows to simplify the procedure for obtaining immunogenic compositions; 4) an adjuvant based on the midrange is much more effective than VMP.

Claims (2)

1. A method of enhancing the immune response to an antigen consisting of one or more epitopes by applying for parenteral immunization of animals or humans a mixture of the aforementioned antigen and adjuvant, characterized in that spherical particles consisting of a thermo-denatured shell protein of plant spiral viruses are used as adjuvant , mainly tobacco mosaic virus (TMV). 2. A method of enhancing the immune response to an antigen consisting of one or more epitopes by using the aforementioned antigen for parenteral immunization of animals or humans, which is fixed on the surface of spherical particles consisting of a thermo-denatured protein of the envelope of spiral plant viruses, primarily TMV.

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