RU2440140C1 - Immunogenic composition, containing foreign antigens on surface of spherical carriers, obtained by thermal denaturation of spiral viruses - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, biology, nanothechnology, and deals with obtaining immunogenic compositions. Claimed immunogenic composition contains 1) platform-carrier, for which used are spherical particles, obtained by thermal structural restructuring of viruses belonging to group of tobacco mosaic virus (tobamoviruses) or other plant viruses with spiral structure and 2) foreign proteins/antigens/epitopes, bound with surface of spherical particles. Foreign antigen/epitope in composition of complex with spherical particles demonstrates high immunogenicity and preserves antigenic specificity.

EFFECT: claimed compositions are biologically safe, stable.

10 ex, 13 dwg

Description

The scope of the invention.

The invention relates to the field of production of immunogenic complexes based on the use of carrier platforms - spherical particles (MF), products of thermal denaturation of spiral viruses, including viruses of the tobacco mosaic group (TMV) (tobamoviruses) and other plant viruses with a spiral structure. These carrier platforms are nanoparticles with a diameter of up to 100 nm (VLF) and microparticles with a diameter of up to 800 nm or more (VMS) obtained in the process of thermal denaturation, structural rearrangement of particles of the native virus.

The objective of the present invention was to create immunogenic compositions based on the binding of various targets (in particular, foreign antigens / epitopes) with the surface of midrange carriers. These MFs can be used to create compositions with foreign target antigens, antigenic determinants (epitopes), enzymes, antibiotics, synthetic polymers, metals and other objects.

These compositions are intended for use in medicine, veterinary medicine, immunology, virology, virus-free agriculture and technology (the latter, if necessary, the creation of bio-inorganic complexes or complexes with synthetic polymers based on midrange platforms).

In the present invention, compositions were created that are immunogenic complexes of midrange platforms with foreign proteins or epitopes of a number of viruses. It has been shown that all spherical platform particles are coated with molecules of foreign proteins / epitopes packed on the surface of MF. These complexes can be used to obtain vaccines and diagnostic preparations for combating viral diseases and diseases of a different nature.

The present invention is directly related to the production of vaccines and diagnostic antibodies, opening up the possibility of rapid assembly in vitro of antigenically active immunogenic complexes consisting of midrange platforms, the surface of which is associated with foreign antigens / epitopes of the target pathogen.

Terminology

Technical and scientific terms used in the description of the invention have the same meaning and meaning, which are usually applied in the relevant fields of science and technology.

The terms "platform, carrier" refer to the spherical particles described in the present invention.

The term “spherical nanoparticles” (ELFs) refers to spherical particles up to 100 nm in size. The term “spherical microparticles” (SMP) refers to larger spherical particles (800 nm or more).

The term "spherical particles" (MF) refers to both types of spherical particles.

The terms “heterologous”, “foreign” (for example, “heterologous or foreign proteins”) are proteins that are antigenically unrelated to MF proteins used to create a complex (composition) with an MF carrier.

Foreign proteins are usually (but not necessarily) encoded not in the genome of the virus from which the MF is obtained, but in the genome of another virus or an alternative pathogen other than the virus (bacteria, fungi, etc.).

The term "antigen", as used in the invention, refers to foreign (target) proteins that are used to form nanocomplexes of midrange proteins.

Used in the invention, the terms "epitope" and "antigenic determinant" refers to the short region (plot, in the present invention, comprising 12-32 amino acid residues) of a foreign protein used to form the midrange epitope nanocomplex. In principle, the size of the epitope can vary significantly.

The term “immunogenic” as used herein refers to the ability of MFs to induce antibody production in mice, other animals, and humans.

The level of technology.

The so-called affinity conjugated antigenic systems are known. Such systems include: 1) chimeric viruses with BO carrying the binding sites of foreign target proteins, antigens, receptors, antibodies specifically recognized by the modified foreign protein. In these cases, the envelope viral protein gene was genetically modified as part of a full-genome copy or in the isolated BO gene by inserting inserts or deletions to obtain affinity binding sites for target substances of protein nature. For example, reactive lysine was introduced into the N-terminal portion of the BTH TM to bind biotin to the viral capsid. In independent experiments, the target protein was covalently linked to streptavidin. As a result of specific avidin-biotin interactions, virus particles decorated with the target antigen protein were obtained (Smith ML, Lindbo JA, Dillard-Telm S., Brosio PM, Lasnikk AB, McCormick AA, Nguen LV, Palmer KE, 2006. Modified tobacco mosaic virus particles as scaffords for display of protein antigens for vaccine applications. Virology 348, 475-488. A biologically active complex consisting of TMV linked to a water-soluble polypeptide is known (US Patent, 20090053261). This complex uses native TMV with covalently bound biotin. In this case, the polypeptide containing covalently bound av When the above components are mixed in an aqueous medium, a single biologically active complex is formed due to the interaction of biotin and avidin groups. A disadvantage of the known vaccine preparation is the complexity and high cost of the procedure for producing TMV-biotin and epitope-avidin platforms, as well as its relatively low biological activity US Patent Pub. N. 2009/0053261 A1, Feb. 26, 2009; Lindbo JA, Palmer KE, Owensboro KY, Smith ML. This system includes the step of affinity assembly of compositions based on the interaction of a chemically modified recombinant carrier virus and a modified foreign antigen. Despite the significant difference, this system is relatively close to the claimed invention and can be considered as a prototype. Affine conjugated antigenic systems are also discussed in a later patent (Leclerc, D. 2010, Immunogenic affinity-conjugated antigen systems based on Papaya Mosaic Virus and used thereof. US Patent Application Publication, 2010/0047264 A1, Feb.25). (Leclerc, D.2010, Immunogenic affinity-conjugated antigen systems based on Papaya Mosaic Virus and used thereof. US Patent Application Publication, 2010/0047264 A1, Feb.25). The affinity-conjugated antigenic system discussed above includes the step of affinity assembly of compositions based on the interaction of chemically modified carrier virus and a foreign antigen. Despite the significant difference, this system is relatively close to the claimed invention and can be considered as a prototype.

Disadvantages of using affinity-conjugated systems: 1) relatively low biological activity; 2) the complexity and multi-stage procedure, including: a) preparation of a modified biotin or streptavidin (66 kDa protein) BO virus and b) biotin or streptavidin-labeled target protein / antigen, followed by c) mandatory three-stage purification, i.e. separation of biotin labeled protein from streptavidin-modified particles and isolation of the virus-streptavidin-biotin-protein complex.

3) The use of this system is complicated by genetic instability and other difficulties characteristic of the accumulation of chimeric viruses, discussed above.

4) Another disadvantage of affinity-conjugated antigenic systems is the lack of universality - strict specificity due to the specificity of the interaction of its two components: a modified carrier platform (BO virus) and a modified target protein (for example, antigen). In certain experiments, the high specificity of the interaction of the carrier with the target protein is useful, facilitating the purification of the created composition from unnecessary impurities. However, in the present invention, that is, under conditions of absence of impurities due to the use of a high degree of purification of both components of the composition, specificity should sharply limit the capabilities of the carrier (MF) to one type of target protein / antigen. The ability of MF to form compositions with various structurally and functionally unrelated proteins is shown below.

5) In contrast to the prototype, once obtained, MFs can be used to adsorb a variety of CBs. The circumstance is that, unlike viral particles, the surface of the midrange has a high sorption capacity with respect to substances of various chemical nature. This property of the midrange is illustrated by examples 2, 3, 4, 5 of the present invention. Due to this feature of MF, the method of preparing a composition based on them consists in simple mixing of MF and CB in a ratio that ensures the formation of a monolayer of CB on the surface of the MF. Mentioned in a number of examples, the fixation of CB on the surface of the midrange using formaldehyde is aimed at increasing the stability of the composition when it is used for immunization. Thus, an important result of the invention is the creation of universal carriers (platforms) for creating various complexes and immunogenic compositions.

It is known that RNA-free virus-like multisubunit nanoparticles (HPVs) bearing foreign epitopes on their surface can be obtained by polymerization of chimeric subunits of BO (the BO-foreign epitope) or by genetic modification of a full-sized viral genome containing a coat protein gene combined with a foreign epitope. The result of the practical implementation of these principles is the creation of chimeric viral particles carrying the antigenic determinant (epitope) of the pathogen or other functionally active polypeptide as their target peptide (McCormick A.A. and Palmer K.E. (2008) Genetically engineered Tobacco mosaic virus as nanoparticle vaccines. Expert Rev Vaccines. 7 (1), 33-41; Steinmetz NF, Lin T., Lomonossoff GP and Johnson JE (2009) Structure-based engineering of an icosahedral virus for nanomedicine and nanotechnology. Curr. Top. Microbiol . Immunol. 327, 23-58; Denis, N. Majeau, E. Acosta-Ramirez, C. Savard, M.-C. Bedard, S. Simard, K. Lecours, M. Bolduc, C.Pare, B. Willems, N. Soukry, P. Tessier, P. Lacasse, A. Lamarre, R. Lapointe, CL Macias, and D. Leclerc (2007) Immunogen icity of papaya mosaic virus-like particles fused to a hepatitis C virus epitope: evidence for the critical function of multimerization. Virology 363, 59-68; Turpen HT et al. US Patent 5977438, Nov. 1999. Production of peptides in plants as viral coat protein fusions; Leclerc. US Patent 7641896 B2, Jan. 5 2010 Adjuvant viral particle; Werner S. et al. International Patent Application PCT / EP2006 / 009029 (WO / 2007/031339). It should be noted that the number of foreign protein molecules attached to an individual TMV particle is very small (S. Werner, S. Marillonnet, G. House, V. Klimyuk and Yu. Gleba (2006) Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A. Proc. Natl. Acad. Sci. USA 103 (47), 17678-17683).

annotation

It is important to emphasize that in all cases when genetically modified viruses are used as the platform used to obtain vaccines or other complexes, the morphology and general architecture of the native virus remains unchanged. In other words, the natural envelope of the virus (capsid) serves as a platform particle created in this case by a chimeric BO.

In contrast to the examples discussed above, in the present invention, the indicated MF carriers were used to obtain antigenically active immunogenic compositions and to bind foreign antigens / epitopes to their surface (schemes of Figs. 1-2) formed as a result of thermal denaturation and structural rearrangement of TMV in the MF . To obtain the MF, the initial virus solution was diluted to the desired concentration and distributed 50 μl in 0.5-ml polypropylene thin-walled tubes (for PCR). To prevent evaporation of the solution (and therefore, a change [increase] in the concentration of the virus during heat treatment), 30 μl of mineral oil was applied to the surface of the virus solution. Further, the tubes were placed in the cells of the Tercik thermal cycler (DNA-Technology, Russia) and a program was launched that regulates the temperature in each cell.

The invention shows that MFs are promising platforms with advantages for assembling immunogenic complexes in vitro (Examples 2-5 and Figs. 3-6 and 8). The present invention is the first representing MF as nanoparticle platforms for creating immunogenic compositions.

Description of the invention. The objective of the present invention was to create biologically active compositions consisting of the above spherical particles (midrange platforms), capable of associating with various targets, in particular, to bind foreign antigens / epitopes to the surface of these midrange. In accordance with this aspect of the present invention, we have shown that several types of antigens / epitopes of different origins are capable of forming antigenically active and immunogenic compositions with an MF carrier, including 1) potato virus X protein (Figure 3, Example 2), 2) The N-terminal epitope M2e of the transmembrane protein M2 of the human influenza A virus (Fig. 4, Example 4), 3) the rubella virus E1 glycoprotein epitope (Fig. 5, Example 3), 4) the hemagglutinin tetraepitope (HA) of H5 human influenza A virus ( 6), 5) epitope 12 of the envelope protein of the smallpox virus plum (Fig. 8, Example 5 )

The MF antigen / epitope complexes were stabilized using 0.05% formaldehyde (FA). Data in favor of the need to use FA in the preparation of compositions was obtained by the example of smallpox virus epitopes (sharks) plum (BOC) (potivirus) (Figs. 7-8, Example 7).

To obtain a composition with MF, a 1N recombinant protein was used, including BVKV with a delegated N-terminus and a 10-membered BOC BOC epitope (Figs. 7 and 8). The invention shows that the immunogenicity of the MF + antigen / epitope complex is very high, however, it increases significantly after stabilization of the complex by formaldehyde treatment. As a result of the treatment of the FA complexes, the antiserum titer was about 1: 1000,000 (see FIG. 10 and Example 7).

It is important that the surface of all MFs involved in the experiment was coated with the antigen / epitopes used in the experiment. The latter follows, in particular, from figure 3 and figure 6 and indicates a high efficiency of antigen binding in the assembly of immunogenic compositions based on midrange. These compositions, consisting of the midrange platform and foreign antigen / epitopes associated with the surface of the midrange platform, were immunogenic and antigenically active. Foreign antigen / epitopes bound to the surface of the midrange platforms specifically reacted with antibodies to the corresponding antigen / epitope, indicating that the specificity of the midrange antigen / epitopes is preserved in the nanocomposition.

The results presented in the present invention indicate the possibility of using midrange platforms for the assembly in vitro of antigenically active immunogenic complexes of nano-vaccines. Compositions of MFs carrying alien epitopes on their surface provide a number of advantages compared to attenuated, chemically inactivated, and subunit vaccines:

(i) The above approach eliminates the possibility of recombination and reversal of pathogens.

(ii) Vaccines obtained by the method described above, based on thermal denaturation of plant viruses, are biologically safe, since plants and animals do not have common pathogens.

(iii) The procedure for producing midrange platforms is based on heating the virus at 94-98 ° C, that is, it does not require additional sterilization of the platform particles.

(iv) MF donor virus (in particular, VTM) is readily available since it accumulates in leaves of infected plants in very large quantities.

(v) The antigenically active immunogenic complexes described in the invention can also be used for the production of low-cost diagnostic monoclonal antibodies and as diagnostic preparations for the detection of infectious diseases in humans and animals.

(vi) The use of spherical immunogenic complexes stabilized with formaldehyde simplifies the technology for the preparation and use of vaccines or diagnostic preparations.

(vii) The creation of a method of “block” assembly of immunogenic complexes outside the cell on the MF platform carrying pathogen epitopes on its surface will make it possible to fundamentally simplify and accelerate the production of vaccines and diagnostic preparations of various nature for use in medicine, veterinary medicine and crop production.

(viii) The present invention allows the "assembly" of immunogenic complexes containing more than one type of foreign antigens, that is, to obtain polyvalent antisera.

Thus, an important result of the invention is the creation of universal carriers (platforms) for creating various types of complexes and immunogenic compositions.

The proposed biologically active complexes are not described in the literature and are new, which significantly expands the range of biologically active complexes.

Brief Description of the Drawings

Figure 1. is a schematic representation of one or two model antigens (Ag1 and Ag2) associated with the midrange platform

Figure 2. is a schematic representation of detection by fluorescence microscopy of the Ag1 antigen bound to the surface of the MF-Ag1 complex. Primary antibodies have been shown to react with Ag1 molecules on the surface of the midrange nanoplatform.

Figure 3 illustrates that the surface of all midrange platforms is coated with BVC BO molecules, (a) labeled with fluorescein isothiocyanate (FITC); (b) control passing light. Label size 3 microns. Confocal microscopy.

Figure 4 illustrates that dehydrofolate reductase fused to the M2e epitope (23 amino acids) of the human influenza A virus matrix protein M2 (DHFR-M2e) binds to the surface of the MF. The M2e epitope is detected by murine anti-M2e antibodies and secondary chicken anti-mouse antibodies labeled with Alexa 488 green fluorophore. Label size 5 microns. Fluorescence microscopy.

Figure 5 illustrates that the rubella virus E1 protein tetraepitope bound to the surface of the midrange platform is detected by primary murine anti-E1 protein antibodies and secondary chicken anti-mouse antibodies conjugated with Alexa 488 green fluorophore. Label size 3 microns. Fluorescence microscopy.

6 illustrates that the surface of all midrange platforms is covered with tetraepitopes consisting of 4 conserved monoepitopes of human influenza A virus hemagglutinin (HA). Detection by primary mouse anti-HA antibodies and secondary chicken anti-mouse antibodies conjugated to a fluorophore. Control passing light (a); fluorescence microscopy (b). Label size 3 microns.

Figure 7 illustrates the structure of two recombinant BVCV BO proteins fused to two different BOC BOC epitopes: 1) a 12-member VNTNRDRDVDAG (P2) and 2) a 10-membered SMLNPIFTPA (1N) containing 6 histidines (6HIS) and an epitope from BOC BOC at the C-end. The designation IEGR corresponds to the site of action of proteinase Xa during the release of "epitope-BO".

Fig. 8 illustrates that a 12-membered epitope of plum poxvirus (BOC 12) expressed in bacteria, fused to the PVA envelope protein, binds to the MF surface and is detected using primary rabbit anti-BOC antibodies (BOC 12) and secondary donkey anti-rabbit antibodies conjugated with fluorophore Alexa 555 red. Label size 5 microns. Fluorescence microscopy.

Fig.9 is a graph illustrating the results of determining the titer of mouse antisera to TMV and to MF and showing a higher immunogenicity of MF compared to native TMV particles. Enzyme-linked immunosorbent assay (ELISA) (Example 6).

Figure 10 illustrates the immunogenic activity of the composition "MF + IN BOC BOC" (protein 1N contains a 10-membered epitope BOC BOC, see Fig.7) and a significant increase in this activity when the composition is stabilized with formaldehyde. Comparison of the titers of mouse antisera to the BOC epitope in indirect ELISA: 1) antiserum obtained by immunizing mice with recombinant 1N protein; 2) antiserum obtained by immunization of MF, on the surface of which 1N protein was placed on the basis of electrostatic interactions; 3) the antiserum obtained by immunization of mice with antigenic complexes "mid-protein 1N", stabilized with formaldehyde; control is normal mouse serum. The antiserum was titrated relative to BOC at a concentration of 10 μg / ml. Axis-X - dilution of antiserum; Axis-Y - optical density at a wavelength of 405 nm.

11 illustrates the immunogenic activity of the composition "MF + BO CVC", when the composition is stabilized with formaldehyde, and is a graph showing the results of determining the number of specific antibodies in mouse antisera when immunizing animals with an antigen per se or an antigen fixed on the surface of MFs of various sizes .

Fig is a graph illustrating the results of determining the titer of mouse antisera to the virus obtained by immunization of animals BV PVA in a mixture with MF or fixed on their surface. IFA.

13 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

Production of proteins / epitopes in bacterial cells. In the present invention, recombinant antigens / epitopes expressed in bacterial cells have been used to bind to the surface of MF. All recombinant DNA manipulations were performed according to standard procedures (Sambrook J., Ftitsch EF and Maniatis T. (1989) Molecular cloning: A laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). For cloning the created constructs and for their superexpression, E. coli DH5ot and M15 [pREP4] strains were used, respectively. Recombinant constructs expressing proteins fused to (His) ₆ - peptide were obtained by cloning PCR fragments into the plasmid vector pQE. Restriction PCR fragments were ligated at the corresponding sites into an expression vector as previously described (Ivanov K.I., Ivanov R.A., Timofeeva E.K., Dorokhov Yu.L. and Atabekov JG (1994). The immobilized movement proteins of two tobamoviruses form stable ribonucleoprotein complexes with full-length viral genomic RNA. FEBS Lett. 346, 217-220). Cells of Escherichia coli strain M 15 transformed with the recombinant vector were grown in culture fluid at 37 ° C to OD ₆₀₀ equal to 0.8-0.9. 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 (H) ₆ recombinant proteins on Ni ²⁺ nitrilotriacetate agarose (Ni-HTA) was performed according to the manufacturer's protocol (QIAGEN).

Example 2

The formation of the complex "SC-FITZ-labeled protein of the membrane X of the potato virus"

The potato X virus envelope protein (BO) (CVC) was isolated by treating the viral suspension in Tris-HCl buffer, pH 7.5 with neutral saline solution of LiCl, bringing it to a final concentration of 2M, placed at -20 ° С overnight, centrifuged at 10000 g. The supernatant was dialyzed against distilled water. The dialysis protein was ultracentrifuged at 100,000 g. To 1 mg BO PVA with a concentration of 1 mg / ml was added 18 μg of fluorescein isothiocyanate (FITC) and incubated for 24 hours at 4 ° C, dialyzed against water to clear the unbound fluorescent label. The resulting protein labeled with FITC was used to form a complex with an MF carrier. For this, 50 μg of MF were incubated with 1 μg of protein in water. The complex was stabilized by the addition of formaldehyde to a concentration of 0.05% (10 minutes, 20 ° C). The binding of MF to BO was recorded using a fluorescence microscope (LeicaDRMB) equipped with an integrated ORCAII-ERG2 camera (Hamamatsu, Japan).

The formation of a complex of MF with the FITC-labeled envelope protein of the virus X potato PVA is shown in Fig.3.

Example 3

The formation of the complex "mid-carrier polyepitope A rubella virus"

For the formation of a complex with MF, the antigenic determinant A of the rubella virus glycoprotein E1 was used. The following is the amino acid sequence (32 amino acid residues) of the E1 glycoprotein E epitope A:

The epitope region A is indicated in bold.

A recombinant protein (molecular weight about 21-22 kD) containing six histidine amino acid residues (His ₆ ) at the N-terminus and representing four repeats of the epitope A (polyepitope A) E1 glycoprotein was constructed on the basis of plasmid pQE-BT-4A. Polyepitope A in the complex with MF was detected by immunofluorescence.

The MF-polyepitope A complex was incubated at room temperature for 2 hours on coverslips pretreated with poly-L-lysine. To fix the samples, they were treated for 15 minutes with 4% paraformaldehyde at room temperature, then three washes with water were carried out for 5 minutes each. After washing, the samples were incubated for 30 minutes in a solution consisting of 1% bovine serum albumin (BSA), phosphate buffer (PBS, composition: 7 mm Na ₂ HPO ₄ , 1.5 mm KH ₂ PO ₄ , pH 7.4, 137 mm NaCl, 2.7 mM KCl) and 0.05% Tween-20. Further, the preparations were incubated with primary mouse antibodies to rubella virus E1 glycoprotein for 30 minutes in a humid chamber in PBS with 0.25% BSA and 0.05% tween-20. Control samples were incubated in blocking solution without the addition of primary antibodies. Unbound antibodies were washed with a washing solution (PBS with 0.25% BSA and 0.05% Tween-20) on a shaker 3 times for 5 minutes. The binding of primary antibodies to antigenic complexes was detected using secondary anti-mouse antibodies conjugated with Alexa-488 fluorophore (Molecular Probes, Eugene, OR). Co secondary antibodies were incubated for 30 minutes in a humid chamber; they were washed from unbound antibodies 3 times for 5 minutes in a washing solution and once in PBS for 5 minutes. After that, the samples were enclosed between coverslips and slides using a photoprotective additive of 1,4 diazobicyclo [2.2.2] octane (DABCO), incubated for 30 minutes at + 4 ° C. The resulting preparations were analyzed using a fluorescence microscope (LeicaDRMB) equipped with an integrated ORCAII-ERG2 camera (Hamamatsu, Japan).

The formation of a complex of MF with the rubella virus polyepitope A is shown in FIG. 5.

Example 4

Binding of the MF carrier with the recombinant DHFR protein fused to the M2e epitope of influenza A.

The next to form the MF-epitope M2e complex was a recombinant protein consisting of a dehydrofolate reductase (DHFR) molecule fused to the M2e epitope of human influenza A virus with 23 amino acid residues in length (J.Fan, X. Liang, M. Horton, N. Perry , M. Citron, G. Heidecker, T. Fu, J. Joyce, C. Przysiecki, P. Keller (2004) Preclinical study of influenza virus A M2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys. Vaccine 22, 2993 -3003). The ability of monoclonal antibodies obtained against the M2 protein (Mab) to weaken the replication of the virus suggests that M2, and in fact the external domain of M2e, can be used as a target for vaccines (Tompkins SM, Zhao Z-Sh., Lo, Ch-Yu., Misplon JA, Liu T., Ye Z., Hogan RJ, Wu Zh., Benton KA, Tumpey TM and Epstein SL (2007) Matrix Protein 2 Vaccination and Protection against Influenza Viruses, Including Subtype H5N1. EmergInfect Dis . 13, 426-435).

In the present invention, DHFR serves as a carrier for the M2e epitope (DHFR-M2e).

In order to establish the fact of the placement (landing) of M2e peptide on the surface of the MF, the immunofluorescence method was applied, schematically shown in Figures 1 and 2. The M2e epitope was detected by mouse antibodies to M2e and secondary chicken anti-mouse antibodies labeled with Alexa 488 green fluorophore. Figure 4 of the present invention illustrates that the M2e epitope associated with MF has retained its antigenic properties, and that the use of the recombinant DHFR-M2e protein, where DHFR plays the role of a carrier, provides the presentation of a foreign target epitope (M2e) on the surface of spherical nanoparticles.

Example 5

Binding to the surface of the MF complex "BV CVC-epitope 12-member" (vntnrdrdvdag) of smallpox virus plum (BOC)

Epitope "12", 12 amino acid residues long, smallpox envelope protein (scab) plum (vntnrdrdvdag) (Fernández-Fernandez MR, Martinez-Torrecuadrada JL, Roncal F., Dominguez E., Garcia JA (2002) Identification of immunogenic hot spots within plum pox potyvirus capsid protein for efficient antigen presentation. J. Virol. 76, 12646-12653) was coupled with BO PVA, and chimeric protein P2 "BO PVA-epitope 12 BOC" (see Figure 7) was expressed in cells E. coli. The recombinant protein "BO CVC-epitope 12" was covalently linked to the surface of the SC using formaldehyde, as described above. In order to detect the binding of the “12” epitope of plum pox virus by confocal fluorescence microscopy, primary rabbit antibodies to BO of plum pox virus and secondary donkey anti-rabbit antibodies conjugated with Alexa 555 red fluorophore were used (Fig. 8).

The gene for the chimeric protein "BO CVC-epitope" 12 "BOC was constructed on the basis of plasmid pQE30. 6His are marked in bold, underlined is the site of factor Xa protease hydrolysis, in italics and frame - antigenic determinant - epitope 12 of plum pox virus.

Amino acid sequence of the chimeric protein "BO CVC-epitope 12 of plum pox virus":

Using fluorescence microscopy, it was shown that the surface of the midrange is covered with the BOC 12 foreign epitope (Fig. 8).

Example 6

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 HPO ₄ , 0.1 M NaCl, pH 7.4) was sorbed in a volume of 100 μl 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 1/20000 working dilution 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 was added - ABTS with hydrogen peroxide in 0.05 M citrate-phosphate buffer pH 4.4. 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.9 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 7

The immunogenicity of the composition “MF-epitope 10-membered SMLNPIFTPA BOC) increases when the complex is stabilized with formaldehyde.

The invention contemplates the activity of an immunogenic composition consisting of a partially deleted from the N-terminus of BV CV, fused to the BOC BOC epitope (see 1N in FIG. 7). The possibility of producing antibodies specific for the BOC epitope (SMLNPIFTPA) located at the N-terminus of recombinant BVC CVC - 1N was investigated. BALB / c mice were used for immunization. In the experiment there were 4 groups of mice. The first group of mice was immunized with a solution of recombinant 1N protein, the second group of mice was immunized with a suspension of MF, on the surface of which, based on electrostatic interactions, a recombinant protein 1N was planted. Electrostatic interactions between MF and recombinant 1N protein could be unstable under physiological conditions. In this regard, the third group of mice was immunized with antigenic complexes consisting of MF and recombinant 1N protein, further stabilized by the addition of formaldehyde. The amount of recombinant 1N protein in all cases was 28 μg. The immunogenic MF-AG complex was formed upon incubation of 100 μg MF with 28 μg of recombinant CVC BO carrying the BOC epitope at the N-terminus. As a control, a PBS solution was used. Three intraperitoneal immunizations were carried out with a two-week interval between them. One week after the last immunization, the obtained serum was used to determine the titer of antibodies to the BOC epitope using the indirect ELISA method.

The immunogenicity of the low molecular weight protein 1N was small (Figure 10; 1N). Slightly higher antibody titers were obtained by immunization of MF mice, on the surface of which 1N protein was planted by electrostatic interactions (Figure 10; C4 + 1N).

The highest titers were obtained during immunization of animals with immunogenic complexes stabilized with formaldehyde. In this case, the titer of antisera was significantly higher (Figure 10; MF + 1N (FA) and amounted to about 1: 1000,000. This suggests that formaldehyde significantly increased the stability of the compositions, sharply stimulating their immunogenicity.

Apparently, under physiological conditions, MF-1N complexes could partially dissociate into MF and recombinant 1N protein, which was manifested in a noticeable decrease in serum titer (Figure 10).

Example 8

Immunogenicity of the composition "SCH-BO CVC".

Outbred white mice were immunized intraperitoneally with MF 100 or 500 nm in diameter, the surface of which was coated with a monolayer of antigen - BVCV. The dose of MF was 500 mcg. The control group of mice was immunized according to the same scheme with one BO. 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, individually from each mouse, was taken from the tail vein a week after the last immunization. Blood was held for 2 h at room temperature until a clot formed, placed in a refrigerator overnight, and the next day the clot was separated by centrifugation 10,000 g for 10 min. The obtained serum was used to determine the titer of antibodies to BO.

The titer was determined in a pool obtained by mixing equal volumes of antisera from each animal using an indirect enzyme-linked immunosorbent assay. On tablets MaxiSorp (Nunc) sorbed BV CVC (6 μg / ml in deionized water) in a volume of 100 μl per well. The plates were allowed to stand overnight in the refrigerator, washed with PBST (0.05 M K ₂ HPO ₄ , 0.1 M NaCl, 0.1% Tween 20, pH 7.4) 5 times in 200 μl per well and the tested antisera were added in double serial dilutions prepared on PBST starting from 1/1000 dilutions. 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 1/20000 working dilution 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 was added - ABTS with hydrogen peroxide in 0.05 M citrate-phosphate buffer pH 4.4. 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 last dilution was considered the antiserum titer, the optical density of which was three times higher than the corresponding value of preimmune mouse serum.

The results of determining the titers of mouse antisera are presented in Fig.11. These results show that MFs of different sizes enhance IO by an antigen fixed on their surface. Immunization of animals with MF coated with BO leads to a fifteen-fold increase in the concentration of antibodies to BV CVC in the blood of an immunized animal compared to immunization with one BO. The antibody titer on day 7 after the last immunization was 1/128000.

Example 9

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

Antisera to BO PVA obtained as described in example 8 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 8.

The results of determining the titers of antisera are presented in Fig.12. 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 10

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 8.

The results of determining the titers of murine antisera shown in Fig. 13 showed that when animals are co-immunized with a mixture of BO and MF in a weight ratio of 1: 1, antibodies to BO and to the intact virus appear in the blood of the 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.

Claims (1)

An immunogenic composition comprising: 1) as a carrier platform, spherical particles (MFs) obtained by thermal structural rearrangement of the viruses of the tobacco mosaic virus group (tobamoviruses) or other plant viruses with a spiral structure and 2) foreign proteins / antigens / epitopes associated with midrange surface.

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