RU2529959C1 - Immunological adjuvant based on nanoparticles for vaccines against highly pathogenic influenza viruses strains - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: immunoadjuvant is claimed representing hydroxyapatite nanoparticles with adsorbed synthetic peptide - CXCR ligand of 1 2 and receptors. The vaccine against influenza is presented, comprising the claimed immunoadjuvant and chimeric protein NS-ESAT. FIELD: On inorganic nanoparticles the adsorbed peptide Pro-Pro-Gly-Pro-His (PPGPH) which represents the ligand of CXCR 1 and 2. The base of solving the problem set was obtaining complexes of hydroxyapatite nanoparticles with the chimeric protein NS-ESAT. The protocols of obtaining these complexes are developed, as well as the effectiveness of their generation was quantitatively studied. As the results of tests of the complexes obtained on laboratory animals the data is obtained giving evidence that these complexes are capable to stimulate a cellular link of immune response more effectively than the commercial adjuvant. On this account the resulting complexes may be considered as a potential vaccine component, particularly anti-influenza, particularly against such highly pathogenic strains of influenza virus as H5N1, H1N1, and others. As a result of the studies carried out the affinity of hydroxyapatite nanoparticles to chimeric protein NS-ESAT-6 is revealed. A fragment of the influenza virus NS1 protein (125 amino acid residues long) having high affinity for phosphate groups, provides an increased affinity of the fusion protein to hydroxyapatite nanoparticles. The immunological adjuvant is proposed to use as nanoparticles of hydroxyapatite, which, according to the results of tests on laboratory animals is able to stimulate more effectively than the commercial adjuvant the cellular link of immune response. In addition, the use of the sorption properties of hydroxyapatite is proposed for creation of nanocompositions comprising individual immunomodulators stimulating the immune response to the target antigens.

EFFECT: increased immunogenicity of vaccines with use of inorganic nanoparticles.

3 cl, 4 dwg

Description

Application area

The invention relates to nano-immunobiotechnology and, in particular, to the creation of immunogenic preparations and vaccines that can be used to prevent infectious diseases, in particular, seasonal and highly pathogenic pandemic strains caused by influenza virus.

Relevance

The 2009/10 influenza pandemic caused by the H1N1 influenza virus, of a combined, mainly swine origin, showed that massive protection of the population against high pathogenicity influenza viruses can be achieved only by vaccination. Moreover, the analysis of the age structure of the incidence rate during this period showed that people born before 1957 were much less likely to suffer or had the disease in a mild clinical form. These indicators were directly correlated with the presence of a relatively high titer of antibodies to H1N1 viruses that circulated from 1918 to 1955. It is known that the HA and NA of these viruses are close in antigenic structure to modern strains of the H1N1 viruses that caused the 2009-10 influenza pandemic.

These observations stimulated studies of typical and universal determinants for the construction of pandemic vaccines and helped formulate requirements for the protective properties of a new generation of influenza vaccines. The solution to the problem of obtaining highly protective vaccines is impossible without the choice of appropriate technologies and functional adjuvants [Kiselev OI Progress in creating pandemic influenza vaccines and their production technology. Biotechnology 2010 No. 2, pp. 1-25; Atmar R.L., Keitel W.A. Adjuvants for pandemic influenza vaccines. Curr. Top Microbiol. Immunol. 2009, Vol. 333, p. 323-44. doi: 10.1007 / 978-3-540-92165-3_16]. Therefore, the use of new approaches based on nanotechnology with the search for fundamentally new immunoadjuvants can contribute to solving this problem.

Currently, the following types of vaccine preparations are available in Russia and other countries that produce influenza vaccines:

1. Live influenza vaccine.

2. Whole-virion inactivated vaccine.

3. Split vaccine.

4. Subunit vaccine.

Much later, a influenza-type polymer-subunit vaccine was developed. However, the main progress in this area should be attributed to the 1970-80s. It should also be noted that the main direction of improving influenza vaccines was to increase their safety. That is why the transition from whole-virion vaccines to split-vaccines, and from split-vaccines to subunit ones, was made. With high purification of antigens, the problem of a decrease in immunogenicity arose. In this regard, the search for immunoadjuvants began. As a result, about 15 years ago, “polyoxidonium” was used as an adjuvant. The polyoxidonium-based vaccine is called the polymer subunit. However, it is clear that this approach is also outdated. Polyoxidonium is a weak immunoadjuvant and its mechanisms of action are still unknown. In recent years, many new adjuvants have been tested, the effect of which is based on the mechanism of stimulating the immune response through Toll receptors [Takeshi Ichinohe T., Iwasaki A., Hideki Hasegawa N. Innate sensors of influenza virus: clues to developing better intranasal vaccines. Expert Rev Vaccines. 2008; 7 (9): 1435-1445._doi: 10.1586 / 14760584.7.9.1435.]. In fact, it has now been found impossible to create effective and safe vaccines without the use of adjuvants [WHO. Squalene-based adjuvants in vaccines. December 3, 2008 Matyas G, Rao M, Pittman P, Burge R, Robbins I, Wassef N et al. Detection of antiboides to squalene III. Naturally occurring antibodies to squalene in humans and mice. JIM, 2004, 286: 47-67]. The capabilities of nanotechnology make it possible to use the new aggregate state of known adjuvants or to create fundamentally new adjuvant compositions for the production of vaccine preparations characterized by low antigenic load and high safety. The concept of nanoadjuvants appeared, formulated in our and a number of other studies [O. Kiselev. Progress in creating pandemic influenza vaccines and their production technology. Biotechnology 2010 No. 2, pp. 1-25; Narasimhan, B. Novel Biodegradable Nanoadjuvants for Immunotherapy of Pancreatic Cancer. National Institutes of Health, 10/1/2006 to 9/30/2008].

State of the art

1) Adjuvants in the composition of modern influenza vaccines

In modern influenza vaccines, adjuvants based on aluminum hydroxide, polyoxidonium and squalene are mainly used [O. Kiselev. Progress in creating pandemic influenza vaccines and their production technology. Biotechnology 2010 No. 2, pp. 1-25; Erofeeva M.K., Nikanorov I.Yu. 9.1. Specific prophylaxis. Modern domestic and foreign vaccines. In the book: Influenza. Epidemiology, diagnosis, treatment, prevention. Ed. MIA (Medical News Agency). Ed. O.I. Kiseleva, L.M. Tsybalova and V.I. Pokrovsky. M. 2012. S.443-466]. With regard to adjuvants based on aluminum hydroxide, it should be noted that in recent years, substantiated criticism of its use in the production and use of vaccine preparations has progressively increased [Tomljenovic L., Shaw S.A. Aluminum vaccine adjuvants: are they safe? Curr Med Chem. 2011; 18 (17): 2630-7]. Even the safety of aluminum hydroxide adjuvants is being called into question. First of all, these fears are argued by the fact that soluble alumina from the injection site diffuses into the lymph nodes and bloodstream. As a result, the toxic effect of the adjuvant appears on the functions of brain neurons. Repeated administration of vaccines during a person’s life can accelerate or initiate the development of diseases such as Alzheimer's disease [Shawa S.A. Petrikc M.S. Aluminum hydroxide injections lead to motor deficits and motor neuron degeneration. J. of Inorg. Biochem. 2009, Volume 103, p. 1555-1562]. The fundamental difference between nanoparticles based on aluminum derivatives or the replacement of these adjuvants with a physiologically compatible hydroxyappatite from aluminum hydroxide lies in their state of aggregation and the inability to diffuse into the lymphatic system and especially into the bloodstream, therefore their application is aimed at solving two main problems of the invention: improving safety by the maximum possible reduction in antigenic load and decrease diffusion at the locus of administration. The second problem is solved by using the unique adsorption properties of hydroxyappatite for the adsorption of functional immunoadjuvants.

Thus, the advantages of using adjuvants based on inorganic (hydroxyappatite) nanoparticles can be systematized as follows:

1. High inertness of inorganic substances as the main material.

2. High adsorption ability, allowing you to create a depot of antigen using small amounts of adjuvant (10-20 μg / ml), which makes it possible to enhance the immune response with less antigenic load.

3. The high adsorption capacity of nanoparticles can be used to solve the problem of the formation of a depot of biologically active components (proteins, peptides, nucleic acid fragments) to stimulate the corresponding functions of the immune response.

4. Technologically simpler and faster production compared with the main adjuvants of organic nature.

2) Immunological adjuvants based on nanoparticles

Aluminum hydroxide in an aqueous medium is microcrystals of aluminum oxide-hydroxide with dimensions of 4.5 nm × 2.2 nm × 10 nm, however, these particles form larger aggregates 1-10 μm in size. The synthesis of stable aluminum hydroxide nanoparticles of a given size is apparently quite complicated, the latest publication describes a process using organosilicon molecules as centers of nanoparticle formation (Preparation of Amorphous Aluminum Oxide-Hydroxide Nanoparticles in Amphiphilic Silicone-Based Copolymer Microemulsions; Yana Berkovich et al. Journal of Colloid and Interface Science. 2002.245, 58-67).

An alternative material in the development of a new generation of adjuvant may be hydroxyapatite. This substance is a natural component of bone tissue, accounting for about 50% of its mass. The biocompatibility of hydroxyapatite, demonstrated by extensive experience in implantology, will avoid side effects, such as, for example, macrophagic myofascitis, associated with pathological accumulation of aluminum in cells. The possibility of using hydroxyapatite, including in nanoform, began to be studied only in the last 2-3 years.

It must be borne in mind that an immunoadjuvant with a wide range of applications is optimal, in particular, combined with vaccines of various types. In the case of influenza vaccines, the most important is the stimulation of the formation of neutralizing antibodies, which helps to stop the development of infection at the very beginning, however, the induction of a T _H2 -dependent immune response is also relevant: it is important at the later stages of the development of influenza infection, in the elderly or with weakened immunity. The presence of their own non-specific immunostimulatory effects is desirable as an addition to enhancing the immunogenicity of the vaccine antigen, taking into account the genetic variability of the influenza virus. In addition, all these qualities must be combined with safety when used as part of a vaccine. The immunoadjuvant should also be sufficiently stable in the composition of the vaccine. Finally, the simplicity of the synthesis technology, which makes vaccine production fast and scalable, is also of great importance. Inorganic nanoparticles, in particular, based on hydroxyapatite, combining the biocompatibility of hydroxyapatite and the unique properties of materials in the nanoform, are a promising candidate that combines all these characteristics.

Disclosure of invention

The present invention was tasked with creating an immunological adjuvant based on hydroxyapatite nanoparticles for use in creating vaccines. In fact, the problem was solved by:

a) selecting the type of inorganic nanoparticles with the best sorption properties with respect to the chimeric protein NS-ESAT (a fusion protein comprising the sequence of the NS protein of influenza A virus and the antigenic protein ESAT-6 Mycobacterium tuberculosis [Sereinig S., Stukova M. Zabolotnyh N. et al Influenza Virus NS Vectors Expressing the Mycobacterium tuberculosis ESAT-6 Protein Induce CD4 Th1 Immune Response and Protect Animals against Tuberculosis Challenge. Clinic, and Vaccine Immunol. 2006, Vol.13, p.898-904, 1556-681 1/06 / $ 08.000 doi: 10.1128 / CVI.00056-06]). The choice of antigen was determined by the fact that it is fully characterized in structure and primary antigenic properties. NS1 proteins of influenza A and B viruses are key factors in the control of signaling systems and carriers of pathogenicity factor in highly pathogenic type A influenza viruses. In the structure of the NS1-ESAT6 fusion protein, the sequence of the viral component is represented by the 125 N-terminal amino acid sequence including the dsRNA-binding site. This site plays a key role in inhibiting the synthesis of type I interferons through the blockade of activation of dsRNA-dependent protein kinase (Kiselev OI Genome of the pandemic influenza virus H1N1v-2009. "Company Dimitrade Graph Group" 2011. P.168). The dsRNA binding sites are of considerable interest in terms of the design of combination vaccines and the ability to interact with exposed phosphate groups and corresponding nanoparticles;

b) assessing the immunogenic properties of nanoparticles “loaded” with antigen by determining the degree of proliferation of lymphocytes after immunization of laboratory animals with NS-ESAT protein in combination with a hydroxyapatite nanoparticle preparation. The immunogenicity of nanoparticles was compared with the immunogenicity of a commercial adjuvant. The results of the evaluation of the immunological properties of the nanocomposite indicate that the greatest effect, ceteris paribus, was manifested when NS-ESAT was introduced in combination with hydroxyapatite nanoparticles with a synthetic peptide — the ligand of CXCR 1 and 2 receptors adsorbed on them.

The first aspect of the invention relates to the production of a complex of hydroxyapatite nanoparticles and NS-ESAT protein. It was shown that hydroxyapatite nanoparticles had the highest adsorption activity with respect to the NS-ESAT protein.

A second aspect of the invention relates to the study of the immunoadjuvant properties of hydroxyapatite nanoparticles. Twice immunization with the chimeric NS-ESAT protein in combination with the hydroxyapatite nanoparticle preparation stimulates the cellular immune response to a greater extent than immunization with the NS-ESAT protein without adjuvant or the NS-ESAT protein in combination with a commercial aluminum hydroxide gel adjuvant and magnesium.

The third aspect of the invention is directed to solving the problem of enhancing the adjuvant properties of hydroxyappatite by adsorption of short peptides related to CXCR ligands 1 and 2 receptors on hydroxyappatite nanoparticles [Weathington N.M. van Houwelington A.N., Noerager B.D. et al. A novel peptide CXCR ligand derived from extracellular matrix degradation during airway inflammation. Nature Med. 2006, Vol. 12, p. 317-323]. Ligands for CXCR chemokine receptors for the first time stimulate the immune response to individual antigens.

Brief Description of the Drawings

Figure 1. Electron micrographs of hydroxyapatite nanoparticles. Negative contrast, 1.5% phosphoric tungsten acid. The scale segment is 50 nm.

Figure 2. The proportion of sorbed protein after incubation of four different proteins: human albumin, human insulin, chicken lysozyme and NS-ESAT chimeric protein (initial concentration of each protein is 40 μg / ml) with three types of nanoparticles: titanium oxide, aluminum oxide and hydroxyapatite (5% suspension).

Figure 3. The splenocyte stimulation index of mice after double immunization (intraperitoneally) with NS-ESAT protein (10 μg / dose) in combination with hydroxyapatite nanoparticles or a commercial aluminum and magnesium hydroxide adjuvant.

Figure 4. The stimulation index of mouse splenocytes after double immunization (intraperitoneally) with NS-ESAT protein (10 μg / dose) in combination with hydroxyapatite nanoparticles loaded with a CXCR ligand — Pro-Pro-Gli-Pro-Gis peptide.

The implementation of the invention

The implementation of the invention is illustrated by the following examples.

Example 1. Electron microscopic studies of the morphology of hydroxyapatite nanoparticles.

Hydroxyapatite nanoparticles were examined under a JEM-100S electron microscope (JEOL, Japan). The results are presented in figure 1. It was determined that most nanoparticles have a diameter of 60-100 nm.

Example 2. Obtaining a complex of hydroxyapatite nanoparticles with the chimeric protein NS-ESAT.

To study the various properties of the complexes of nanoparticles with the NS-ESAT chimeric protein, we used a solution of this protein in phosphate-buffered saline and a 5% aqueous suspension of hydroxyapatite nanoparticles (Sigma-Aldrich). The nanoparticle suspension was dispersed by sonication and then mixed with a solution of the chimeric NS-ESAT protein in a 1: 1 ratio. The protein solution was pre-filtered through a bacterial filter with a pore diameter of 0.3 μm. After this, incubation was carried out at 37 ° C for 2 hours with stirring.

Example 3. The study of the sorption ability of various nanoparticles and a standard adjuvant in relation to the chimeric protein NS-ESAT and other proteins.

Solutions of four different proteins were used to study sorption: a chimeric protein NS-ESAT, human albumin, human insulin, chicken lysozyme in phosphate-buffered saline (the initial concentration of each protein was 40 μg / ml) and a 5% aqueous suspension of titanium oxide nanoparticles , alumina and hydroxyapatite. Sorption properties were evaluated as the ratio of the concentration of total protein in the sample after incubation with nanoparticles to the concentration of total protein before incubation. Incubation was carried out under the conditions described in example 2. After incubation, the nanoparticles were precipitated by centrifugation at 13,500 rpm in an Eppendorf centrifuge, and the protein content was determined in the supernatant. Total protein was determined spectrometrically by Bradford. The results of the experiment showed that the largest among the investigated nanoparticles of different chemical nature sorption with respect to the NS-ESAT protein is precisely the hydroxyapatite nanoparticles (Figure 2). However, with respect to other proteins studied in the experiment, the sorption ability of hydroxyapatite nanoparticles was not always maximum and, in general, was highly dependent on a particular protein (Figure 2).

Example 4. The study of the immunoadjuvant properties of hydroxyapatite nanoparticles.

In an experiment to evaluate the adjuvant effect, double immunization of outbred laboratory mice (females) with a solution of the chimeric NS-ESAT protein in phosphate-buffered saline was carried out. For comparison, in one group of animals, immunization was carried out with the chimeric protein NS-ESAT in combination with a commercial adjuvant based on a gel of aluminum and magnesium hydroxide (Imject Alum, Thermo Scientific). The second immunization was carried out two weeks after the first, the dose of 10 μg of protein. One month after the first immunization, mouse spleen lymphocytes were isolated and stimulated with the ESAT protein, after which the degree of lymphocyte proliferation (defined as the stimulation index) and, accordingly, the immunization efficiency were assessed by the radioactive label activation rate. It is important that, in this case, the efficiency of stimulation of the cellular component of the immune response was determined, which is insufficient when using standard adjuvants based on aluminum. The greatest effect, under these conditions, manifested itself upon the introduction of NS-ESAT in combination with hydroxyapatite nanoparticles (Figure 3), and the stimulation of the immune response was greater than in groups immunized with the NS-ESAT protein without adjuvant or the NS-ESAT protein in combination with commercial adjuvant. This makes hydroxyapatite nanoparticles a promising adjuvant that stimulates the cellular immune response, in particular, in the composition of influenza vaccines.

Example 5. The study of the adjuvant properties of peptides - ligands CXCR 1 and 2 receptors.

As already mentioned, hydroxyappatite is a highly effective sorbent. To stimulate the immune response in recent years, Toll receptor agonists have been actively studied [Tomai M.A., Miller R.L., Lipson K.E., et al. Resiquimod and other immune response modifiers as vaccine adjuvants. Expert Rev. Vaccine 2007, Vol.6 (5), p.835-847]. Despite its high efficiency, this approach has a number of fundamental disadvantages. First, among these adjuvants, low molecular weight substances (Resiquimod) are considered, which possess rapid diffusion at the locus of administration. The second drawback is that the effect on the receptors involves the activation of their signaling systems, which is individual in nature and not always effective (for example, when immunizing children or the elderly).

Another approach seems to be more productive: stimulation of cytokine receptors with a wide functional profile. Such receptors include CXCR 1 and 2 receptors [Murdoch C. CXCR4: chemokine receptor extraordinaire. Immunol Rev. 2000, Vol. 177, p. 175-84].

In this regard, it is of interest to use peptide chemoattractants in the composition of adjuvant nanoparticles, which, on the one hand, could initiate the migration of immunocompetent cells to loci of the appearance of foreign antigens, and, on the other hand, stimulate the presentation of antigens in the most functional way. Such chemoattractants include ligands of the CXCR 1 and 2 receptors, in particular the short peptide Pro-Pro-Gli-Pro-Gis (PPGPH). A domain homologous to this peptide was found in most chemokines and IL-8 [Weathington N.M. van Houwelington A.N., Noerager B.D. et al. A novel peptide CXCR ligand derived from extracellular matrix degradation during airway inflammation. Nature Med. 2006, Vol. 12, p. 317-323]. To obtain a new highly effective adjuvant, we adsorbed this peptide on hydroxyappatite nanoparticles.

The synthetic peptide Pro-Pro-Gli-Pro-Gis (PPGPH) was adsorbed on hydroxyappatite nanoparticles in 0.1 M phosphate buffer, pH 6.8. After adsorption of the peptide, the nanoparticles were washed at 10,000 xg and mixed with the antigen (see examples 1 and 2, figure 2). The results of immunogenicity testing are presented in FIG. 4. As can be seen from the data presented, the immunogenicity of the NS-ESAT antigen in the case of the use of a complex of hydroxyappatite nanoparticles with a peptide increases significantly, which is an unexpected effect and indicates a high level of stimulation and the possibility of using this approach to design nanovaccines.

Claims (3)

1. Immunoadjuvant, which is a hydroxyapatite nanoparticle with an adsorbed synthetic peptide - a ligand of CXCR 1 and 2 receptors. 2. The immunoadjuvant according to claim 1, wherein the peptide is Pro-Pro-Gly-Pro-His (PPGPH). 3. The use of the immunoadjuvant according to claim 1 for creating vaccines.

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