RU2661028C2 - Pharmaceutical composition for passive rabies immunization, pharmaceutical kit, pharmaceutical kit application method - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine, namely to immunology, and can be used to produce a pharmaceutical kit for passive rabies immunization. Pharmaceutical kit placed in an acceptable package comprises: 1) a pharmaceutical composition comprising virus neutralizing anti-rabies single-domain antibodies in an effective amount and a pharmaceutically acceptable buffer solution up to 1 ml, wherein the virus neutralizing anti-rabies single-domain antibodies have an amino acid sequence SEQ ID NO.1; wherein an effective dose of virus neutralizing anti-rabies single-domain antibodies is a dose of not less than 150 ME activity, in lyophilized form; 2) a pharmaceutical composition comprising recombinant pseudo-adenovirus particles carrying a gene of virus neutralizing anti-rabies single-domain antibodies in an effective amount, a pharmaceutically acceptable buffer solution up to 1 ml; recombinant pseudo-adenovirus particles carrying a gene for virus neutralizing anti-rabies single-domain antibodies have a nucleotide sequence of a genetic insert SEQ ID NO.3, and an effective dose of recombinant pseudo-adenovirus particles carrying a gene of virus neutralizing anti-rabies single-domain antibodies is at least 5×10⁸ PFU in 1 ml of a pharmaceutically acceptable solvent; 3) instructions for the administration of this pharmaceutical kit. Group of inventions also relates to the use of a pharmaceutical kit for passive rabies immunization.

EFFECT: use of this group of inventions provides emergency prevention of rabies through passive immunization by using the produced pharmaceutical compositions, action of which begins with 6 hours of administration and lasts up to 7 days.

2 cl, 12 ex, 4 tbl, 6 dwg

Description

Technical field

The present invention relates to the field of virology, immunology, biotechnology and medicine. The proposed pharmaceutical composition for passive immunization against rabies, neutralizing the rabies virus, allows for effective emergency post-exposure prophylaxis of rabies in bitten animals.

State of the art

Rabies is an acute infectious disease of animals and humans caused by the rabies virus Rabies virus belonging to the genus Lyssavirus of the family Rhabdoviridae. Rabies is found in more than 150 countries and territories and, according to WHO, is among the five infectious zoonoses that cause the greatest economic damage. Annually, 45-60 thousand people die of rabies in the world, mainly in Asia and Africa.

The source of rabies virus is sick animals (foxes, wolves, dogs, bats, cats, rats, livestock and other warm-blooded animals). Rabies virus can enter the human body when it is bitten by a sick animal, most often by stray or domestic dogs and cats, and if saliva enters the skin affected by microtrauma. In rare cases, infection is possible by inhalation of aerosols containing the virus or by transplantation of an infected organ [11].

In the vast majority of cases of people dying from rabies, dogs are the source of infection (99%). About 10 million people injured by animals seek anti-rabies care, while 40% of those who have been bitten by rabid animals are children under the age of 15 [11]. For example, in countries in Asia and Africa, postexposure prophylaxis helps prevent 330,304 deaths [10].

In Russia, according to Rospotrebnadzor, annually about 400 thousand people apply for anti-rabies help (an average of 300.0 per 100 thousand of the population), of which about 250 thousand people need specific treatment. The proportion of children under 17 years of age affected by animal bites remains constant at 28-30% annually. In 2015, the number of people who came up for attacks and animal bites amounted to 392586, of which children under 17 years old - 114622. At the same time, 9950 people were affected by wild animals.

According to rough estimates by experts of Rospotrebnadzor, in 2015 the economic damage to the country from bites, saliva, and animal scratching amounted to 3542144.2 thousand rubles. In 2015, 6 cases of rabies were recorded in 5 constituent entities of the Russian Federation (Lipetsk, Yaroslavl, Vladimir, Nizhny Novgorod regions and the Stavropol Territory) (3 in 2014, 6 cases in 2013), including one - in a child in the age group of 3 to 6 years

Unfortunately, to date, effective means and methods of treating the already developed rabies disease have not yet been developed. However, even after contact with a suspicious animal, medical specialists can still reliably prevent rabies by using a special technique called “post-exposure prophylaxis” (or emergency, PEP). The possibility of its implementation is based on a long incubation period, during which, after the introduction of anti-rabies drugs, immunity is developed and human disease does not occur with rabies.

The incubation period is a feature of the pathogenesis of rabies and averages 1-3 months, but sometimes occurs less than 1 week or more than one year [11]. The proximity of the site of the virus to the central nervous system increases the likelihood of a shorter incubation period. So, the shortest incubation period is observed with bites of the face and head, and the longest - with bites of the lower extremities and trunk. The estimated virus migration rate is 15–100 mm per day [17].

Thus, based on knowledge of pathogenesis, the most important condition for effective post-exposure prophylaxis is its immediate onset and compliance with the drug administration regimen for a bitten person, which can prevent the onset of disease symptoms and death.

According to the WHO position, the main components of post-exposure prophylaxis are:

- a course of immunization with a powerful and effective rabies vaccine that meets WHO recommendations,

- if there are indications (contacts of categories II and III), the introduction of rabies immunoglobulin (passive immunization) [11].

When vaccination is carried out, it takes time to induce a humoral response, which does not meet the urgency requirements for post-exposure rabies prophylaxis. Therefore, the introduction of an anti-rabies immunoglobulin is added to the PEP scheme, the effect of which occurs almost from the moment of administration, since the drug contains ready-made anti-rabies antibodies.

Used PEP regimens include anti-rabies immunoglobulin (AIG), which is used for all category III contacts and category II contacts (in individuals with immunodeficiency). For Category III contacts, AIG is administered to the patient in the wound or around it and 4-5 doses of the vaccine.

Contacts mean:

- Category II - small bites of uncovered skin, minor scratches or abrasions without bleeding, desalination of damaged skin;

- Category III - a bite or scratch or multiple bites or scratches with skin damage, animal saliva on the mucous membranes, contact with bats [10].

For contacts of categories II and III, the recommended treatment and prophylactic course provides for the introduction of 5 doses of rabies vaccine on days 0, 3, 7, 14 and 28 of the course of post-exposure prophylaxis. The anti-rabies immunoglobulin for passive immunization is administered only once, preferably at the beginning of immunization (on day 0) or, if possible, immediately after its start. WHO pays special attention to the fact that after the 7th day the use of AIH is not indicated, since it is assumed that active antibodies have developed in the body in response to the rabies vaccine [5, 8].

The most important factor determining the protection against rabies after immunization is the presence of a minimum protective titer of rabies antibodies. According to WHO recommendations, the minimum titer of rabies antibodies in the blood serum of the immunized should be 0.5 IU / ml (determined in the test for rapid inhibition of fluorescence foci (RFFIT) or in the reaction of neutralizing the virus with fluorescent antibodies (FAVN)).

Existing anti-rabies immunoglobulin preparations are based on the use of human or animal sera containing a high titer of rabies virus-specific immunoglobulins. The recommended dose of anti-rabies human (homologous) immunoglobulin is 20 IU / kg body weight, and equine (heterologous) anti-rabies immunoglobulin is 40 IU / kg body weight [16].

A known preparation of equine (heterologous) anti-rabies immunoglobulin (RF Patent 2339401) for intravenous and intramuscular administration, in which highly specific anti-rabies serum obtained from immunization of animal producers with purified rabies virus grown on a Vero cell culture or PLC is used as feedstock. The content of γ-globulin fraction in the preparation is not less than 90%, molecular weight distribution: aggregates not more than 1.0%, dimers and monomers less than 10.0%, F (ab) ₂ fragments not less than 84.0%, Fab - and Fc fragments of less than 5.0%, low anti-complementary activity, specific activity of at least 150 IU / ml. The osmolarity of the finished product is in the range of 250-350 mOsm / kg. The invention provides a highly purified heterologous anti-rabies immunoglobulin that is stable during storage and transportation.

The described analogue has a significant drawback that does not allow to consider heterologous (equine) immunoglobulin as the preferred drug in the implementation of post-exposure prophylaxis of rabies. Being a foreign protein for the human body, heterologous anti-rabies immunoglobulin is reactogenic and can cause severe allergic reactions, including anaphylactic shock and serum sickness.

Human (homologous) immunoglobulins are deprived of the above disadvantages.

Known human anti-rabies immunoglobulin highly purified (patent CN 101450967A "High-purity rabies human immunoglobulin and raw material production method thereof" - Anti-rabies human immunoglobulin highly purified and raw materials for its production, methods of use).

This technical solution, as the closest to the claimed by the composition of the active substance and the method of its use, is selected by the authors of the present invention for the prototype.

Obtained by this method, the drug is characterized in that the content of immunoglobulin is more than 98% of the total protein content; the titer of rabies antibodies is equal to or greater than 150 IU / ml, the protein content is less than 120 g / l. The content of the monomer and dimer of immunoglobulin Ig G is more than 99%. The process of obtaining such immunoglobulin is rather laborious and involves several stages: 1) preparation of raw materials based on blood with a titer of rabies antibodies of 10 IU / ml of blood; 2) cold ethanol fractionation, purification; 3) ion exchange chromatography; (4) inactivation of viruses by incubation at low pH; (5) membrane filtration to remove viruses; (6) ultrafiltration, dosing, sterilization.

The presented prototype has significant disadvantages due to the content in its composition of human immunoglobulins.

- rabies globulins from human blood serum is a scarce and expensive drug, immunized human donors are required;

- since the drug is obtained from the blood of donors, the potential for contamination with infectious agents is not ruled out;

- passive rabies antibodies reduce the effectiveness of rabies vaccines, which can lead to rabies and, ultimately, to death.

It is known that the presence of passive anti-rabies antibodies for more than 7 days in the blood of an immunoglobulin immunized reduces the vaccination efficiency due to the inhibitory effect of passive antibodies on the formation of active antibodies [18]. The data indicate that the formation of active antibodies occurs already on the 7th day after the vaccine [1, 2], and the half-life of human anti-rabies immunoglobulin is about 21 days [3].

- due to the inhibitory effect of classical antibodies on the induction of vaccines, the administration of immunoglobulins is not allowed later than 7 days after the previous rabies vaccination, which is a direct contraindication to the administration of immunoglobulins to a bitten patient if he received a preventive vaccine less than 7 days before the incident [5, 8];

- virus-neutralizing effect of human anti-rabies immunoglobulins is realized more slowly, within 24 hours with a peak at 2-13 days, compared with single-domain antibodies, the concentration of which reaches a peak at the 4th hour after intramuscular injection [9].

To date, in Russia for emergency rabies prophylaxis, only two types of drugs are used - equine (heterologous) anti-rabies immunoglobulin, as well as human (homologous) imported. Both drugs have been used for a long time and have certain disadvantages. Based on the current state of the art, the need for the development of innovative drugs for emergency rabies prophylaxis that can neutralize the rabies virus and replace the classic rabies immunoglobulins in the standard treatment and prophylactic immunization is obvious.

Disclosure of invention

The objective of the invention is aimed at creating a drug for the rapid neutralization of rabies virus in the body of an infected person, safe and not reducing the effectiveness of the rabies vaccine in the scheme of emergency post-exposure prophylaxis of rabies.

The technical problem is solved due to the fact that a pharmaceutical composition was created for passive immunization against rabies, in the form of lyophilisates in vials and an injection solution, placed in an acceptable package, including virus-neutralizing anti-rabies single-domain antibodies and recombinant pseudo-adenovirus particles, carrying the gene for virus-neutralizing anti-rabies single-domain antibodies in pharmacologically effective doses.

Virus neutralizing anti-rabies single-domain antibodies have the amino acid sequence of SEQ ID NO.1.

An effective dose of virus-neutralizing anti-rabies single-domain antibodies is a dose of not less than 150 ME activity, in lyophilized form.

Recombinant pseudo-adenoviral particles carrying the gene for virus-neutralizing anti-rabies single-domain antibodies have the nucleotide sequence of the genetic insert SEQ ID NO.3.

At the same time, the effective dose of recombinant pseudo-adenovirus particles carrying the gene of virus-neutralizing anti-rabies single-domain antibodies is at least 5 × 10 ⁸ act.ed. in 1 ml of a pharmaceutically acceptable solvent.

Pharmaceutical kit for passive rabies immunization, placed in an acceptable package, includes a pharmaceutical composition in the form of lyophilisates in vials and an injection solution, contains virus-neutralizing anti-rabies single-domain antibodies and recombinant pseudo-adenovirus particles containing the gene for neutralizing anti-rabies single-domain antibodies in pharmacologically for the simultaneous administration of the components of this pharmaceutical kit.

A method for using a pharmaceutical kit for passive rabies immunization is as follows: lyophilisate of a virus-neutralizing anti-rabies single-domain antibody is reconstituted by mixing the lyophilisate with a pharmaceutically acceptable solvent, then administered to a subject infected with rabies virus at a dose of not less than 20 IU / kg, immediately thereafter administered recombinant pseudo-adenovirus particles carrying the gene of virus-neutralizing anti-rabies single-domain antibodies - not less than 5 × 10 ⁸ active units in solvent.

From the literature data it is known that the disadvantages indicated in the prototype of the disadvantages inherent in traditional immunoglobulins are preparations of recombinant single-domain antibodies obtained on the basis of antibodies from animals of the camelid family. These antibodies consist of a dimer of only one shortened (without the first constant region of CH1) immunoglobulin heavy chain and are fully functional in the absence of an immunoglobulin light chain. For specific recognition and antigen binding, only one variable domain of this antibody is necessary and sufficient. A known example of the use of a recombinant single-domain antibody for specific binding and neutralization of rabies virus with high efficiency in vitro (RF patent No. 2533802 C1 "Trimerised single - domain antibody that specifically binds with glycoprotein G of rabies virus, neutralizing rabies virus" - "Trimerized single-domain antibody, specific binding to rabies virus glycoprotein G, neutralizing rabies virus ").

The full equivalent of the term “single-domain antibody” is the widely used designation “nanobody”, “NANOBODY”, as well as “nanoantibody” and “single-domain nanoantibody”.

The advantages of recombinant single-domain antibodies in comparison with the prototype classical immunoglobulins are:

1) The presence of a highly efficient method of generation and selection; donor people are not needed to obtain single-domain antibody preparations.

2) The small size, ~ 2 × 4 nm, 13-15 kDa, allows unlike classical antibodies to penetrate into nerve cells, which are the localization of rabies virus. Single domain antibodies are able to be in effective concentration in the nervous tissue and neutralize the rabies virus there, which is extremely important for bites near the head, when the time for the virus to penetrate is shortened and can elude the effects of classical antibodies located in tissues other than the nervous one.

3) The absence of inhibitory effect on the formation of active (vaccine) antibodies, since the body eliminates single domain antibodies earlier than 7 days and does not overlap the period of their induction.

4) The absence of an inhibitory effect on the induction of vaccine antibodies also occurs due to the absence of an Fc fragment in single domain antibodies, which allows people who are recently vaccinated with a prophylactic purpose to administer them, even after 7 days after vaccination.

5) With the introduction of single-domain antibody preparations to experimental animals (or patients), the antiviral effect is immediate, in contrast to classical antibodies, the maximum concentration of which is observed only after 2-3 days [15, 6, 13]. This difference allows single-domain antibodies to more quickly realize a virus-neutralizing function in the body of an infected person, which is incredibly important in the implementation of emergency rabies prophylaxis.

6) Structural features (the ability to form paratopes unusual for classical antibodies, allowing them to bind to recesses and active centers of proteins); can be used to identify “hidden” epitopes or epitopes that cannot be recognized by significantly larger conventional antibodies.

7) High solubility and stability (in a wide range of temperatures and acidity of the medium) allows you to develop various solutions of finished dosage forms.

8) High expression yield, cost-effective production in large quantities. Typically, single-domain antibodies are produced in the periplasm of E. coli bacteria (in an amount of 1-10 mg from 1 liter of culture). The possibility of their effective production in yeast, plants and mammalian cells is demonstrated.

9) The simplicity of all kinds of genetic engineering manipulations, adaptations for specific tasks, the ability to create multivalent and multifunctional derivatives.

10) Low immunogenicity; the ability to economically "humanize" antibodies without a noticeable loss of their specific activity.

Being highly effective drugs, single-domain antibodies, however, have a short life time in the body (no more than 2-4 days), which does not allow the use of the rabies antibody preparation in the rabies PEP scheme as a replacement for classical immunoglobulins. Repeated injections of single-domain antibodies increase the cost of immunization, require labor costs of medical staff, repeated visits to the clinic by the patient and muscle injury at the injection site. To increase the period of action of the drug based on single-domain antibodies, it is proposed to prolong their action through the use of recombinant adenovirus, which provides continuous synthesis of rabies antibodies directly in the cells of the body. A single injection of this drug reduces the cost of the PEP, (production and administration of ready-made antibodies is not required, they are produced by the body itself), multiple injections are replaced by a single injection of the drug recombinant pseudo-adenoviral particles on day 0 of the course. The period of production of the target protein by them is several days and fits well into the general scheme of emergency rabies prophylaxis, since it is present in the body up to the formation of vaccine antibodies. A similar approach to successfully prolonging the action of a protein preparation with recombinant pseudo-adenovirus particles was described in RF Patent No. 2488406 (Pharmaceutical composition for the treatment of acute toxic conditions). The safety and effectiveness of this type of vectors has been proven in a number of preclinical and clinical trials conducted worldwide, including in Russia [14]. In addition, to date, fast and flexible technologies for producing this type of vectors have been developed that allow the implementation of large-scale production of various therapeutic agents based on them (RF Patent Application 2012107702; RF Patent 2219241). A particularly important ability is the ability to maintain an effective concentration of anti-rabies single-domain antibodies directly in the nervous tissue due to the penetration of the adenoviral vector and expression of single-domain antibody genes [7].

Based on the foregoing, the authors proposed as a means for emergency prophylaxis of rabies, a pharmaceutical composition for passive immunization against rabies, in the form of lyophilisates in vials and injection solution, placed in an acceptable package, including virus-neutralizing anti-rabies single-domain antibodies (Preparation 1), specific for rabies virus glycoprotein G (valid from 0.5 hours to 12 hours) and recombinant pseudo-adenovirus particles carrying the virus-neutralizing anti-rabies single-house gene antibodies in pharmacologically effective doses (Drug 2), the effect of which begins from 6 hours after administration to the body and lasts up to 7 days.

A method for producing a pharmaceutical composition for passive rabies immunization for emergency post-exposure rabies prophylaxis comprises the following principal steps:

1) Obtaining a preparation of anti-rabies single-domain antibodies (Preparation 1) by obtaining a library of variable domains of noncanonical single-chain antibodies, selecting single-domain antibodies that specifically recognize rabies virus (glycoprotein G), their trimerization (modification, formatting) and their further development in E. coli (strain BL21), with the activity in the finished form of the Preparation 1 from 150 to 250 ME per bottle.

2) Preparation of a recombinant pseudo-adenovirus particle carrying a gene of virus-neutralizing anti-rabies single-domain antibodies (Preparation 2) by constructing a recombinant pseudo-adenovirus particle carrying a gene of virus-neutralizing anti-rabies single-domain antibodies and their preparation with activity in the finished form of Preparation 2 from 5 × 10 ⁸ to 5 × 10 ⁹ PFU / ml (dose).

3) The layout of the finished pharmaceutical kit for passive rabies immunization, placed in an acceptable package, including the pharmaceutical composition in the form of lyophilisates in vials and an injection solution containing virus-neutralizing anti-rabies single-domain antibodies (Preparation 1) and recombinant pseudo-adenovirus particles carrying the virus- neutralizing anti-rabies single-domain antibodies (Preparation 2) in pharmacologically effective doses and instructions for the simultaneous administration of components of this of a pharmaceutical kit.

4) A ready-made pharmaceutical kit for passive immunization against rabies with a set of drugs for emergency rabies prophylaxis consists of preparations 1 and 2. The number of bottles of drug 1 is regulated by the required effectiveness, and can consist of 9 bottles with activity from 150 to 250 ME per bottle or less the number of vials with increasing activity and volume of vials, for example 300-500 ME per vial; or 1500-5000 ME per bottle, but not limited to these variations. The drug 1 is presented as a lyophilisate. The drug 2 is included in the kit in the form of a solution in the amount of 1 bottle with a volume of 1 cm ³ .

5) A method for using a pharmaceutical kit for passive rabies immunization is as follows: lyophilisate of a virus-neutralizing anti-rabies single-domain antibody is reconstituted by mixing the lyophilisate with a pharmaceutically acceptable solvent, then administered to a subject infected with rabies virus in a dose of at least 20 IU / kg, immediately after This introduces recombinant pseudo-adenovirus particles that carry the gene of virus-neutralizing anti-rabies single-domain antibodies - not less than 5 × 10 ⁸ act.ed. in solvent.

Each of the drugs has a number of positive properties.

The anti-rabies single-domain antibodies obtained according to the invention are characterized by the following improved properties [13]: 1) The method developed by the applicant for the production and selection of single-domain antibodies, which is based on the production of nucleotide sequences of genes of non-canonical antibodies of animals of the camelidae family (Camelidae); 2) The possibility of economical operating time in large quantities, since the producer is the bacterium E. coli, which indicates the industrial applicability of the invention; 3) The biological safety of antibodies, since the culture of the E.coli bacterium cannot be contaminated by eukaryotic pathogens; 4) Low immunogenicity, since a single-domain antibody consists only of the variable fragment of the heavy chain responsible for binding to the antigen and does not contain other domains to which undesirable immune reactions are formed; 5) Single-domain antibodies have improved permeability to cells, including nerve cells, due to their small size (~ 2 × 4 nm, 13-15 kDa), which is very important for rabies virus infection; 6) There is no effect on the vaccine immune response, which increases the effectiveness of rabies vaccination in the composition of PEP; 7) High solubility and stability (in a wide range of temperatures and acidity of the medium) allows preparing single-domain antibodies based on them for medical use; 8) The possibility of prolonging the action through the use of recombinant adenovirus particles with the insertion of the gene for anti-rabies single-domain antibodies expressing the transgene directly in the patient's body.

The main advantage of the use of drug 2 based on recombinant pseudo-adenovirus particles is the prolongation of production in the body and the maintenance of the protective level of single domain antibodies from 6 hours to 7 days after injection of drug 2 into the body. This effect is achieved by the fact that the introduction of recombinant pseudo-adenovirus particles containing the gene for anti-rabies virus-neutralizing mini-antibodies leads to the expression of the anti-rabies single-domain antibody gene directly in the cells of the body, and therefore additional injections of the single-domain antibody preparation are not required to maintain an effective concentration of single-domain antibodies in the body , including in the nervous tissue [7].

Description of figures

In FIG. one

SEQ ID NO: 1 is the amino acid sequence of a selected trimerized single domain antibody capable of specifically binding and neutralizing rabies virus.

SEQ ID NO: 2 The nucleotide sequence encoding the amino acid sequence of a trimerized single domain antibody capable of specifically binding and neutralizing rabies virus.

In the indicated sequence, SEQ ID NO: 1 is marked in bold and underlined (from left to right, from the N- to C-terminus) of the hypervariable regions of CDR1, CDR2 and CDR3, which are crucial (CDR3 is especially important) for specific recognition of the surface protein of the virus by this single domain antibody rabies. In the amino acid sequence, two cytosine residues are also highlighted (in gray), which, based on many previous structural studies of such molecules, can form an additional non-canonical Cys-Cys bond and thus significantly stabilize the structure of the antibody. The sequences added during modification (adaptation) of the initial single-domain antibody obtained contain a linear region (the linker region corresponding to the long hinge region of the special camel antibody (“hinge”, h) without cysteine residues, which is crucial for enhancing the virus-neutralizing function, the trimerizing domain (variant of the “leucine” motif) lightning bolts ", ILZ, grayed out), as well as (at the very end) sections encoding two oligopeptide tags: HA tag and (His) ₆ tag. At the end there is a TAA termination codon (in amine the acid sequence corresponds to an asterisk).

In FIG. 2

Data are presented on the analysis of purified trimerized single-domain antibody specifically binding to rabies virus using Lammley electrophoresis in a separating 14% polyacrylamide gel under denaturing and reducing conditions (with sodium dodecyl sulfate, SDS, and with dithiothreitol, DTT). A molecular weight marker is applied in the left lane.

A trimerized single-domain antibody was generated in the bacterial periplasm and then purified (including using affinity metal-chelate chromatography and subsequent gel filtration chromatography) to an almost individually protein product migrating in the gel in a protein zone of approximately 28 kDa (actual size is approximately 23.4 kDa; the antibody moves slowly in the gel due to structural features, in particular due to the presence of terminal positively charged six histidine residues (His tag)).

In FIG. 3

Shown is a gel filtration chromatography profile of purified trimerized single domain antibody passed through a Sephacryl S-100 HR 1.5x50 column in standard PBS buffered saline (under normal physiological conditions without denaturation). Proteins with known molecular weights were used as markers: Bovine serum albumin, BSA - 67 kDa; Ovalbumin, OVA - 45 kDa; Lysozyme, LC - 14.4 kDa. The antibody preparation elutes in fractions corresponding to the trimerized antibody.

In FIG. four

The results of an enzyme-linked immunosorbent assay (ELISA) of the binding of the created anti-rabies single-domain antibody to the rabies virus glycoprotein G protein preparation immobilized in the plate well are illustrated.

Antigen (glycoprotein G) was immobilized in wells in a volume of 50-100 μl from solution (in PBS) with a concentration of about 5 μg / ml. The test mini-antibody was used in dilutions of 4 (1: 4) multiples, starting at 40 μg / ml (1) and ending at about 40 ng / ml (6). In one of the controls, a well without a primary antibody was used (7). In the area of the most reliable analysis of antibody activity, in well (4), where a mini-antibody was used at a concentration of about 0.6 μg / ml. the resulting optical density at 405 nm was about 0.85 p.u.

In FIG. 5

SEQ ID NO: 3 is a nucleotide sequence of an expression cassette of a recombinant pseudo-adenovirus particle (RPAN) carrying the gene of a trimerized virus-neutralizing anti-rabies single-domain B3 antibody. Additional design elements are ATG - the start codon, igk leader - the leader peptide transporting the produced peptide from the cell, hinge-ILZ-HA tag - the linear section (the linker region corresponding to the long hinge region of the special camelid antibody ("hinge", h) without cysteine residues, a trimerizing domain (variant of the leucine zipper motif, ILZ), an oligopeptide tag: HA tag, which ends with the TAA stop codon, is crucial for enhancing the virus-neutralizing function.

In FIG. 6

The cumulative pharmacokinetic curve of the level of virus-neutralizing anti-rabies antibodies is shown after a single intramuscular administration to mice of the anti-rabies drugs 1 and 2 of the invention in the emergency rabies prophylaxis scheme. On day 0, virus-neutralizing anti-rabies single-domain antibodies (Drug 1) and immediately recombinant pseudo-adenovirus particles carrying the virus-neutralizing anti-rabies single-domain antibodies (RPAN) - (Drug 2) were first injected into different muscle groups. Immediately after this, the official rabies vaccine was administered. On the horizontal axis, the protective level of rabies antibodies according to the WHO standard, corresponding to 0.5 ME per ml of blood, is plotted.

The shape of the curve indicates that the protective titer of virus-neutralizing anti-rabies antibodies (above 0.5 ME) in the blood serum of the immunized subject is observed with the proposed schedule for the administration of drugs 1 and 2 sets in the period from 0.5 hour to 168 hours (7 days), after what is the effective formation of vaccine anti-rabies antibodies. Thus, the inhibitory effect of single-domain antibodies on the formation of classical anti-rabies antibodies is not observed.

с седьмого дня начинают образовываться вакцинные антитела

Moreover, due to the inability to distinguish single-domain antibodies of Preparation 1 and Preparation 2, their level is marked by the same dashed line

from the seventh day vaccine antibodies begin to form

The implementation of the invention

The implementation of the present invention is illustrated in the examples below.

Example 1

Obtaining single-domain antibodies that neutralize rabies virus (drug 1).

The process of obtaining single-domain antibodies for Preparation 1 is well known to a person skilled in the art and is described in detail in a number of open sources (for example, RF Patent 2533802. A trimerized single-domain antibody that specifically binds rabies virus glycoprotein G to neutralize rabies virus). Its main stages are:

1) Obtaining a library of variable domains of noncanonical single domain antibodies.

2) Selection of single-domain antibodies that specifically recognize rabies virus, their trimerization (modification, formatting).

3) The accumulation of anti-rabies single-domain antibodies that specifically recognize rabies virus.

During the implementation of these works, a unique single-domain antibody was obtained that specifically binds to rabies virus.

The amino acid sequence of the selected anti-rabies single-domain antibody is shown in Figure 1, SEQ ID NO: 1.

In order to increase the efficiency of isolation and detection of a single-domain antibody, as well as to significantly enhance the biological activity of the initially selected single-domain antibody, the previously developed effective trimerization procedure (formatting, modification of the amino acid sequence of an antibody) was used, which was described in detail by Tilib et al. [12]. This procedure involved attaching to the C-terminus of the initially selected antibody four additional amino acid sites (domains) shown in FIG. 1: linear linker region (corresponding to the upper elongated hinge region of camel IgG2a special immunoglobulins), - a special trimerizing domain - "ILZ" ("isoleucine zipper domain", or, in translation, "isoleucine zipper"), - HA tag (peptide from hemagglutinin protein, to which there are commercial antibodies) and His-tag (six histidine residues at the C-terminus of the protein for effective protein purification using metal chelate chromatography.

As a result of the work, a unique single domain antibody of the invention was obtained that binds to rabies virus.

The resulting preparation 1 of a rabies single-domain antibody at various stages was analyzed by electrophoresis in 14% SDS-polyacrylamide gel (Fig. 2). The purified antibodies were stored in aliquots at 4 ° C or, after adding 50% glycerol, at -20 ° C. For longer storage, antibodies were lyophilized.

After gel filtration chromatography of the purified trimerized single domain antibody passed through a Sephacryl S-100 HR 1.5x50 column in standard PBS buffer solution (under normal physiological conditions without denaturation), it was found that the single domain antibody was eluted in fractions corresponding to the trimerized antibody (Fig. 3).

Example 2

Demonstration of the specific binding of an anti-rabies single-domain antibody obtained according to the invention with rabies virus in vitro (preparation 1).

Demonstration of the specific binding of an anti-rabies single-domain antibody obtained according to the invention with rabies virus in vitro (preparation 1).

The ability of the obtained single-domain antibody to bind rabies virus protein, glycoprotein G, and the activity analysis of the created trimerizing single-domain antibody were studied by ELISA.

For ELISA, cells of a standard 96-well immunological plate (polystyrene, Microlon 600 hi-binding, Greiner bio-one) applied in a volume of 50-100 μl diluted (in PBS at a concentration of about 5 μg / ml) antigen material. Immobilization was performed overnight at + 4 ° C. Then the cells were washed (in PBS) from unbound antigen and the wells were blocked with a solution of 1% bovine serum albumin (BSA) or 1% casein in PBS for 2 hours at room temperature. The cells were again washed in PBS and PBS with 0.05% Tween-20, after which solutions with verified mini-antibodies (containing HA-tag at the C-terminus) were added in successive dilutions of 4-fold, starting from a concentration of 40 μg / ml. After incubation (with stirring) for 2 hours at room temperature, the cells were washed again, as described above, and then incubated with 2000-fold diluted PBS with 0.1% BSA) chicken antibodies to the HA tag conjugated with horseradish peroxidase ( CHGT-45P-Z, ICL, Inc., USA). Incubation was carried out for 1 hour at room temperature. After thorough washing, the activity of the horseradish peroxidase enzyme was detected using ABTS (2,2'-azino-bis (3-ethylbenzothiazolin-6-sulfonate) as a chromogenic substrate. The optical density was measured at a wavelength of 405 nm using a flatbed fluorimeter or scanned and saved the image in TIF format. The control wells did not contain the test antigen or contained another immobilized protein, they were blocked and processed in parallel with the main experimental cells. In Fig. 4 it can be seen that in the ibolee reliable analysis of antibody activity in the well (4), wherein use mini-antibody at a concentration of 0.6 ug / ml. the resulting absorbance at 405 nm was about 0.85 pu

The presented results indicate the specific binding of a trimerized anti-rabies single-domain antibody to rabies virus.

Example 3

Demonstration of the virus-neutralizing activity of anti-rabies single-domain antibodies (Preparation 1) in vivo.

The aim of the study was to assess the ability of anti-rabies single-domain antibodies, which are the active substance of drug 1, to neutralize rabies virus.

Quantitative assessment and determination of the virus-neutralizing activity of anti-rabies single-domain antibodies was carried out in the virus-neutralization reaction according to the method of P. Atanasiu widely known to specialists [20].

The essence of the method is to neutralize a constant dose of a previously-treated infected virus and inoculate the test drug to mice.

The experiment was carried out as follows - a preparation of anti-rabies single-domain antibodies was taken in a volume of 50 μl (based on a predefined in vitro activity), 250 μl of PBS was added and combined with 300 μl of a CVS virus suspension with an activity of 250 LD ₅₀ .

The commercial drug Rebinolin (Kamada Ltd., Israel) with an activity of 180 IU / ml, which is a polyclonal human rabies immunoglobulin, was taken as a positive control. The main disadvantages of classical globulins are: a low-tech and expensive production method using human donors with the possibility of contamination with infectious agents; inhibitory effect on the induction of vaccine antibodies, leading to a decrease in the effectiveness of rabies vaccination; inability to penetrate into nerve tissue.

Commercial human immunoglobulin was taken in a volume of 300 μl and combined with 300 μl of a suspension of the virus. A negative control was phosphate buffered saline (PBS).

A mixture of the virus with commercial immunoglobulin or anti-rabies single-domain antibodies was kept at 37 ° C for 1 hour to carry out the neutralization reaction.

After incubation, each mixture was injected into 10 mice weighing 8-10 g intracerebrally, in a volume of 30 μl.

Two weeks after infection, the results of the reaction were recorded.

The results of the experiment to assess the neutralizing activity in mice are shown in table 1.

As follows from the table, when studying the virus-neutralizing properties of anti-rabies single-domain antibodies in a virus-neutralizing reaction in mice, it was found that anti-rabies single-domain antibodies have virus-neutralizing (protective) properties against the rabies virus of the CVS strain (culture) with an activity of 250 LD ₅₀ , out of 10 mice all 10 survived in the experiment, i.e. survival was 100%, which is comparable to the positive control. In the negative control group, all animals died of rabies because they were not given either a commercial anti-rabies immunoglobulin or anti-rabies single-domain antibodies.

Thus, it was found that the anti-rabies single-domain antibodies included in the composition of the preparation 1 have virus neutralizing (protective) properties against rabies virus.

Example 4

Obtaining recombinant pseudo-adenovirus particles carrying the gene of a single domain antibody against rabies virus (preparation 2).

The main stages of work on the preparation of recombinant pseudo-adenovirus particles carrying the gene of a single domain antibody against rabies virus are:

1) Construction of a recombinant pseudo-adenovirus particle carrying the gene of a virus-neutralizing anti-rabies single-domain antibody.

2) Growing on a specialized culture of eukaryotic cells (in particular, on a cell culture HEK293),

3) Purification and formulation.

The construction of a recombinant pseudo-adenovirus particle (RPAN), with the insertion of a virus-neutralizing anti-rabies single-domain antibody gene, was carried out on the basis of the human serotype 5 adenovirus genome (size 70-80 nm).

The preparation of the RPAN construct with the insertion of a virus-neutralizing anti-rabies single-domain antibody gene is carried out by methods known to the average person skilled in the art. Moreover, the obtained design of the recombinant pseudo-adenovirus particle should ensure, when the subject is introduced into the body in an effective amount, the expression of the virus neutralizing anti-rabies single-domain antibody gene in a titer of at least protective (0.5 ME per ml of blood serum immunized) for at least 7 days. Examples of such obtaining effective RPAN constructs with transgene inserts can be patents RF 2523599, 2507258 (for example, Patent No. 2488406. Pharmaceutical composition for the treatment of acute toxic conditions; Patent No. 2507258).

When constructing such a particle, the transgene was the nucleotide sequence of a single-domain antibody SEQ ID NO: 2, previously subjected to codon optimization by a well-known technique to enhance expression in human cells. During the construction process, additional elements were also incorporated into the expression cassette that influenced the expression process of the anti-rabies single-domain antibody. Thus, the result was a recombinant pseudo-adenovirus particle having the nucleotide sequence of SEQ ID NO: 3 shown in FIG. 5, expressing a single domain anti-rabies antibody with SEQ ID NO: 1.

The number of recombinant pseudo-adenovirus particles capable of transducing (activity) HEK293 cells was determined using the standard plaque titration method, resulting in a particle titer expressed in plaque forming units per ml (PFU / ml). A synonym for PFU is the unit of activity - act.

Subsequently, to obtain the finished form of Preparation 2 with the aim of forming a kit, recombinant pseudo-adenovirus particles were multistage purified, formulated, poured and transferred to the packaging stage. This process is described in detail in well-known sources, for example, in RF patent 2489168.

The obtained sterile concentrate of RPAN expressing the anti-rabies single-domain antibody gene fully met the requirements of pharmaceutical quality. Subsequently, it was bottled and was the drug of 2 sets for emergency (post-exposure) prophylaxis of rabies.

Example 5

Demonstration of the virus-neutralizing activity of anti-rabies single-domain antibodies expressed by recombinant pseudo-adenovirus particles (Preparation 2)

The aim of this study was to determine the virus-neutralizing properties of anti-rabies single-domain antibodies expressed by recombinant pseudo-adenovirus particles.

The preparation 2 of the kit contains recombinant pseudo-adenovirus particles with the insert of the gene for anti-rabies single-domain antibodies as the active substance. The mechanism of action is the expression of recombinant pseudo-adenovirus particles of anti-rabies single-domain antibodies in the human body after immunization. The accumulated anti-rabies single-domain antibodies neutralize the rabies virus in the body of an infected person and prevent the development of a subsequent rabies disease.

To conduct the study, the well-known virus neutralization reaction in cell culture (FAVN) was used [4].

Before carrying out the reaction, work was carried out to obtain experimental material. For this, cells of the HEK293 line were scattered on culture dishes with a diameter of 3 cm in an amount of 2 × 10 ⁵ cells / cm ² , incubated for 24 hours, and then infected with the obtained recombinant adenoviruses at a dose of 10 PFU / cell. As a negative control, cells infected with recombinant Ad5-null adenovirus that does not carry a transgene were used. Gene expression of anti-rabies single-domain antibodies was determined in the culture medium and lysates of culture cells, samples of which were taken 48 hours after infection. To prepare cell lysates for analysis of antibody expression, the cells were treated with RIPA buffer (50 mM Tris - HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, a set of protease inhibitors (for example, Roche, Switzerland). Before analysis, the concentration of total protein was measured in samples spectrophotometrically using a Bradford reagent (for example, Sigma-Aldrich, USA). The resulting preparations were examined at the same concentration. Comparison was made with the WHO standard.

The experimental results are presented in table 2.

Результаты в таблице свидетельствуют о наличии экспрессии гена вируснейтрализующего антирабического однодоменного антитела рекомбинантными псевдоаденовирусными частицами в супернатанте среды и в супернатанте клеточного лизата культуры клеток HEK293 в высоких титрах, в отрицательном контроле однодоменных антител не было обнаружено.

The results in the table indicate the presence of gene expression of the neutralizing anti-rabies single-domain antibody by recombinant pseudo-adenovirus particles in the supernatant of the medium and in the supernatant of the cell lysate of HEK293 cell culture in high titers; no single-domain antibodies were detected in the negative control.

Thus, in the example, it was shown that the Kit 2 preparation contains recombinant pseudo-adenovirus particles with the insertion of the anti-rabies single-domain antibodies gene, which, when infected with eukaryotic cells, can produce anti-rabies single-domain antibodies that have virus-neutralizing properties against rabies virus.

Example 6

Pharmacokinetics

The aim of the study was to study the pharmacokinetics of drugs 1 and 2 in laboratory animals to select effective doses and administration regimens.

To evaluate the test results, the well-known neutralization reaction in cell culture (FAVN) was used [4].

Evaluation criteria were the titer of rabies virus antibodies in the blood serum of immunized mice. The minimum titer of 0.5 IU per ml of blood serum required by WHO was taken as the widely used reference level.

The study consisted of two parts:

1) the study of the pharmacokinetics (duration of stay) of virus-neutralizing anti-rabies single-domain antibodies in the body of immunized mice.

2) the study of the pharmacokinetics (accumulation) of virus-neutralizing anti-rabies single-domain antibodies expressed by recombinant pseudo-adenovirus particles.

1) Pharmacokinetics of virus-neutralizing anti-rabies single-domain antibodies (Preparation 1).

The study was conducted on linear Balb / c mice, females. The groups included 6 animals. Blood was taken from the tail vein.

The preparation of 1 kit was administered once intramuscularly at a dose of 20 IU / kg, taking into account the equivalent transfer to the mouse. 2 experiments were conducted with different contents of anti-rabies single-domain antibodies - 150 ME and 250 ME in a bottle. The lyophilized preparation was dissolved in 1 ml of water for injection before administration. Control animals were injected with water for injection as a negative control.

According to the results of the study, shown in table 3, the period of persistence of virus-neutralizing single-domain antibodies in the blood of mice is from 0.5 hours to 96 hours after administration of drug 1, with the content of anti-rabies single-domain 150 ME per bottle. The protective titer of rabies antibodies in this case (more than 0.5 IU / ml of blood serum) was observed from 0.5 hours up to 12 hours after administration, which was taken into account when developing the administration schedule.

When using a drug with an activity of 250 ME in a vial, a smaller volume of the drug was injected - 125 μl dissolved in 1 ml of water for injection in order to comply with the dosage of the drug, and the results on the duration of persistence of single-domain antibodies were obtained that were significantly similar to the drug 150 ME. Thus, the drug with an activity of 150 ME and 250 ME are effective at a dosage of 20 IU / kg of the patient.

As a result, a pharmacokinetic curve was obtained for the preparations of virus-neutralizing single-domain antibodies when using a drug with different activity in the vial: 150 ME and 250 ME. In both cases, the persistence of anti-rabies virus-neutralizing single-domain antibodies lasted no more than 96 hours with a peak at the 4th hour after administration of the drug 1 set. A protective titer of anti-rabies single-domain antibodies of more than 0.5 IU / ml of blood serum was observed for 12 hours after administration.

2) The study was conducted on linear Balb / c mice, females. The groups included 6 animals. Blood was taken from the tail vein.

The drug 2 sets was administered once in doses of 5 × 10 ⁸ PFU, 5 × 10 ⁹ PFU, and 7 × 10 ⁹ PFU in equivalent mouse doses, intramuscularly. Control animals were injected with phosphate-buffered saline as a negative control.

The results of the study shown in table 4 show the presence of expression and accumulation of virus-neutralizing anti-rabies single-domain antibodies with recombinant pseudo-adenovirus particles in the blood of immunized animals with the introduction of the drug 2:

at a dose of 5 × 10 ⁸ PFU - for 4 days,

in doses of 5 × 10 ⁹ PFU and 7 × 10 ⁹ PFU - for 7 days.

However, when using a dosage of 7 × 10 ⁹ PFU, reversible swelling occurred at the injection site in experimental animals, therefore, in the future this dose was not considered as effective.

The resulting anti-rabies antibody titers were not lower than the WHO standard, which defines a minimum protective titer of anti-rabies antibodies of 0.5 ME per ml of blood serum of the immunized subject.

Thus, it was found that the drug is effective in the dose range of 5 × 10 ⁸ -7 × 10 ⁹ PFU per person. The data obtained were taken into account when developing the dose of the finished form of the drug and its administration schedule.

Example 10

The layout of the pharmaceutical kit for passive rabies immunization. Composition of a set of drugs for anti-rabies single-domain antibodies for emergency rabies prophylaxis

When setting up the kit for emergency rabies virus prophylaxis, the pharmacokinetic profiles and effective doses of drugs 1 and 2 obtained in the previous example were taken into account.

Thus, the composition of the ready-made kit for emergency rabies prophylaxis was selected, which includes:

1) Preparation 1 - a preparation of single-domain antibodies that neutralize rabies virus, containing a virus-neutralizing anti-rabies single-domain antibodies - in an effective dose. The number of vials of drug 1 in the kit is regulated by the required efficiency, and can consist of 9 vials with activity from 150 to 250 ME per vial, or fewer vials with a corresponding increase in activity and volume of vials, for example 300-500 ME per vial; or 1500-5000 ME per bottle, but not limited to these variations. The drug 1 can be presented in the form of a lyophilisate. The drug 2 is included in the kit in the form of a solution in the amount of 1 bottle with a volume of 1 cm ³ . The dosage of single-domain antibodies per vial was chosen from the convenience for use by people of different ages and weights. It is possible to complete with two bottles of the Preparation 2.

2) Preparation 2 - a preparation of recombinant pseudo-adenovirus particles carrying the gene of virus-neutralizing anti-rabies single-domain antibodies - 5 × 10 ⁸ -5 × 10 ⁹ PFU in 1 ml of a pharmaceutically acceptable solvent - 1 bottle. It is possible to complete with two bottles of the Preparation 2.

3.) The kit is placed in acceptable packaging.

4.) The kit also contains instructions for the simultaneous administration of the components of this pharmaceutical kit.

Each of the preparations obtained, after passing through quality control, met the requirements of the Russian Pharmacopoeia.

The drug 1 in appearance was an amorphous mass of white; reconstituted preparation from lyophilized - colorless or slightly yellowish slightly opalescent solution. The anti-rabies antibody specifically binds to a homologous antigen and does not bind to non-specific antigens. When determining transparency, the reconstituted preparation stood comparison with standard No. IV and was colorless. By the presence of mechanical impurities, it met the requirements of RD 42-501-98, the number of invisible particles with a size of ≥5 μm - not more than 6000; size ≥25 μm - not more than 600). The pH of the reconstituted solution was within the range of pH 7.0 to 7.5. The drug was sterile, non-toxic, pyrogen-free. Contained from 150 to 250 IU of rabies antibody in a vial. The loss in mass upon drying was not more than 2.5%. The contents of the ampoule were completely dissolved in 1 ml of water for injection with shaking for 2 minutes.

The drug 2 in appearance was a colorless or yellowish slightly opalescent solution. It contained the human adenovirus genome of the fifth serotype and the gene sequence of a single domain antibody against rabies virus, the molecular weight of the amplified DNA fragment in the preparation corresponded to the molecular weight of the amplified DNA fragment in the positive control. When determining the transparency withstood comparison with standard IV. It was colorless. By the presence of mechanical impurities, preparation 2 withstood the requirements of RD 42-501-98, the number of invisible particles with a size of ≥5 μm - no more than 6000; size ≥25 μm - not more than 600). The pH of the solution ranged from 6.0 to 8.0. Recoverable volume of the drug 2 - not less than nominal. It was sterile. Contained less than 7 × 10 ³ replicatively competent adenoviruses per dose. It was not toxic; pyrogen-free. Contained 5 × 10 ⁸ -5 × 10 ⁹ PFU per 1 ml (dose).

The finished pharmaceutical kit for emergency rabies prophylaxis was stored in a dark place at a temperature of +2 to + 8 ° C. Do not freeze. Shelf life is determined at 2 years.

Example 11

Safety of pharmaceutical kit drugs.

Since the purpose of the pharmaceutical kit is the emergency prevention of rabies in humans, all kit preparations must be safe.

Preparations 1 and 2 sets passed the required range of preclinical safety studies, which included studies: acute toxicity, chronic toxicity, reproductive toxicity studies, immunotoxicity and allergenicity studies, mutagenic activity.

During the research were used: Preparation 1 with the content of anti-rabies single-domain antibodies 150-250 ME in a bottle; The drug 2 - containing recombinant pseudo-adenovirus particles with the insertion of the gene for anti-rabies single-domain antibodies - 5 × 10 ⁸ -5 × 10 ⁹ act.ed. in 1 ml of buffer solution.

Doses of the drug in the study of acute toxicity in mice and rats exceeded the recommended immunizing dose for humans up to 308 times, in chronic - up to 31 times.

Conducted preclinical safety studies did not reveal negative effects that impede the clinical study in the declared finished dosage form.

Thus, the pharmaceutical set of drugs for emergency rabies prophylaxis complies with safety requirements - does not have toxicity and reactogenicity, meets the requirements of hazard class 4 “low-hazard substances”.

Example 12

Mode of application.

A method for using a pharmaceutical kit for passive rabies immunization is as follows: lyophilisate of a virus-neutralizing anti-rabies single-domain antibody is reconstituted by mixing the lyophilisate with a pharmaceutically acceptable solvent, then administered to a subject infected with rabies virus at a dose of not less than 20 IU / kg, immediately thereafter administered recombinant pseudo-adenovirus particles carrying the gene of virus-neutralizing anti-rabies single-domain antibodies - not less than 5 × 10 ⁸ active units in solvent.

This example shows a method of using a pharmaceutical kit of anti-rabies single-domain antibodies in a post-exposure (emergency) rabies prophylaxis scheme.

The provision of emergency rabies prophylaxis in the Russian Federation is carried out in strict accordance with WHO requirements and regulatory documents. The purpose of the registered rabies immunoglobulins is emergency rabies prophylaxis in combination with rabies vaccine in the condition of contact category III (single or multiple transdermal bites or scratches, licking of damaged skin; mucosal contamination with saliva when licking, contact with bats) and category II contacts (small bites uncoated skin, minor scratches or abrasions without bleeding, desalination of damaged skin), if we are talking about eggs with immunodeficiency, with a sick or suspicious rabies animal.

To justify the use of drugs 1 and 2 of the pharmaceutical kit, an experiment was conducted in which, in the generally accepted scheme of post-exposure rabies prophylaxis, traditional rabies immunoglobulins were replaced by single-domain antibodies according to the invention. The effectiveness of the course was evaluated by the level of anti-rabies single-domain antibodies that persist in the blood serum of a laboratory animal.

To evaluate the test results, the well-known neutralization reaction in cell culture (FAVN) was used. (Cliquet F. et al. Development of a fluorescent antibody virus neutralization test (FAVN test) for the quantitation of rabies-neutralizing antibody, Journal of Immunological Methods, 212, 1998, 79-87 - Improving the virus neutralization response with fluorescent antibodies (FAVN test ) [four].

The selected dose of anti-rabies single-domain antibody preparations of 20 IU / kg corresponds to the applied dose for human immunoglobulins, which also showed its effectiveness in studying the immunogenic properties of Preparation 1 in experiments in mice.

The study was conducted on linear Balb / c mice, females. The groups included 6 animals. Blood was taken from the tail vein.

The progress of the study was as follows:

on day 0, the mice of the experimental group were administered three drugs in turn:

1) The drug 1 set of rabies single-domain antibodies - at a dose of 20 IU / kg intramuscularly;

2) Immediately after, the Preparation 2 sets with recombinant pseudo-adenovirus particles carrying the gene for anti-rabies single-domain antibodies - at a dose of 20 IU / kg intramuscularly;

3) Immediately after that, the official rabies vaccine (for example, Rabipur, Germany) in a dose, according to the instructions for use, intramuscularly.

4) It is possible to additionally administer the drug 2 sets with recombinant pseudo-adenovirus particles containing the gene for anti-rabies single-domain antibodies - at a dose of 20 IU / kg intramuscularly, if prolongation of the period of presence of passive antibodies in the case of vaccines is required, a protective titer in response to which is formed on the 14th day after immunization on day 0. In this situation, use the Pharmaceutical kit, which contains two bottles of the drug 2.

Intramuscular injections of drugs were carried out in various places. Further, blood samples were taken from the mice in the hours: 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, 168, 336 to determine the ranges of rabies antibodies. The obtained experimental results are shown in FIG. 6.

The indicated method of use of Preparations 1 and 2 sets corresponds to the generally accepted scheme of emergency prophylaxis of human rabies. An obligatory component of the anti-rabies emergency prevention regimen is the introduction of an official anti-rabies vaccine after the introduction of the classic anti-rabies immunoglobulin. In the case of using the kit for emergency rabies prophylaxis, Preparations 1 and 2 serve as an alternative to official (classic) immunoglobulin. Thus, the administration of the rabies vaccine is carried out as soon as possible after the administration of the Preparation 1 and the Preparation 2 of the kit, but no later than 15 minutes after their administration. Further administration of the vaccine is carried out according to generally accepted clinical regimens and is not considered within the framework of this invention, since it does not in any way affect the use of the claimed invention.

Vaccine antibodies formed in response to the administration of an official vaccine appear in the protective titer on day 7.

The method of use according to the invention does not inhibit the induction of vaccine antibodies, which, as is known, can appear already on the 7th day after the introduction of the rabies vaccine [1, 19].

In the emergency rabies prophylaxis scheme, the introduction of the Drug 1 and the Drug 2, carried out before the administration of the official anti-rabies vaccine, allows to achieve a protective level of virus-neutralizing anti-rabies antibodies (at least 0.5 IU / ml) in the blood serum of the immunized patient from the moment of administration for 7 days. Further, from the 7th day, the appearance of a protective level of antibodies was observed, which were formed in response to the administration of an official rabies vaccine. Thus, it is shown that the introduction of a set of drugs according to the invention does not reduce the effectiveness of vaccination and does not create an "immunological hole" in the patient. At the same time, the level of protective antibodies does not fall below the protective (0.5 ME per ml of blood) during the considered period of post-exposure (emergency) rabies prophylaxis.

Therefore, a method has been invented for passive immunization against rabies by the use of a pharmaceutical kit of drugs for neutralizing anti-rabies single-domain antibodies for emergency rabies prophylaxis, which can prevent rabies in a bitten suspicious animal.

Industrial applicability

All these examples confirm the solution of the problem, namely, the creation of a drug for the rapid neutralization of rabies virus in the body of an infected person, which does not safely and does not reduce the effectiveness of the rabies vaccine in the scheme of emergency post-exposure prophylaxis of rabies with rapidly onset and prolonged action, as well as the industrial applicability of the claimed invention a set of drugs for emergency rabies prophylaxis.

List of abbreviations

BSA - Bovine Serum Albumin

CVS - rabies virus culture strain

FAVN - neutralization of the virus with fluorescent antibodies

ILZ - "isoleucine zipper domain", "isoleucine lightning"

PBS - phosphate buffered saline

RFFIT - rapid inhibition of fluorescence foci test

RIPA - radio deposition buffer

SDS - sodium dodecyl sulfate

AIG - anti-rabies immunoglobulin

PFU - units of activity - act.

WHO - World Health Organization

DNA - deoxyribonucleic acid

DTT - dithiothreitol

LD50 - 50% lethal dose

ME - international units

PEP - post-exposure (emergency) prophylaxis

RD - guidance document

RPAN - Recombinant Pseudo Adenovirus Particle

LIST OF USED LITERATURE

1. Bose A. et al. A randomized non-inferiority clinical study to assess post-exposure prophylaxis by a new examined vero cell rabies vaccine (Rabivax-S) administered by intramuscular and intradermal route, Vaccine, 2016 Sep 14, 34 (40), 4820-4826 - Randomized, incomplete clinical trial to evaluate post-exposure prophylaxis with a new purified Vero cell rabies vaccine (Rabivax-S) for intramuscular and intracutaneous applications.

2. Both L. et al. Passive immunity in the prevention of rabies, Lancet Infect Dis., 2012, May, 12 (5), 397-407 - Passive immunity in the fight against rabies.

3. Cabasso V.J. et al. Rabies immune globulin of human origin: preparation and dosage determination in non-exposed volunteer subjects, Bull World Health Organ, 1971, 45, 303-315 - Anti-rabies immunoglobulin of human origin: obtaining and dose determination in volunteers who were not exposed to rabies.

Development of a fluorescent antibody virus neutralisation test (FAVN test) for the quantitation of rabies-neutralising antibody, Journal of Immunological Methods, 212, 1998, 79-87 - Усовершенствование реакции нейтрализации вируса флуоресцентными антител (FAVN тест) для количественного определения антирабических нейтрализующих антител.4. Cliquet F.,

Development of a fluorescent antibody virus neutralization test (FAVN test) for the quantitation of rabies-neutralizing antibody, Journal of Immunological Methods, 212, 1998, 79-87 - Improvement of the virus neutralization response by fluorescent antibodies (FAVN test) for the quantification of anti-rabies neutralizing antibodies .

5. Final for WER Jul 9, 2010 Anti-rabies vaccines WHO position paper fs099 / en /.

6. Hassanzadeh-Ghassabeh Gh. et al. Nanobodies and their potential applications, Nanomedicine, 2013, 8 (6), 1013-1026. - Nanobody and the possibility of their application.

7. Hui Kuo et al. Retrograde transfer of replication deficient recombinant adenovirus vector in the central nervous system for tracing studies. Brain Research, Volume 705, Issues 1-2, 24 December 1995, Pages 31-38.

8. Khawplod P. et al. What is an acceptable delay in rabies immune globulin administration when vaccine alone had been given previously? Vaccine, 1996, 14, 389-391 - How much is an acceptable “waiting time” for administering rabies immunoglobulin with early single vaccine administration?

9. Nigg A.J., Walker P.L. Overview, prevention, and treatment of rabies, Pharmacotherapy, October 2009, 29 (10), 1182-1195.

10. Rabies vaccines WHO position paper, Weekly Epidemiological Record, 2007, Vol. 82, NO. 49/50, 7 December 2007, 425-436 - WHO Position on Rabies Vaccines fs099 / en /

11. Rabies, Fact Sheet, March 2016, No. 99. - Rabies, Newsletter. http://www.who.int/mediacentre/factsheets/fs099/en/.

12. Tillib et al. Formatted single-domain antibodies can protect mice against infection with influenza virus (H5N2), 2013, Antiviral Res, 97, 245-254 - Formatted single-domain antibodies can protect the mouse from infection with the influenza virus (H5N2).

13. Tillib S.V. "Camel Nanoantibody" is an efficient tool for research, diagnostics and therapy, Molecular Biology, 2011, 45, No. 1, 66-73. “Camel Antibody” is an effective tool for research, diagnosis and therapy.

14. Thomas S.E., Ehrhardt A., Kay M. Progress and problems with the use of viral vectors for gene therapy, Nature Reviews Genetics, May 2003, 4, 346-358. - Progress and problems when using viral vectors for gene therapy.

15. Wesolowski J. et al. Single domain antibodies: Promising experimental and therapeutic tools in infection and immunity, Med. Microbiol. Immunol., 2009, Vol. 198, P. 157-174. - Single domain antibodies: A promising tool for the diagnosis and treatment of infections and immunity.

16. WHO Expert Consultation on Rabies. WHO Technical Report Series 982. Second report. Geneva, Switzerland, WHO, 2013 - WHO Expert Consultation on Rabies. WHO Technical Report Series. Second report.

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20. Kaplan M., Koprowski H. Laboratory techniques in rabies, 3rd ed., Geneva, WHO, 1973, 314-318 - Methods of laboratory studies of rabies.

Claims (8)

 1. Pharmaceutical kit pharmaceutical compositions for passive immunization against rabies, placed in an acceptable package, including: - a pharmaceutical composition comprising: virus-neutralizing anti-rabies single-domain antibodies in an effective amount, while virus-neutralizing anti-rabies single-domain antibodies have the amino acid sequence of SEQ ID NO.1, and an effective dose of virus-neutralizing anti-rabies single-domain antibodies is a dose of at least 150 ME activity, in lyophilized form, - a pharmaceutical composition comprising: recombinant pseudo-adenovirus particles carrying the gene for virus-neutralizing anti-rabies single-domain antibodies in an effective amount, while recombinant pseudo-adenovirus particles carrying the gene for neutralizing anti-rabies single-domain antibodies have the nucleotide sequence of the genetic insert SEQ ID NO.3, and an effective dose of recombinant pseudo-adeno-virus anti-rabies single-domain antibodies is antibodies is at least 5 × 10 ⁸ PFU in 1 ml of pharmaceutically acceptable Voritor, pharmaceutically acceptable buffer solution up to 1 ml; - instructions for administering this pharmaceutical kit. 2. The method of using the pharmaceutical kit of pharmaceutical compositions for passive immunization against rabies according to claim 1, which consists in administering in turn a pharmaceutical composition containing virus-neutralizing anti-rabies single-domain antibodies and immediately after the pharmaceutical composition containing recombinant pseudo-adenovirus particles containing the gene for virus-neutralizing anti-rabies single-domain antibodies, wherein the lyophilisate of a pharmaceutical composition containing one neutralizing anti-rabies Antibody antibodies are reconstituted by mixing the lyophilisate with water for injection in a volume of 1 ml, then administered to a subject infected with rabies virus at a dose of at least 20 IU / kg, immediately after that a pharmaceutical composition containing recombinant pseudo-adenovirus particles containing the gene for neutralizing anti-rabies single-domain antibodies is administered in a pharmaceutically acceptable solvent, within 5 × 10 ⁸ -5 × 10 ⁹ PFU; Immediately after this, an official rabies vaccine is administered in accordance with the instructions for its use.

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