UA143248U - METHOD OF OBTAINING VACCINE FOR PREVENTION AND TREATMENT OF PSEUDOMONOSES - Google Patents

Abstract

Method of producing multistage purulent vaccine by photodynamic inactivation of pathogen. First, take the required number of relevant strains of pseudomonads, incubate them with Pseudomonas aeruginosa bacteriophage, then add riboflavin mononucleotide and menadione sulfate. The solution is then irradiated with light, the precipitate is centrifuged and the resulting vaccine in the form of a supernatant is used as a vaccine without purification.

Description

The useful model belongs to microbiology and biotechnology and can be used to create vaccines for the treatment and prevention of pseudomonosis caused, among other things, by antibiotic-resistant strains of Pseudomonas aeruginosa (Rvendotopaz aegidipovza).

The search for new approaches to the creation of immune drugs continues in various countries of the world.

In Ukraine, there are no diagnostic drugs for the identification of the causative agent and the determination of specific antibodies, as well as vaccine drugs for the prevention and treatment of pyogenic infection. Obtaining such drugs of domestic production is promising, relevant and socially and economically justified. The problem of combating pyogenic infection can be divided into several components: firstly, treatment of patients with pyogenic infection; secondly - emergency prevention of complications caused by R. ayegidipozva (in patients with wounds, burns, open injuries, with invasive methods of treatment); thirdly - prevention of infection by R. Ayehidipov of risk groups (medical personnel of hospitals, intensive care, surgical, burn departments, ATO fighters, patients before planned surgical interventions, etc.). When using traditional methods to create vaccines, when it is necessary to remove protective antigens from the bacterial cell, a number of problems arise.

First of all, the percentage ratio of protective antigens is insignificant, therefore large volumes of biomass are required for cultivation, as well as multiple methods of purification and concentration, which leads to high production costs. Secondly, there is no guarantee of the complete absence of impurities, chemical reagents (phenol, merthiolate), which can cause unwanted reactions in the body of the vaccinated. Over the past 20 years, the attention of researchers has been focused on obtaining various fractional vaccines - based on proteins of the outer membrane, plasmids incorporated into eukaryotic cells, DNA vaccines with fragments encoding flagella B and extracellular protein RE. Research on DNA vaccines is focused on the extraction of the most characteristic fragments encoding specific virulence factors, as well as on the basis of the introduction of such vaccines.

The downside of using such vaccines is the unpredictability of possible consequences due to the presence of foreign material. Most of the previously created vaccines passed only the stage of preclinical studies or were stopped, for a number of reasons, in the early stages of preclinical studies.

Of the greatest success, the "Pseudovac" vaccine, which includes structural and extracellular antigens of inactivated 7 immunotypes of R. aegidipoza strains, has achieved the greatest success.

The effectiveness of this vaccine was studied in children and adults with burns, surgical interventions, in intensive care unit patients on ICU. Although the vaccine is evaluated as sufficiently effective, there is no data in the literature regarding the prospects of its use in patients with cystic fibrosis, in various forms of chronic non-specific lung diseases, etc.

The vaccine "Aegidep" (USA and Switzerland), which includes polysaccharides purified from 12 strains, conjugated with toxin A, received a positive evaluation for the treatment of cystic fibrosis. It should be noted that as a result of the reactogenicity of these vaccines and their insufficient effectiveness in as a result of monospecificity, they did not find practical application.

For various reasons, no drug designed for immunoprophylaxis and immunotherapy of pyelonephritis has found a place for use in clinical practice.

The main problem of creating highly effective vaccines against blue pus infection is obtaining vaccine antigens that would cause a protective response regardless of the serotype (or even subtype) of the pathogen, and remain low-toxic and non-reactogenic. A separate direction of research by scientists from different countries is the application of technologies for obtaining immunogens by inactivating the original producer by the photodynamic method.

When photoinactivated against biomolecules (by visible light), flavins, thanks to the formation of specific flavin aptamers, are able to specifically and irreversibly fragment DNA and RNA of pathogens exactly at the locations of flavin aptamers. Previously, it was believed that the mechanism of action of riboflavin is related to the action of singlet oxygen, but recently this statement has been questioned due to the lack of characteristic reactions to singlet oxygen upon irradiation of riboflavin solution. Recently, the mechanism of action of flavins and quinones has been associated with radical photocatalysis.

A known method of creating therapeutic vaccines that contain infectious particles that can be viral, bacterial and/or cellular in nature. The method includes the stages of adding an effective non-toxic amount of an endogenous photosensitizer to the bacterial suspension and the subsequent exposure to this solution of light irradiation sufficient to inactivate the genetic material of the infectious agent, but not sufficient to destroy its antigenic characteristics (05 Raiepi 875909282 ). The disadvantage of the method is the low efficiency of inactivation of microbial pathogens under the influence of the spectrum of visible light together with photosensitizers - flavin derivatives. In most cases, after incubation for 6-36 minutes of the solution of the suspension of Pseudomonas aeruginosa in the presence of the photosensitizers specified in the nearest analogue, it did not lead to 100 95 death of the pathogen. In addition, the vaccine samples obtained by the authors were monostrain and could protect the vaccinated against only one strain of Pseudomonas aeruginosa. The effectiveness of vaccination also decreased due to the corpuscular nature of the vaccine. The photosensitizers used in the presented analogue had low solubility and, accordingly, photoinactivation efficiency for most microorganisms. These shortcomings are eliminated by using many relevant strains of Pseudomonas aeruginosa at once, and as sensitizers - more soluble riboflavin mononucleotide and menadione sulfate, which were not used in the closest analogue. To increase the protective efficiency and reduce the reactogenicity of the vaccine obtained by the proposed method, a specific polyvalent or adapted to specific strains of Pseudomonas aeruginosa bacteriophage is used, which allows to increase the number of available antigens of microorganisms as a result of phagolysis. At the same time, the received vaccine is not a corpuscle (suspension), as in the closest analogue, but is water-soluble and does not require additional purification.

The basis of a useful model is the task of developing a method of inactivating the causative agent (Rzendotopavzh aegidiposis) to obtain a vaccine for the prevention and treatment of pseudomonosis.

The task is solved by the fact that the obtained suspension of the required number of relevant strains of the Pseudomonas aeruginosa culture in a nutrient medium is first incubated with the Pseudomonas polyvalent bacteriophage, then riboflavin mononucleotide and menadione sulfate are added, after which the solution is irradiated with light, the sediment is centrifuged, and the vaccine obtained in in the form of supernatant liquid is used as a vaccine without purification.

Another condition for the implementation of a useful model describes the addition to the solution as a photosensitizer of riboflavin mononucleotide in concentrations from 0.01 to 1 95.

Another condition for the implementation of a useful model describes the addition to the solution as a photosensitizer of menadione sulfate in concentrations from 0.01 to 1 Vol.

Another condition for the implementation of the useful model describes the application for the inactivation of the stimulus light in the region of 260-400 nm and intensity from 1 to 100 W/cm3.

Another condition for the implementation of a useful model describes the use of light in the 260-400 nm region with an intensity of 1 to 100 W/cm3 and/or irradiation in the 400-600 nm region with an intensity of 10 to 100 W/cm3 for inactivation, and irradiation solution with light lasts from 3 to 30 minutes.

Under the specified conditions, an effective vaccine is obtained, which has protective properties against blue pus infection.

Options for implementing a useful model

The parameters for the inactivation of the vaccine strains of Reecidotopaz aegidipoz were worked out experimentally: culture concentration, dose of bacteriophage, photosensitizers and their doses, nature and type of irradiation, stability of culture disinfection (Table 1).

Table 1

Study of the efficiency of inactivation of pathogens under different processing modes

Sample Sample ingredients Type of exposure Availability Availability

Mo rad r d opr growth: 24 h. | growth: 5 days: 1x 107! without radiation 6 1 |R. aegidipova (106 CFU/ml) radiation-free 05 LO CUS/ML| kusuml

And physical solution of exposure to the world 400 - in 2x107 600 nm 2 ho KUs/ml| kuo/ml - - - Z

R. aegidiposis VM light 400 7 x 101 CFU/ml 2. t physiological solution nm : KUS/ml zhxynyopurulent bacteriophage VM light 210 - Z x 10! CFU/ml 102 CFU/ml ! exposure to the world 400 - bh 102

R. aegidiposis 600 nm 8 x 10' CFU/ml CFU/ML 3. "Physical solution of light irradiation 210 and 102 riboflavin mononucleotides and ! h

R. aegidipoza in physiological solution of exposure to the world 400 - and 4. the same riboflavin mononucleotide | 600 nm 1 x 10 CUb/ml menadione sodium sulfate

R. aegidiposis (105 CFU/ ml) and riboflavin radiation from the world 400 - 5. mononucleotide and . 600 nm of menadione sodium sulfate xynopurulent bacteriophage

As can be seen from Table 1, the proposed technology of inactivation of vaccine strains of the pathogen is most effective for option 5, where both photoinactivators and blue-pus bacteriophage are used.

Example 1. Study of the protective properties of candidate vaccines on the pseudomonosis model in mice

The protective properties of the candidate vaccine, which was made on the basis of the proposed method, were evaluated in the experiment.

20 relevant regional strains of R. aeepidiposa are previously cultivated on a nutrient medium for 24-48 hours, a culture suspension is prepared in STB with 1 95 glucose at a concentration of 106 CFU/ml according to Ms-Rapap, a suspension of phagolysate (polyvalent or adapted bacteriophage) is added in a ratio of 1 : 6 volume units, keep the solution for 30 minutes at 35"C.

The liquid in the reactor is regularly stirred until a homogeneous suspension is obtained. Sodium menadione sulfate and riboflavin mononucleotide are added to the suspension in the reactor in such a way that the final concentration of each component is 1.095. The solution is kept with photosensitizers for 60 minutes, after which it is irradiated with ultraviolet light (260 - 280 nm) for 30 minutes, constantly stirring the solution. After that, the solution is irradiated with visible light (400-600 nm) for 30 minutes and centrifuged at 8,000 rpm. for 20 minutes, after which the supernatant is used as a vaccine.

Inbred mice weighing 20.0 - 24.0 g were used (Table 2).

The following vaccine samples were used for immunization of animals: Mo 1 - thermally inactivated corpuscular vaccine (positive control); Me 2 - unpurified polystrain vaccine, inactivated by riboflavin-vicasol photodynamic method (hereinafter - "B";

Mo Z - supernatant obtained after centrifugation of vaccine sample "B" for 20 minutes at 8000 rpm;

Mo 4 - sediment after centrifugation of vaccine "B" for 20 minutes at 8000 rpm, resuspended in physiological solution.

Each of the samples was tested on inbred mice (20 animals per sample), by injecting 0.2 ml subcutaneously (10 animals each) and 0.5 ml intraperitoneally (10 animals each).

The scheme of administration was as follows: vaccine samples were administered on the first, third and seventh day.

Supervision of research animals allowed us to draw certain conclusions about the toxigenicity and reactogenicity of the samples

First, none of the vaccinated mice died during the entire observation period (25 days).

There was no redness, swelling, or necrosis at the injection site; general behavior, appetite, physical activity also did not change.

In order to determine the protective properties of vaccine preparations, 14 days after vaccination, the animals were injected with an infectious dose of a mixed suspension of daily cultures of 8 strains of R. aegidipoza 109 CFU/ml, 0.5 and 1.0 ml intraperitoneally.

In parallel, in the unvaccinated control group, 10 unvaccinated animals (5 per dose) were infected by a similar method.

As the research results showed, in the group of vaccinated animals, one animal died on the 11th day after infection, while in the control group - b animals (60.0 Fo) - died in the first 2-6 days, and the rest - in a period of 8 days .

Symptoms of intoxication were observed in all animals of the control group (lethargy, loss of appetite, impaired coordination of movements).

During dissection, there was swelling and necrosis of the peritoneum in the abdominal cavity of the animals.

When inoculating the washing waters of the abdominal cavity, a culture of R. aegidiposa was removed in large quantities (107 - 108 CFU/ml).

In the group of vaccinated animals, no pseudomonads were detected during abdominal puncture followed by inoculation of the exudate on the selective culture medium.

Table 2

The effectiveness of the developed vaccine according to the criterion of protective properties

Mo N. Number of animals in the group, n (n died, t (t name of candidate vaccine variant |

Mo 1 - thermally inactivated 1 corpuscular vaccine by heating 20h10/10) 10/11) positive control);

Mo 2 - unpurified polystrain vaccine, inactivated by riboflavin-2 |vicasol photodynamic 20h10/10) 0" method (table 1, Sample Mo 5, hereinafter - "B"))

Mo Z supernatant obtained

C after centrifugation of the sample 2010710) 0 vaccine "B" for 20 minutes at 8000 rpm;

Mo 4 - sediment "B" after centrifugation of vaccine "B" 4 | for 20 minutes at 8000 rpm, 20h10/10) 0" resuspended in physiological solution.

Control - infected, but not I I khr«0.001; the Chi-square test was used

The obtained experimental data indicate the absence of toxigenicity and reactogenicity of the candidate vaccine obtained by the recommended method, while preserving protective properties (90 95), which confirms the perspective of this method of obtaining vaccine preparations.

Claims (7)

USEFUL MODEL FORMULA 1. The method of obtaining a multistrain anti-pneumococcal vaccine by photodynamic inactivation of the pathogen, which is characterized by the fact that, firstly, the necessary number of relevant pseudomonad strains is taken, incubated with the pneumococcal polyvalent bacteriophage, then riboflavin mononucleotide and menadione sulfate are added, after which the solution is irradiated with light , the precipitate is centrifuged and the resulting vaccine in the form of supernatant is used as a vaccine without purification. 2. The method according to claim 1, which differs in that riboflavin mononucleotide is added in concentrations from 0.01 to 1 95. 3. The method according to claim 1, which differs in that menadione sulfate is added in concentrations from 0.01 to 1 95. 4. The method according to claim 1, which differs in that the light is radiation in the range of 260-400 nm and intensity from 1 to 100 W/cm3. 5. The method according to claim 1, which differs in that the light is radiation in the range of 400-600 nm and an intensity of 10 to 100 W/cm3. 6. The method according to claim 1, which differs in that the light is continuous irradiation in the range of 260-400 nm and intensity from 1 to 100 W/cm? and irradiation in the region of 400-600 nm and intensity from 10 to 100 W/cm3. 7. The method according to claim 1, which differs in that exposure to light lasts from 3 to 30 minutes.