Application of the photodynamic method in vaccinology

Abstract

Intriduction. Currently, toxic substances are used to inactivate vaccine antigens, which enter into a chemical reaction with the antigen and change its chemical structure or configuration, as a result of which, after a covalent reaction with inactivators, abnormal antigenic determinants appear that increase the reactogenicity and allergenicity of vaccine preparations.  One of the modern trends in the development of science related to the improvement of vaccine preparations is the use of substances that are safe and do not lead to the appearance of abnormal antigenic determinants for the inactivation of pathogens.  Such substances can include photosensitizers.  The use of photoinactivated pathogens provides a basis for the creation of the latest vaccine preparations.  Flavins are substances capable of photomodification, i.e. able to transfer charge upon absorption of photons, which mediates cell signaling or gene expression in endogenous protein complexes, such as light-oxygen-voltage sensing domains in bacteria and plants.  Unlike other substances for inactivating vaccines, flavonoids used in the medical and pharmaceutical industry are classified as "GRAS" (generally regarded as safe) by the Food and Drugs Administration (FDA) toxicity class. Materials and methods. The object of the study is the bacteriophage polyvalent Piophage®. Reference strains of bacteria (Staphylococcus aureus ATCC 25923 (F-49), Escherichia coli ATCC 25922 (F-50) and Pseudomonas aeruginosa ATCC 27853 (F-  51). Bactericidal irradiators (bactericidal wavelength 253.7 nm) were used as a source of ultraviolet light. Riboflavin solution in polysorbate-80 0.02%-1.0% (Fluka, Austria) was used as a photoinactivator. Results & Discussion. Phage sensitivity was determined in 122 clinical strains of E. coli. It was established that among the clinical strains of Escherichia coli, 4.7% were not sensitive to Piofag, 47.6% had sensitivity at the level of individual negative colonies (from 10 to more than 20) - weak and moderate, 47.7% had sufficient sensitivity ( semi-fluid and fluid lysis) to the indicated commercial polyvalent bacteriophage. Among 33 clinical strains of Pseudomonas aeruginosa, the percentage of strains that were not susceptible to commercial polyvalent bacteriophage was 31.4%, 61% of clinical strains were susceptible at the level of individual negative colonies, and only 7.6% of clinical strains of P. aeruginosa were highly susceptible ( semi-draining and draining lysis) to Piofag. It was experimentally established that the infectious activity (phage titer) of the commercial Piophage bacteriophage according to the Appelman method is 10^-9. Determined that the average activity of the commercial bacteriophage Piofag to the tested P. aeruginosa test cultures was (3.66 ± 0.62) × 109 IU/ml, to the E. coli test cultures (5.66 ± 0. 41) × 109 IU/ml and (4.33 ± 0.64) × 109 IU/ml. to test cultures of S. aureus. It was experimentally determined that UV irradiation for 60 minutes led to a virulicidal effect in all experiments, regardless of the use of FS. Solutions of riboflavin in concentrations from 0.5% to 0.05% partially protected the bacteriophage from UV radiation - after 45 minutes of irradiation, individual negative colonies were recorded, in contrast to the control. Inactivation of bacteriophages at the level of controls was determined under the influence of two concentrations of photoinactivator - 0.02% and 0.01%. Conclusion. The obtained results indicate that ultraviolet rays cause, most likely, local changes in the Piofag® viral nucleic acid, leading to the replacement of individual bases, thus the reversion of bacteriophage strains included in Piofag® did not occur.  According to the results of the experiment, the best concentrations of riboflavin for further experiments were found to be 0.02% and 0.01% at UVF for 15 minutes.
Keywords: phytoinactivators, bacteriophage, bacteria, vaccines.