Corresponding author: Illya Podolsky ( illya.podolsky@nuph.edu.ua ) Academic editor: Georgi Momekov
© 2021 Mykola Rybalkin, Natalia Khokhlenkova, Yuliia Azarenko, Olha Kaliuzhnaia, Illya Podolsky.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Rybalkin M, Khokhlenkova N, Azarenko Y, Kaliuzhnaia O, Podolsky I (2021) The study of the therapeutic effectiveness of the associated inactivated and subunit vaccines based on Candida albicans and Candida tropicalis fungi. Pharmacia 68(1): 89-91. https://doi.org/10.3897/pharmacia.68.e49384
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Candidiasis can be in various forms, the most dangerous of them are systemic and visceral candidiases. Many researchers believe that using drugs that are able to stimulate a protective immune response against candidal infections, i.e. immunobiological drugs, is a promising direction in the fight against candidiasis, and that these drugs are an alternative to antifungal agents. The aim of the work was to compare the therapeutic properties of the inactivated and subunit vaccines of cells of C. albicans and C. tropicalis fungi. Previously, animals were infected with the candidal infection. In 5 days, after infection the vaccines studied were injected to mice intramuscularly in the volume of 0.2 ml in the upper part of the rear right paw. In 14 days, these vaccines were injected to mice in the volume of 0.2 ml in the upper part of the rear left paw. In 14 days, the body protection functions were determined by the titer of C. albicans specific antibodies when performing the enzyme-linked immunosorbent assay (ELISA). According to the results of the research conducted it has been found that the use of the inactivated and subunit vaccines stimulates the eight times growth of the titers. Therefore, both vaccines are effective. However, the subunit vaccine has the maximum purification from ballast substances; therefore, it is promising to use exactly this vaccine for further studies.
antigen, candidiasis, immunity, therapy, vaccine
The number of patients with candidiasis dramatically increases; it is associated with the irrational use of antibiotics, hormones, steroid drugs, as well as with deterioration of the environment, increased pathogenicity of Candida genus fungi and development of comorbidities (
In modern practice of domestic physicians the species of Candida fungus as a causative agent of the disease are identified very rarely. Candidiasis caused by Candida genus fungi is most often diagnosed without closer definition of its species. It should be noted that, even if required, the identification of the species of Candida fungus is not always possible to do because of the lack of reagents, equipment and experience of laboratory technicians. Moreover, the long-term use of the same antifungal drugs has led to the loss of sensitivity of many species of Candida fungi or their separate epitopes to them (
Some researchers believe that using drugs that are able to stimulate a protective immune response against candidal infections, i.e. immunobiological drugs, is a promising direction in the fight against candidiasis (
Was to compare the therapeutic properties of the inactivated and subunit vaccines of cells of C. albicans and C. tropicalis fungi.
The therapeutic effect of the associated inactivated vaccine of C. albicans and C. tropicalis fungi with the concentration of 10 + 10 (×106 of cells/ml) and the associated subunit vaccine of C. albicans fungi with the protein concentration of 3 mg/ml and C. tropicalis with the protein concentration of 5 mg/ml in the ratio of 1:1 was studied in healthy two-month white mice with the body weight of 18–22 g. There were 6 animals in the control and test groups; they were kept in the same conditions on a standard diet.
Before the studies the animals acclimatized themselves under the experimental room conditions. The animals were infected intraperitoneally by the suspension of C. albicans fungi of ССМ 335-867 strain in the amount of 20×106 of cells and C. tropicalis of АТСС 20336 strain in the amount of 60×106 of cells in the volume of 1 ml. In 5 days, the inactivated vaccine was injected intramuscularly in the volume of 0.2 ml in the upper part of the rear right paw to mice of one group, while the second group received the subunit vaccine. In 14 days, 0.2 ml of the inactivated vaccine was injected again to mice of one group in the upper part of the left rear paw, while the second group was injected the subunit vaccine. The animals of the control group were injected with the physiological saline solution. In 14 days, the body protection functions were determined by the titer of C. albicans specific antibodies when performing the enzyme-linked immunosorbent assay (ELISA) according to the State Pharmacopoeia of Ukraine (SPhU, ed. І, art. 2.7.1, p. 55–57). For this purpose, the immunoassay kit for detection of antibodies from G to C. albicans class with the “Vector-Best” ELISA test system was used.
The results of the study showed that before infection the antibody titers of healthy animals were in the range of 1:400. It can be explained by the possible contact with the fungus of Candida genus during the life of mice or a possible carrier state of the genus fungi since they are part of the normal microflora of animals.
After infection of mice the antibody titers were 1:800, indicating development of the immune response of animals to cells of C. albicans and C. tropicalis fungi.
With the first injection of the associated inactivated vaccine the antibody titers of C. albicans fungi were observed at the level of 1:800, indicating the insufficient immune response of a single injection since the antibody titers remained at the level of infected animals without their growth. After a double injection of the associated inactivated vaccine with an interval of 14 days there was an increase in the antibody titers by eight times (1:3200) compared to the titers in healthy animals and by four times compared to the infected animals. In 1 and 3 months of the studies the antibody titers remained at the same level and were 1:3200. The results of the studies are presented in Table
After the first injection of the associated subunit vaccine the antibody titers of C. albicans fungi were observed at the level of 1:800, indicating the insufficient immune response of a single injection. After a double injection of the associated subunit vaccine with an interval of 14 days there was an increase in the antibody titers by eight times (1:3200) compared to the titers in healthy animals and by four times compared to the infected animals. The studies conducted in 1 and 3 months demonstrated that antibody titers were eight times increased compared to the original data and were 1:3200 (Table
Comparison of the therapeutic action of the associated inactivated and subunit of cells of C. albicans and C. tropicalis fungi.
Associated vaccines | After the 1st injection | After the 2nd injection | In 1 month | In 3 months | Control |
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Antibody titers | |||||
Inactivated vaccine | 1: 800 ± 173 | 1: 3200 ± 792 | 1: 3200 ± 758 | 1: 3200 ± 761 | 1: 800 ± 152 |
Subunit vaccine | 1: 800 ± 168 | 1: 3200 ± 731 | 1: 3200 ± 723 | 1: 3200 ± 749 | 1: 800 ± 171 |
Therefore, both vaccines provide the increase of the titers by eight times. For further studies the associated subunit vaccine was selected since it had the maximum purification and was without ballast substances. Ballast substances can provoke adverse immune reactions that will have a negative impact on the vaccine.
To determine the vaccine priority the study on the effectiveness of its action should be conducted, i.e. determine the number of animals remained alive after introduction of the vaccine.
The antibody titers in the control group increased twice. The experimental animals showed no signs of the disease. The animals of the control groups showed the signs of a moderate form of the disease – adynamia, unkempt appearance, refusal to eat, the body weight loss, contractures of the neck muscles, the lateral location of the body, dysfunctions of the excretory organs; when examining the mucous membranes of natural orifices the signs of pathological processes, plating of fungi with feces were revealed. In an advanced form of the disease there was adynamia, unkempt appearance, refusal to eat, the body weight loss, contractures of the neck muscles, paralysis of the limbs, convulsions, the lateral location of the body, and dysfunctions of the excretory organs. During the autopsy when examining the mucous membranes of natural orifices and internal organs of the animals the signs of such pathological processes as microabscesses in the renal cortical layer, lungs, spleen, liver, etc., isolation of retrocultures of fungi from the animals’ organs were observed.
According to the results of the studies conducted it has been found that the use of the associated subunit and inactivated vaccines stimulates the eight times growth of titers compared to the original data, and these titers are 1:3200.
Thus, both vaccines provide immune responses. However, the subunit vaccine has the maximum purification from ballast substances, which can give adverse immune reactions. Therefore, it is promising to use the associated subunit vaccine of C. albicans fungi with the protein concentration of 3 mg/ml and C. tropicalis with the protein concentration of 5 mg/ml in the ratio of 1:1 for further studies.