Determination of Candida albicans fungus proteins concentration by Elisa method at Intramuscular Introduction in Candidiasis Therapy

Mykola Rybalkin¹, Tatiana Diadiun²*, Natalia Khokhlenkova¹, Yuliia Azarenko¹,

Serhiy Stepanenko³

¹Biotechnology Department, National University of Pharmacy, Kharkiv.

²Commodity Science Department, National University of Pharmacy, Kharkiv.

³Technologies of Pharmaceutical Preparations Department, National University of Pharmacy, Kharkiv.

*Corresponding Author E-mail: diadiunscience@gmail.com

ABSTRACT:

The objective of this work was to determine the C. albicans fungus protein concentration at intramuscular introduction in therapy of candidiasis. Therapeutic effect of C. albicans fungus proteins in concentrations 1, 2, 3, 4 and 5mg/mL has been examined in white mice. Animals were infected intraperitoneally with suspension of C. albicans fungus strain CCM 335-867 in the amount of 20 million cells per 1mL volume. After 5 days and repeatedly after 14 days mice were injected intramuscularly in the upper back hind paw with proteins of Candida fungus cells of volume 0.2mL. 14 days after each injection, the determination of the protective functions of the animal body has been carried out by the titer of specific C. albicans antibodies during immunoassay. According to the data obtained during studies on the treatment of candidiasis, it has been found that in the intramuscular route of administration after the first injection with C. albicans protein concentration of 3 mg/mL antibody titers increased 2 times, and after the second - 8 times. Proteins of C. albicans fungus at a concentration of 3 mg/mL activate the body's defence mechanisms.

KEYWORDS: Candidiasis, Antigen, Vaccine, Immunity, Therapy.

INTRODUCTION:

Fungi of the genus Candida are found on the skin and mucous membranes. At reduced immunity, fungi can penetrate the skin through the stages of adhesion, colonization and invasion^1,2. Candidiasis manifests itself in various forms. Systemic and visceral candidiasis pose the greatest danger^3–5. These forms of candidiasis are characterized by a long course of the disease, have a variety of clinical manifestations and frequent recurrences. Such forms of candidiasis are difficult to treat with modern medicines, including antifungal antibiotics^6-7.

Due to the difficult situation with the treatment of candidiasis, many researchers propose to use vaccines as an alternative to antifungal preparations for the prevention and treatment of candidiasis^8-9. Similar studies have been conducted in many countries^10-12. Candidiasis vaccines cover a wide range of different vaccine variants with different compositions ranging from whole inactivated and virulently attenuated fungal cells to subunit vaccines based on a single recombinant protein, or peptide fragment, or glycoconjugate: inactivated cells of the pathogen, attenuated live pathogen cells, Candida fungal cell enolases, Candida fungal cell surface iC3b receptors, Candida fungal cell mannans, Candida fungal cell β-glucans, Candida cell Hsp90 heat shock protein, SAP protein genes, ALS protein genes, glycoconjugates (mannans and β-glucans) of Candida fungal cells.

At this stage of research, it was necessary to select the most promising vaccines for clinical trials from a variety of potential variants of anti-candidiasis vaccines. This requires an analysis all the available options for vaccines and choice of those having high reliable efficacy and safety, as well as those that can be standardized in composition and production technology and produced with minimal financial costs, minimal use of technological stages and equipment.

It should be borne in mind that some vaccines developed by different researchers are extremely difficult to manufacture, and some of the proposed vaccines are difficult to characterize by a mixture of antigens. It is also necessary to consider the strengths and weaknesses of each specific vaccine. For example, whole inactivated cells are chemically quite complex, which creates serious problems with safety and standardization. Inactivated cells showed weak immune responses compared to live attenuated fungal cells. Live vaccines are usually one of the best immunogens, but have the disadvantage of limited use in immunocompromised patients, because even weakened cells can sometimes cause disease in immunocompromised individuals. On the other hand, for subunit vaccines, which may be the best option for production, standardization and safety, there is a greater likelihood of further clinical trials. However, subunit vaccines have their drawback - weak immunogenicity that usually requires the addition of an adjuvant to induce long-term protective immunity. Although this is not a problem in experimental animal models, there is a shortage of good adjuvants suitable for use in clinical practice. An alternative to adjuvants is the use of liposomes, virusomes, inert nanoparticles, derived PAMP components and other bioengineered drugs.

It should be noted that in Ukraine at the moment no domestic vaccine against candidiasis is produced and no imported one is registered. Therefore, the development of a vaccine for candidiasis infection is a pressing issue of modern pharmacy and medicine.

Previous studies have substantiated the method of disintegration of C. albicans fungal cells, which provides the release of proteins and carbohydrates. The proposed potential composition of the protein-carbohydrate vaccine provides the widest effect on all determinants of the pathogen, which provides greater efficiency, in contrast to individual antigens^10-13. This method provides the release of antigens which, when injected intramuscularly in healthy mice, activate the protective mechanisms against candidiasis, that is, animals did not get sick with candidiasis when infected with Candida infective matter. It is now necessary to check whether the antigens of the C. albicans fungal cells have therapeutic effect, that is, whether they are able to activate protective mechanisms against candidiasis infection at intramuscular and subcutaneous administration to mice with candidiasis, which will further ensure the recovery of diseased animals.

The aim of this work was to determine the C. albicans fungus protein concentration at intramuscular introduction in therapy of candidiasis.

MATERIALS AND METHODS:

All studies were performed in laminar box supporting aseptic conditions. C. albicans strain CCM 335-867 was pre-cultured in test tubes on Sabouraud agar at 25±2°C for 48 hours and washed the fungal cells with 10mL of sterile isotonic 0.9% sodium chloride solution. The resulting suspensions of fungal cells were transferred to Sabouraud agar mattresses incubated at 25±2ºС for 6 days and washed the fungal cells with 25mL of sterile isotonic 0.9% sodium chloride solution. The microbiological purity of the suspension of C. albicans fungal cells determined visually and by microscopy. Next, centrifugation was carried out at a speed of 3000 rpm for 10 minutes. The obtained precipitate of fungal cells was brought with a sterile isotonic 0.9% sodium chloride solution to (8.5 - 9) x10⁸ in 1mL.

Received suspensions of fungal cells in a volume of 10 mL were subjected to ultrasound for the destruction of fungal cells on the apparatus USUU-21 at a frequency of 22 kHz, intensity 5 W/cm² and at a temperature 25±2°C within 15 min¹⁴. Temperature 25±2°C was constantly monitored for the cell suspensions and maintained by adding cold water to the surrounding tank. Further carried out filtering through the membrane "Vladipore" of MFA - MA No. 3, which provides the separation of biological material with the size of 10 kD and its concentration¹⁵. The filtrate obtained is a mixture of proteins and polysaccharides. In each case, the protein content was determined according to SPU. Considering the fact that Candida fungus cell extract contains proteins and polysaccharides that have antigenic properties, according to the requirements of the SPU, the determination of the active substance is carried out on the substance that has more pronounced properties, i.e. on the protein. Further pre-filtration was performed using filters with a pore diameter of 0.45μm and sterilization filtration using filters with a pore diameter of 0.22μm.

Therapeutic effect of proteins of C. albicans in concentration 1, 2, 3, 4 and 5mg/mL were studied in white mice of two months of age 18 - 22g in weight of 6 animals in the control and experimental groups, which were kept in the same conditions under standard diet. The studies were conducted at the State Institution "II Mechnikov Institute of Microbiology and Immunology". Before the study, the animals were acclimatized in the experimental room. Animals were infected intraperitoneally with suspension of C. albicans fungus strain CCM 335-867 in the amount of 20 million cells of 1mL volume. After 5 days mice were injected intramuscularly in the right upper of hind paw with proteins of Candida fungus cells of volume 0.2mL. After 14 days, the determination of the protective functions of the animal body by titer of specific C. albicans antibodies during enzyme immunoassay has been carried out. For this purpose, a set of reagents was used for the enzyme-linked immunosorbent detection of G antibodies to C. albicans using the Vector-Best ELISA test system. 14 days after the first injection, repeatedly in the upper left hind paw injected intramuscularly 0.2mL of Candida cells proteins and after 14 days, the determination of the protective functions of the animal body by titer of specific C. albicans antibodies has been carried out. Animals in the control group were injected saline.

RESULTS AND DISCUSSION:

The results of the studies have shown that the antibody titers of healthy animals were in the range 1: 200-1: 500. This can be explained by the possible contact with the fungus of the genus Candida during the life of mice or the possible carriage of this type of fungi, since they are part of the normal microflora of animals.

At the intramuscular introduction to candidiasis animals after the first injection of C. albicans fungal proteins in concentration 1, 2, 3, 4 and 5mg/mL antibody titers of C. albicans fungus also doubled compared to titers in healthy animals. The results of the studies are summarized in Table 1.

Table 1: Therapeutic effect of C. albicans cell antigens

Animals	*C. Albicans* protein content mg/mL	Method of administration	*C. Albicans* ab titles in ELISA
Animals	*C. Albicans* protein content mg/mL	Method of administration	healthy animals	ill after 1st injection	ill after 2nd injection
Mice	1	Im	1:200	1:400	1:800
Mice	2	Im	1:300	1:600	1: 1200
Mice	3	Im	1:250	1:500	1: 4000
Mice	4	Im	1:300	1:600	1: 2400
Mice	5	Im	1:500	1:1000	1: 4000

Note: n = 6

After the second intramuscular injection of C. albicans proteins at an interval of 14 days the antibody titer increases 8 times when using protein concentrations of 3, 4 and 5mg/mL. At using protein concentrations 1 and 2 mg/mL there is a 4-times increase in the antibody titer of C. albicans fungi.

The antibody titer in the control group at intramuscular route of administration did not grow. Comparing the results obtained in the experimental and control groups, it is safe to say that intramuscular administration of C. albicans fungal cell proteins stimulates the production of antibodies responsible for humoral immunity.

Studies have shown the best results in C. albicans fungus proteins concentrations of 3, 4 and 5mg/mL, which provided an increase in C. albicans antibody titers 8 times after the second injection. For further studies, it is promising to use the protein concentration of the fungus C. albicans 3mg/mL, since this concentration provides an increase in antibody titers as well as protein concentrations of 4 and 5mg/mL, and the amount of protein used is smaller. Therefore, the use of lower protein concentration of 3mg/mL is more economically feasible and it reduces the side effects that can be stimulated by high protein concentrations.

CONCLUSION:

According to the data obtained during studies on candidiasis treatment, it has been found that in the intramuscular route of administration after the first injection with C. albicans protein of concentration 3 mg/mL antibody titers increased 2 times, and after the second - 8 times.

Thus, it can be concluded that these antigens of C. albicans fungus activate the body's protective reactions.

REFERENCES:

1. Lisovskaya AM, Aleshukina AV. The study of the biological properties of Candida spp. Isolated from different loci in outpatients. Problems of medical mycology. - 2006. - Vol. 8, No.2. - P. 57.

2. Pappas P. G. Invasive candidiasis. Infect. Dis. Clin North Am. - 2006. – No. 20. –P. 485–506.

3. Saigal S, Bhargava A, Mehra SK, Dakwala F. Identification of Candida albicans by using different culture medias and its association in potentially malignant and malignant lesions. Contemp Clin Dent. 2011;2(3):188-193. doi: 10.4103/0976-237X.86454

4. Kabir MA, Ahmad Z. Candida infections and their prevention. ISRN Prev Med. 2012;2013:763628. Published 2012 Nov 4. doi:10.5402/2013/763628

5. Diekema D., Arbefeville S., Boyken L., Kroeger J.& Pfaller M. The changing epidemiology of healthcare-associated candidemia over three decades Diagn. Microbiol. Infect. Dis. - 2012. 73. P. 45–48.

6. Borshch SK, Masliak TR. Combined use of antifungal agents and probiotics in combustiology for the treatment and prevention of candidiasis and irritable bowel syndrome. Modern Gastroenterology. - 2011. – No. 4. - P. 30–39.

7. LeibundGut-Landmann S, Wüthrich M, Hohl TM. Immunity to fungi. Curr Opin Immunol. 2012;24(4):449-458. doi:10.1016/j.coi.2012.04.007

8. Aditi Grover, BS Bhandari, Nishant Rai, Pramesh C Lakhera. Candida albicans vaccines Biotechnology International. - 2010.-Vol. 3, 1. P. 4-17.

9. Xin H, Dziadek S, Bundle DR, Cutler JE. Synthetic glycopeptide vaccines combining beta-mannan and peptide epitopes induce protection against candidiasis. Proc Natl Acad Sci U S A. 2008;105(36):13526-13531. doi:10.1073/pnas.0803195105

10. Cassone A. Development of vaccines for Candida albicans: fighting a skilled transformer. Nat Rev Microbiol. 2013;11(12):884-891. doi:10.1038/nrmicro3156

11. Edwards JE. Fungal cell wall vaccines: an update. J Med Microbiol. 2012;61(Pt 7):895-903. doi:10.1099/jmm.0.041665-0

12. Han Y, Rhew KY. Comparison of two Candida mannan vaccines: the role of complement in protection against disseminated candidiasis. Arch Pharm Res. 2012;35(11):2021-2027. doi:10.1007/s12272-012-1120-9

13. Zhukova NV, Krivosheeva IM. Modern vaccines: characterization and classification Crimean Therapeutic Journal. - 2013. - № 2. - P. 99–104.

14. Rybalkin MV Determination of the optimal method of disintegration of Candida albicans and Candida tropicalis fungal cells. Topical issues in pharmaceutical and medical science and practice. 2014. № 2 (15). Pp. 71–75.

15. Rybalkin MV, Strelnikov LS Scientific and experimental substantiation of Candida antigen purification technology. Scientific Journal «ScienceRise: Pharmaceutical Science». 2017. № 1 (5). Pp. 62–65.

Received on 30.07.2020 Modified on 28.10.2020

Research J. Pharm. and Tech. 2021; 14(6):3249-3252.

DOI: 10.52711/0974-360X.2021.00565