Scientists from all over the world are looking for the best options to ensure effective and safe specific prevention of animal diseases. Despite the progress made in the vaccine production, searching for a low-cost and safe adjuvant that would enhance the immune response remains a matter of great importance.

Modern adjuvants shall boost immunity (cell-mediated and/or humoral), be easily metabolized and be widely affordable [1][2].

Chitosan properties are being actively studied worldwide [3–20]. Its derivatives are widely used in the pharmaceutical manufacturing and in veterinary medicine. Chitosan-based products have shown a variety of biological properties, including antimicrobial and cholesterol-lowering ones. The chitosan properties increase the dissociation rate of poorly soluble medicinal products, enhance their absorption, influence the drug release and create drugs of prolonged action [2]. It was also noted that chitosan solution boosts both the humoral and cell-mediated immune response after subcutaneous administration of vaccines [15]. It is also possible to inject chitosan as a part of medicinal products and vaccines [21].

The available literature provides information on chitosan as an antigen sorbent and a stimulator of the post-vaccination immune response [3][7][8][9][10][12][13][14][17][18][19][20][22]. Progress made in studying the properties of chitosan derivatives suggests that they may meet the basic requirements for modern adjuvants.

Previously, in order to find optimal concentrations of chitosan-based products for administration to animals, we analyzed the cytotoxicity of various dilutions of the tested samples in the continuous bovine kidney cell line PT-80 [6][11].

The purpose of this research is to study adjuvant properties of chitosan derivatives in various concentrations, administered to mice together with an anti-rabies vaccine.

Chitosan. The following chitosan-containing products manufactured by Bioprogress (Shchelkovo, Russia) on the basis of saline solution (NaCl 0.9%), were taken as starting materials:

– water-soluble chitosan (succinate), 2% solution – Preparation No. 1;

– edible chitosan (water-soluble), 2% solution – Preparation No. 2;

– edible chitosan (acid-soluble), 2% solution – Preparation No. 3.

Anti-rabies vaccines for animals:

– anti-rabies vaccine RABIKOV manufactured by the Shchelkovo Biocombinat (Russia);

– anti-rabies vaccine RABIES (Intervet International, B. V., the Netherlands) was used to compare protective properties with the domestically produced RABIKOV vaccine as part of the implementation of import substitution measures.

Experiment design. Тhirty-seven homogeneous groups of female white lab mice were formed for experimental purposes. The mice weighed 21–35 g and were 85–100 days old. Six mice were included in each group. The mice were injected with the tested products (at a volume of 0.3 cm³), according to the scheme given in the table.

Before the experiment, all the laboratory animals stayed in quarantine for 14 days. The experimental mice were subjected to daily clinical examination and control weighing. On day 28 postadministration, the animals were decapitated and pathological material (organs and blood serum) was taken for further tests.

All animal experiments were conducted in strict compliance with Interstate Standards GOST 33215-2014, GOST 33216-2014 as adopted by the Interstate Council for Standardization, Metrology and Certification, as well as in accordance with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.

The mass index of organs is calculated as the ratio of the organs mass to animal body mass (organ mass index = morgan/manimal).

Calculating titers of rabies virus-neutralizing antibodies. In order to observe adjuvant properties of chitosan-based preparations, we measured the level of rabies virus-neutralizing antibodies in sera from laboratory animals using diffusion precipitation test, according to the instruction for “Diffusion precipitation test kit for rabies diagnosis” produced by the All-Russian Research and Technological Institute of Biological Industry (Russia).

Processing statistics. The obtained statistics were processed using the standard Microsoft Excel 2007 program adopted in biology and medicine. The results were considered reliable at the probability level of p < 0.05.

Preliminary tests in PT-80 cell line have shown that edible chitosan (acid-soluble) at a concentration of 1:4 has a cytotoxic effect [6], therefore, during the experiment 1:64 and 1:10⁸ concentrations were used in the lab animals.

During the observation, no deviations in mice behavior were reported, and there were no specific death cases. Further autopsy revealed no pathological lesions at the injection sites (inflammation, granulation, etc.) or in the internal organs [6].

The liver is an organ that increases in size due to acute toxicity and decreases in size due to chronic toxicity. Therefore, the organ mass index was calculated.

Figure 1 shows that the mean liver mass index in all mice groups ranged from 0.045 to 0.050. This suggests that the tested preparations do not have any acute toxicity effects.

The spleen is the largest lymphoid organ. Therefore, to assess the immunity response to the administration of the tested preparations, the organ mass index was calculated for experimental mice.

The mean spleen mass index calculated (Fig. 2) for groups 1–8 and 10–16, either corresponded to the data obtained for groups 19, 20 and 37, or exceeded them (p ≤ 0.05). This may suggest that the mice’s immune system is stimulated by the chitosan-containing preparations. However, in group No. 9, the mean spleen mass index was lower than in the control groups. It can be assumed that intramuscularly administered edible chitosan (water-soluble) at a concentration of 1:64 does not have any pronounced immunostimulating effect.

To confirm chitosan adjuvant properties, the next step was to measure the level of rabies virus-neutralizing antibodies in the sera from laboratory mice using diffusion precipitation test. The experiment results are given in Figure 3.

The data obtained show that the titres of rabies virus-neutralizing antibodies post-vaccination without the tested preparations (groups No. 19 and 20) were 1:32; whereas those groups that were subcutaneously vaccinated together with water-soluble chitosan (succinate) in all the tested concentrations (groups No. 2, 4 and 6) showed the antibody level of 1:64. Regardless of the administration route, edible chitosan (acid-soluble) at all the tested concentrations (groups No. 13–16) and edible chitosan (water-soluble) at a concentration of 1:10⁸ (groups No. 11 and 12) stimulate antibody production.

Water-soluble chitosan (succinate) was administered intramuscularly at all the tested concentrations (groups No. 1, 3 and 5), edible chitosan (water-soluble) was administered subcutaneously (concentration 1:4, group No. 8) and intramuscularly (concentration 1:64, group No. 9) reduced the level of rabies virus-neutralizing antibodies down to 1:8 – 1:16. In this regard, it can be assumed that the tested preparations (at the given concentrations and administered using the mentioned routes) suppress the immune response, since natural chitosan salts are practically insoluble at pH above 6, which may be problematic for the delivery of vaccine antigens that are soluble and stable at neutral pH or higher [2].

Thus, it has been shown that the tested chitosan-containing preparations do not have a negative impact on the laboratory animals and have immunogenic properties.

The following preparations can be recommended as affordable adjuvants: water-soluble chitosan (succinate) for subcutaneous administration; edible chitosan (acid-soluble) at a concentration of 1:64 and above, as well as edible chitosan (water-soluble) at a concentration of 1:10⁸ (administered subcutaneously and intramuscularly). Alongside it, our test results as well as results provided by other researchers [2] show that water-soluble chitosan (succinate) administered intramuscularly and edible chitosan (water-soluble) administered subcutaneously at a concentrations of 1:4 and administered intramuscularly at a concentration of 1:64 reduce the vaccine efficacy.

Contribution: Dobroskok K. B. – has conducted the experiment, processed resulting statistics, created tables and diagrams, is an author of the article; Yarygina E. I. – a scientific advisor, responsible for experiment design, has conducted the experiment, is an author of the article; Lipatova M. S. – responsible for selection and analysis of the relevant literature, processed resulting statistics, created tables and diagrams; Kalmykova M. S. – responsible for selection and analysis of the relevant literature; is an author of the article.

Вклад авторов: Доброскок К. Б. – проведение исследований, статистическая обработка результатов, составление таблиц и диаграмм, подготовка текста статьи; Ярыгина Е. И. – научное руководство, планирование опытов, проведение исследований, подготовка текста статьи; Липатова М. С. – подбор и анализ литературных источников по теме, статистическая обработка результатов, составление таблиц и диаграмм; Калмыкова М. С. – подбор и анализ литературных источников по теме, подготовка текста статьи.