Cell Envelopes of Francisella tularensis: Immunogenic Activity and Toxicity

At present vaccinal prevention as a priority system of measures aimed at the warning, restriction of distribution and elimination of especially dangerous infectious diseases is of special importance. Live attenuated vaccines developed and introduced in practice still in the XX century including tularemia vaccine are used as vaccinal preparations for specific prophylaxis of these illnesses. In this connection development of new generation vac-cines possessing high immunological and epidemiological efficiency is an actual direction of researches.

The basic antigen determinants are located on surface of Francisella tularensis bacterial cells. So, one of F. tularensis immunodominant antigens is Lipopolysaccharide (LPS). However, there are contradicting data in the literature concerning properties of F. tularensis external membrane components that can be explained by various methods of its isolation and investigation conditions. There are data that F. tularensis LPS does not stimulate immunocompetent cells [1]. Nevertheless, other researchers established activation of mice immunocompetent cells by F. tularensis LPS in case of its dose increase in comparison with standard preparations [2,3]. A number of authors demonstrate that F. tularensis outer membrane proteins possess immunological activity [4,5], more than 30 immunoreactive tularemia agent antigens are included into its composition. It is known that F. tularensis outer membrane proteins influence on specificity of macroorganism immune response, possess unique polyepitope antigenic structure and are considered as perspective components in engineering of subunit chemical vaccines.

The aforesaid has formed the basis for isolation and comprehensive studying of F. tularensis cell envelopes as a perspective component in vaccine developments [6,7].

The aim of this investigation included the detection of toxicity of the resulted CE F. tularensis different subspe-cies and its immunizing dose with subsequent estimation of the functional activity of experimental animal blood cells under the influence of these preparations in vivo and in vitro.

Cell envelopes of F. tularensis: Cell Envelopes (CE) prepared by lysis of live cells from 6 F. tularensisstrains: F. tularensis subsp. novicida Utah 112 [6], F. tularensis subsp. mediasiatica ?-61, F. tularensissubsp. tularensis B-399 A-Cole, F. tularensis subsp. holarctica21/400, F. tularensis subsp. holarctica306, F. tularensis subsp. holarctica15 NIIEG from the collection of Irkutsk Antiplague Research Institute were used in this work.

Experimental animals: The tests were performed using adult certificated outbred white mice (from Scien-tific Production Association “Vektor”, Novosibirsk city) both sexes, weighing 22-24 g. Manipulations with experimental animals carried out in concordance with the international principles of the Helsinki declara-tion concerning to the humane relation to animals.

Immunizing Dose (ID) and protective activity: Immunizing Dose (ID) ? protective activity of CE was detected using 114 white mice. Animals were divided into six experimental groups in 18 white mice in each group and one control in 6 mice. F. tularensis CE preparations were injected subcutaneously into the right leg in volume 0.5 ml in three doses: 3.8; 19 and 95 µg (at recalculation to protein). Control animals were in-jected the same volume of NaCl isotonic solution, ?? 7.2. At 28 days after immunization white mice were injected subcutaneously a bacterial suspension of high virulent F. tularensis subsp. holarctica306 (Dcl 1) strain in dose of 100 CFU per an animal.

Toxicity of CE F. tularensis: Toxicity of CE F. tularensis different subspecies was determined in 125 white mice as experimental models. Animals were divided into 24 experimental and one control groups (5 mice in each group). White mice were immunized once subcutaneously with CE preparations of F. tularen-sis different subspecies in the right leg in volume of 0.5 ml in a dose 6.3; 19.0; 57.0 and 100 µg (at recalcu-lation to protein). Control animals were injected the same volume of NaCl isotonic solution, ?? 7.2.

Flow cytometry: Functional activity of blood cells was examined by a flow cytometry method in vivo and in vitro. Total 120 white mice were used in vitro experiments. Leukocytes isolated from heparinized blood using 3 % gelatin were tested. Cells in concentration of 10⁶ (50 µl) were incubated during 60 min with CE F. tularensis preparations in two doses (19 and 95 µg to protein). Immunogenic activity of CE F. tularensiswas examined in 60 white mice in vivo. In this case the animals were immunized with CE F. tularensispreparations in dose 95 µg/0.2 ml of buffered physiological solution per an animal. Blood sampling was performed in 3 days after injection of the experimental preparations. The phenotype of white mice blood cells was defined using monoclonal antibodies by Becton Dickinson Company (USA) in the following pan-el: CD45-APC/CD3-FITC/CD4-Alexa-700/CD8-APC-Cy7/CD25-PE-Cy7/Annexin-PE/7-AAD. Stained samples were analyzed on flow cytofluorometer BD FACSCanto ™ II. Total activated T-lymphocytes (CD3⁺CD25⁺), activated T-helpers (CD3⁺CD4⁺CD25⁺) and activated B-lymphocytes (CD3^-CD19⁺CD25⁺) were detected. Also viability of the cell populations by percentage of live cells (?nV^-7^-AAD^-), necrotic cells (?nV^-7^-AAD⁺), cells at the stage of early (?nV⁺7^-AAD^-) and late (?nV⁺7^-AAD⁺) apoptosis was estimated in vitro.

Statistical analysis: Statistical data were processed using parametrical t-criterion with the Bonferroni ad-justment. P < 0.05 was considered significant.

It was shown that cell envelope preparations of F. tularensis different subspecies in doses 6.3; 19.0; 57.0 and 100 µg were non-toxic for experimental animals (LD₅₀ is not determined). Dissection at the place of the preparation injections revealed no alterations in kidneys, adrenal glands, lungs, heart and other organs. At the same time, alterations in spleen and lymph nodes were observed in case of F. tularensissubsp. holarctica15 NIIEG, CE F. tularensis subsp. novicida Utah 112, F. tularensis subsp. tularensis B-399 A-Cole and F. tularensis subsp. mediasiatica ?-61 injections indicating immune reorganization of organism.

Experiments for ID (Table 1) detection of the tested preparations showed that CE F. tularensis subsp. holarctica306, F. tularensis subsp. mediasiatica ?-61 and F. tularensis subsp. tularensis B-399 A-Cole in dose of 95 µg protected experimental animals in the infectious process caused by high virulent F. tularensis subsp. holarctica306 strain. Thus only in case of CE F. tularensis subsp. mediasiatica ?-61 application the survival rate of the animals was 83 %, CE F. tularensis subsp. holarctica306 - 33 %, CE F. tularensis subsp. tularensis B-399 A-Cole - 17 %.

Table 1: Immunizing dose and protective activity of cell envelopes after F. tularensis subsp. holarctica306 subcutaneous injection to experimental animals.

The results demonstrated that the experimental F. tularensis preparations in doses of 19 and 95 µg/10⁶leuko-cytes in vitro failed to affect statistically on the expression level of early activation marker (CD25) of T- and B-lymphocytes. Preparations of CE F. tularensis different subspecies in these doses did not influence on necrosis and apoptosis processes in experimental animal blood cells.

Experiments in vivo showed that statistically significant (P < 0.05) increase of activated B-lymphocytes in com-parison with the controls (2.6 ± 0.4 %) was registered in 3 days after white mice immunization with CE F. tula-rensis subsp. mediasiatica ?-61 (4.1 ± 0.6 %) and F. tularensis subsp. tularensis B-399 A-Cole (3.4 ± 0.8 %). At the same time CE F. tularensis subsp. holarctica15 NIIEG and F. tularensis subsp. holarctica306 promoted the percentage increase of T- lymphocytes in 1.2 times (Table 2).

It is known that macrophages are important in development of the infectious process caused by F. tularensis being both a primary target and also a site of bacteria localization and reproduction. Activation of intracellular signal pathways of immunocompetent cells causing the secretion of cytokines and chemokines is mediated by interaction of microbic components with pattern-discriminating macrophage receptors. So, Bacterial Lipopep-tide (BLP), lipoproteins, Lipopolysaccharide (LPS) and also many other proteins (FopA, FopC, TUL4, FslE, FmvB and others) are located on surface of bacterial cells, in particular F. tularensis. Due to outer membrane proteins possess higher immunogenic activity in comparison with LPS [4], studying of CE F. tularensis is a fundamental basis for interpretation of intracellular survival mechanisms, interaction of bacteria with macroor-ganism cells and a perspective direction in search of components for development of tularemia vaccine.

The conducted examinations demonstrated that CE from F. tularensis subsp. novicida Utah 112, F. tularensis subsp. mediasiatica ?-61, F. tularensis subsp. tularensis B-399 A-Cole, F. tularensis subsp. holarctica21/400, F. tularensis subsp. holarctica306, F. tularensis subsp. holarctica15 NIIEG strains in doses 6.3; 19.0; 57.0 and 100 µg were non-toxic for experimental animals. This fact was confirmed by the results of pathomorphological analysis of white mice organs immunized with these preparations and also by the indicators of blood cell viability at interaction with CE F. tularensis in vitro.

The immunizing dose (95 µg) of F. tularensis subsp. holarctica306, F. tularensis subsp. mediasiatica ?-61 and F. tularensis subsp. tularensis B-399 A-Cole protecting white mice from experimental tularemia infection was established.

There are literature data that the preparations containing F. tularensis external membrane proteins possess im-munological activity in 19 µg dose [4,5]. In this connection CE F. tularensis in two doses: 19 and 95 µg was used in our experiments to estimate functional activity of blood cells of the laboratory animals by flow cytome-try method. It was experimentally demonstrated that CE F. tularensispreparations rendered multidirectional influence on activation of T- and B-lymphocytes of white mice blood cells. So, CE F. tularensis subsp. media-siatica ?-61 and F. tularensis subsp. tularensis B-399 A-Cole preparations possessing immunogenic activity (the survival rate was 83 % and 17 % of animals, respectively) resulted in increase of CD3^-CD19⁺CD25⁺-lymphocyte percentage, and F. tularensis subsp. holarctica306 CE (33 %) - CD3⁺CD25⁺- lymphocyte.

The distinctions in protective activity of the preparations and their ability to increase functional activity of white mice lymphocytes determined during the examination can be possibly caused by nucleotide polymorphism of the genes coding outer membrane proteins [8], and variability of LPS structure [9,10] in F. tularensis subspecies that undoubtedly affects on the bacteria virulence.

Formerly we determined that CE F. tularensis stimulated IL-1β, TNF-α, IL-2, GM-CSF and G-CSF production by immunocompetent cells of the experimental aniimmunocompetent cells of the experimental animals in vivo [6,7].

On the basis of the conducted investigations it is possible to conclude that CE preparations of F. tularensissubsp. holarctica306, F. tularensis subsp. mediasiatica ?-61 and F. tularensis subsp. tularensis B-399 A-Cole possess protective activity, ability to increase functional activity of blood lymphocytes and stimulate production of proinflammatory cytokines by white mice immunocompetent cells. At the same time the further complex research of immunogenic properties of these preparations as perspective components in development of tularemia vaccines is required.

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