Preventive efficacy of preparations of vesicles of external membranes of atoxygenic strains of Vibrio cholerae О1 serogroup

Introduction

All Gram-negative bacteria produce outer membrane vesicles (OMVs), spherical structures with a diameter of 20 to 300 nm [1][2]. It has been established that molecules of protein and lipopolysaccharide nature located on the outer membrane of microbes are targets for components of innate (nonspecific) immunity, as well as exhibit the properties of protective antigens and participate in the formation of specific antibacterial immunity [3]. Therefore, studies on the immunogenic and protective properties of vesicles of outer membranes of various pathogens are actively carried out all over the world. To prevent meningococcal infection caused by Neisseria meningitidis serogroup B, the vaccine Bexsero (Italy), consisting of OMVs, has been licensed and is successfully used in clinical practice [3]. The publications provide evidence of the formation of effective immunity to various bacterial infections when using vaccine preparations based on vesicles of outer membranes of Escherichia coli [4], Neisseria gonorrhoeae [5], Burkholderia pseudomallei [6], Salmonella typhimurium [7], Helicobacter pylori [8], Neisseria meningitidis [9], Brucella abortus [10], Klebsiella pneumoniae [11], Bordetella pertussis [12], etc.

It has been shown that vesicular vaccines have a number of important advantages: they are safe, but have the properties of a whole bacterial cell, the antigens on their surface are in the native state. In addition, they do not require a cold chain and buffer solution, which makes their use economically favorable and promising [13].

Like all Gram-negative bacteria, cholera vibrios are capable of secreting OMVs, spherical-shaped formations of 20 to 200 nm that transport various virulence factors of the pathogen, including cholera toxin (CT), to the epithelial cells of the small intestine [14–16].

The effectiveness of using cholera vibrio OMVs as a means of specific prevention of cholera is evidenced by experiments conducted by foreign scientists. Thus, Schild et al. [17] studied mice born from mothers immunized with preparations of OMVs of toxigenic strains of Vibrio cholerae E7946 O1 El Tor Inaba and V. cholerae O395 O1 Ogawa. They revealed the presence of anticholera immunoglobulins (Ig) of class G in an amount sufficient to fully protect newborns from cholera vibrio colonization, which indicated the possibility of passive humoral immunity to the cholera pathogen. At the same time, specific IgA and IgG1 were registered in the feces of immunized females, confirming the induction of immune response in the gastrointestinal tract. Interestingly, the inclusion of a heterologous antigen (periplasmic alkaline phosphatase of E. coli) in cholera vibrio OMVs and subsequent intranasal immunization with them induced a specific immune response to the heterologous antigen in mice. These findings indicate the potential use of vesicles for the delivery of various antigens in the development of new vaccines [17]. Roy et al. [18] found that V. cholerae OMVs were significantly less reactogenic than live and heat-killed bacteria, and oral immunization with such preparations could induce long-term immunity. Bishop et al. [19] found that immunization of mice with vesicles of outer membranes of cholera vibrio O1 serogroup Inaba or Ogawa provided protection of the small intestine of animals from colonization by V. cholerae O1 of both serotypes. The same authors showed that OMVs of V. cholerae O1 or O139 serogroups did not provide cross-serogroup protection, but the use of a mixture of OMVs isolated from cholera vibrios of O1 and O139 serogroups provided protection of animals from infection with these strains. Other scientists found [20] that the administration of OMVs of V. cholerae or enterotoxigenic E. coli (ETEC) induced the formation of species-specific immunity, and the combination of vesicles of these bacteria induced a high immune response to both pathogens.

Sinha et al. [21] created an experimental cholera vaccine based on cholera pentavalent outer membrane vesicles (CPMVs) by mixing vesicles of the outer membranes of five virulent strains of V. cholerae. It was shown that four-fold oral immunization with CPMVs induced specific B- and T-cell responses, production of serum anticholera antibodies of G, A, M, sIgA class on mucous membranes, and secretion of cytokines characteristic of Th2 and Th17 immune responses in mice. Adult mice immunized with this vaccine and their offspring were protected against infection with virulent strains of V. cholerae.

Sedaghat et al. [22] demonstrated that oral vaccination of mice with OMVs, especially in combination with a clinical strain of V. cholerae isolated from a cholera patient (WC-OMVs), provided protection against infection with virulent strains of cholera. According to the authors, the greatest effectiveness of WC-OMVs may be associated either with an increase in the number of protective antigens or with the adjuvant properties of the vesicles.

Thus, all of the above indicates the possibility of using OMVs for specific prophylaxis of cholera. However, experimental developments concerning the creation of vaccines based on cholera vibrio OMVs were carried out using virulent strains of V. cholerae. Working with such strains requires a special regime with enhanced safety measures, which makes it difficult to obtain vaccine preparations. In addition, vesicles obtained from toxigenic strains may contain CT, the main pathogenicity factor of the cholera pathogen. It was shown that the majority of CT is secreted in an OMV-associated form and is located exclusively inside vesicles [16]. Unlike free CT secreted through the type II secretion system, OMV-associated CT is protected from degradation by intestinal proteases, which allows it to remain in the intestinal tract longer and retain toxicity [15].

The aim of the study was to evaluate the efficacy and feasibility of using vesicles of outer membranes of atoxigenic strains of V. cholerae O1 serogroup for the prevention of experimental cholera.

Materials and Methods

V. cholerae O1 El Tor 18950 (stxAV^-tcpA^-), 18780 (stxAV^-tcpA⁺) strains isolated from surface water bodies were used to obtain vesicle preparations. Cultures were grown at 37 ˚C for 18 h on Marten agar (pH 7.6). From the daily agar culture, a 1 bln suspension was made using a turbidity standard in 3 ml of a physiological saline solution (pH 7.8), and 0.5 ml of the suspension was inoculated into 25 ml of LB broth. The strains were cultured with additional aeration (shaking) at 120–130 oscillations per minute on an Incubator Shaker (Incubator Shaker, series 25, USA) for 18 hours at 37 ˚C. From the grown culture, 0.5 ml of each was seeded into four 250 ml bottles of the LB medium and grown for 18 hours with aeration.

After disinfection with sodium merthiolate 1:10,000, the culture grown in nutrient liquid medium was centrifuged at 6000 rpm for 25 minutes in cold on a centrifuge (Weckman, model J2-21, USA). The resulting supernatants were alternately filtered through membranes with pore diameters of 0.45 μm and 0.22 μm. The preparations were concentrated 10-fold on an ultrafiltration unit (Amicon) followed by the centrifugation of samples at 45,000g for 2 hours. For further study, the precipitate was dissolved in 500 μl of a physiological solution (pH 7.8). The amount of protein in the obtained preparations was 400–500 μg/mL. Protein detection was performed on a device (Bio Rad). The preparations were stored at -20 ˚C.

The presence of vesicles in the preparations was confirmed by transmission electron microscopy using a Jeol JEM 1011 electron digital microscope. All manipulations were performed according to MU 1.3.3103-13 [23]. The presence of spherical vesicles with sizes of about 40–200 nm was detected.

Assessment of the specific sterility of preparations by the bacteriological method showed their biological safety.

The proteins of vesicle preparations were analyzed by PAGE electrophoresis in the presence of SDS according to the method of Laemmli [24]. The presence of lipopolysaccharide (LPS) in the vesicle preparations was determined by the direct enzyme-linked immunosorbent assay (ELISA), which was performed by the conventional method in 96-well flat-bottom serological plates (Costar) [25].

The protective ability of the obtained OMVs was evaluated on the model of white mice (16–20 g at the age of 6–10 weeks) and on the model of adult rabbits (1.5–2 kg). When working with experimental animals, the authors were guided by international principles of the “European Convention for the Protection of Vertebrate Animals Used for Experiments and Other Scientific Purposes” ETS N 123 (Strasbourg, 1986) and the Decree of the Ministry of Health of the Russian Federation from April 1, 2016 No. 199N “Approval of the Rules of Good Laboratory Practice”. All experiments received a favorable opinion from the Bioethics Committee of the Federal Clinical Institution of Rostov-on-Don Plague Control Institute of Rospotrebnadzor, Protocol of the meeting No. 2 dated January 17, 2022.

White mice were immunized parenterally (intramuscularly) once and twice (seven days apart) with the obtained preparations of OMVs at doses of 2.5 and 5 μg of protein. Control animals were injected with a physiological saline solution in the same volume (0.2 ml) and according to the same scheme. Two weeks after the last immunization, a generalized form of cholera was induced in experimental and control animals [26]. The results of the experiment were taken into account only when all control animals died within a day.

The protective effect of vesicle preparations was also evaluated on the model of adult rabbits. Animals were immunized parenterally (intramuscularly) once and twice (seven days apart) with vesicle preparations of 150 µg of protein in a volume of 0.5 ml. Control rabbits were injected with a physiologic saline solution in the same volume and according to the same scheme. The protective activity of the preparations was studied using a model of a ligated loop of the small intestine of adult rabbits [27]. The development of experimental cholera was registered by the presence of cholerogenic and enteropathogenic effects in the experimental loop of the small intestine. The enteropathogenic effect was evidenced by the development of edema of the mucosa and submucosa, hemorrhage and necrosis of the covering epithelium of the intestinal villi, and the cholerogenic effect was evidenced by the distended experimental loop (as a result of exudate of tissue fluid and electrolytes into the intestinal lumen). The presence/absence of the cholerogenic effect was determined by calculating the loop distension coefficient using the formula

where K>1.0 indicated the presence of the cholerogenic effect.

The obtained results were analyzed using Microsoft Excel 2010 and StatSoft Statistica Windows 10.01. Experiments were carried out in three replications. Then, the values of confidence intervals of the arithmetic mean (M) were determined for the level of significance (P) of 95%. A comparison of populations by qualitative characteristics was carried out using Fisher’s test. Differences were considered significant at p<0.05.

Results

The presence of the main protective antigens (proteins and LPS) on the membrane of isolated vesicles was assessed. It was shown that the preparations obtained from V. cholerae strains O1 18950 and 18780 contained protein bands at the level of molecular weight markers of 40 and 45 kDa in the first preparation and 38 and 40 kDa in the second, which corresponded to the molecular weight of outer membrane proteins belonging to the omptine family (porin outer membrane proteins OmpT, OmpU). Besides, a minor band around 66.2 kDa was detected in these two preparations. The presence of LPS, according to the ELISA results, was confirmed in all tested vesicle preparations.

When studying the protective properties of the obtained vesicle preparations in the test of active protection of mice, it was revealed that a single immunization with OMVs V. cholerae O1 El Tor 18950 and V. cholerae O1 El Tor 18780 insufficiently protected animals from death after infection with virulent strains of cholera both when using an immunizing dose of 2.5 μg and 5 μg (Table 1).

Double immunization with V. cholerae vesicles at a dose of 2.5 μg significantly increased the protective efficacy of the preparations, preventing the death of about 50% of animals in the group receiving vesicles of V. cholerae strain O1 El Tor 18950 and about 45% of mice immunized with the preparation of OMVs V. cholerae O1 El Tor 18780.

The best results in this experimental model were obtained by the authors when the drugs were administered twice at a dose of 5 μg. This scheme ensured the survival of 100% of white mice from the group primed with V. cholerae OMVs O1 El Tor 18950 and practically all animals vaccinated with V. cholerae O1 El Tor 18780 vesicles (Table 1).

Analysis of the results of the prophylactic efficacy of the studied vesicles of V. cholerae O1 El Tor 18950 and V. cholerae O1 El Tor 18780 on the model of adult rabbits showed that a single administration of the preparations at a dose of 150 µg did not protect the animals from the development of cholera symptoms in the small intestine, just as in the case of a single application of the preparations in the modeling of the generalized form of cholera in mice.

The enhancement of protective activity was registered by the authors only after double immunization of animals. In rabbits of the experimental groups in the experimental and control isolated loops of the small intestine, there were no enteropathogenic and cholerogenic effects (K<1.0). At the same time, in animals of the control group, experimental loops were distended with fluid (cholerogenic effect) and swelling of deeper lying tissues, hemorrhages, and necrosis of the covering epithelium of villi (enteropathogenic effect) were observed (Table 2).

Discussion

The results of the experiments show that vesicles isolated from atoxigenic strains of cholera vibrio have protective properties and protect animals from the development of experimental cholera. The highest efficacy of the obtained preparations in the studied doses is shown by double parenteral administration, preventing the death of all white mice taken in the experiment and the development of pathogenetic processes in the small intestine of adult rabbits.

The obtained data are confirmed by the authors’ previous studies on the protective activity of outer membranes of cholera vibrio isolated from atoxigenic strains of V. cholerae. The presence of immunogenic and protective properties of cholera vibrio outer membranes isolated by sparing methods from the V. cholerae El tor 18950 strain (ctx^-, tcp^-, OmpT⁺, OmpU^-, OmpW⁺) was revealed. Double immunization of white mice (10 µg each) and adult rabbits (700 µg each) with this preparation prevented the development of cholera in experimental animals infected with virulent strains of the pathogen, and this effect persisted up to five months of the postvaccinal period (observation period) [28]. It was shown that the protective ability of V. cholerae O1 El Tor 18950 strain was associated with the increased production of OmpT protein on its membrane, which is highly immunogenic and protective [29]. The OMVs obtained in this work, including those from this strain, had a good protective effect at much lower doses (5 μg/mouse and 150 μg/rabbit). This indicates the preservation of the native structure of the main protective antigens (LPS and outer membrane proteins, which are part of the vesicles [13]) and provides them with greater protective ability compared to outer membrane preparations.

In contrast to foreign authors who received experimental preparations of vesicles from virulent strains of cholera vibrio [17–22], which requires special safety measures and control of the toxicity of preparations due to the presence of CT as the main pathogenicity factor of cholera vibrio, in the present study, OMVs were isolated from avirulent (atoxigenic) strains of cholera vibrio, and their protective effect was shown in experimental cholera. The obtained results indicate the possibility of using these vesicles for specific prophylaxis of cholera and may be useful in the development of new vaccine preparations against this infection.

Conclusion

1. OMVs isolated from cultures of atoxigenic strains of cholerae O1 El Tor 18950 and V. cholerae O1 El Tor 18780 have protective activity against toxigenic cholera vibrio O1 serogroup.
2. When vesicles are used, the immunizing dose and the frequency of administration are important.
3. Double intramuscular administration of OMVs (for white mice at a dose of 5 μg, and for adult rabbits at a dose of 150 μg) prevented the development of experimental cholera in all animals taken in the experiment.
4. The obtained data indicate the possibility and prospects of using these drugs to improve the specific prophylaxis of cholera.