INTRODUCTION:

Salmonella typhi bacteria, which causes typhoid fever, is a global health issue, particularly in underdeveloped nations^1,2. Various treatments have been widely used, ranging from generic drugs, antibiotics, vaccines, to traditional herbal medicines^3,4,5,6. Due to the development of antibiotic resistance mechanisms in these gram-negative bacteria, the current existing vaccinations are no longer effective^7,8,9,10,11. The currently approved vaccine against S. typhi for humans is treated with the following vaccines: (i) Vi polysaccharide capsule-based vaccine; (ii) live attenuated oral vaccination; and (iii) killed whole-cell parental vaccine¹².

The mentioned vaccines rather engender certain immune responses. However, some limitations are still an issue. Although the killed whole-cell parenteral vaccine is safe for immunocompromised individuals and is cold-chain independent, it still has a drawback. Such a whole-cell vaccine often causes local reactions^13,14. The live attenuated oral vaccine strain Ty21a is tolerant (low reactogenicity), low side effect rate, and has cross-protection with S. Paratyphi. The impediments to the development of live-oral vaccines are: some salmonellosis is caused by various serovars and strains¹⁵, and this vaccine cannot be administered to children under 6 years old; in individuals undergoing antibiotic therapy or individuals using steroids and undergoing radiation treatment, a booster dose (repeat dose) is required every 5 years for groups of individuals who are at risk, and there is a possibility of reverse virulence¹⁶. Vi polysaccharide vaccine (Typhim Vi; Aventis) is a Vi antigen-based vaccine, suitable for adults and children over the age of 2 years. However, because this vaccine is a T-independent polysaccharide (no immunologic memory), it can cause a low response in infants and groups of individuals with a high burden of disease¹⁷.

Vaccine subunits contain only the antigenic portion of the pathogen required to elicit a protective immune response¹¹. Like inactivated whole-cell vaccines, subunit vaccines do not contain any living parts of the pathogen and are therefore considered to be very safe and stable. Systemic and local side effects are two to four times less likely to occur with subunit vaccines than with whole-cell vaccines. Side effects are also much less severe with acellular vaccines^13,14.

This study uses three kinds of protein subunit combinations as antigens: outer membrane protein (OMP), fimbriae, and flagellin. Based on previous research, it has been found that Salmonella typhi bacteria's OMP adhesin protein with a molecular weight of 36 kDa (later named AdhO36 protein) contains an immunogenic potential adhesin molecule. Therefore, it can be used as a component of mucosal vaccines to prevent infectious diseases¹⁹. A potential bacterial adhesion mechanism model has been developed into an anti-adhesion vaccine. It is expected to inhibit bacterial colonization and infection^20,21.

Fimbriae on the surface of bacterial cells is a target for antibodies to inhibit bacterial attachment or interaction with the host^20,21. A study by Santoso (2002) explained that the Salmonella typhi strain isolated from typhoid fever patients was known to have fimbrial adhesion (FA) which was classified as fimbriae type-1 with a BM of 36 kDa, and was named AdhF36. When colonization takes place, the AdhF36 protein functions as a virulence factor in the adhesion process. When utilized as a vaccine candidate, it is intended that it will capable of suppressing the initial phases of bacterial adhesion activity. The research also revealed that the AdhF36 protein can prevent mouse enterocyte cells from adhering to one another and possesses immunogenic properties²².

The movement and chemotaxis of bacterial pathogens as well as the invasion of epithelial cells as a virulence factor depend on the presence of bacterial flagella, which are appendages on the cell surface. Many enteric bacteria release flagellin²³, an important component of the flagella, which when it reaches the basolateral membrane of the intestinal epithelium stimulates innate immune signaling pathways mediated by Toll-like receptor 5. According to our hypotheses, any flagellin that penetrates the epithelium and enters the body might also control cells of the immune system²⁴. The vaccination serves its purpose in this situation²⁵.

This study's objective was to evaluate the protein subunit of the Salmonella typhi antigen's immunogenic potential in boosting cellular and mucosal immune responses, with the hope of using it as a viable typhoid fever vaccine candidate.

MATERIALS AND METHODS:

Population and samples:

All experimental animals are obtained from the Experimental Animal Research Laboratory (LPHC) Faculty of Medicine, Universitas Brawijaya with a total of 36 Balb/C mice (female, BW: 25 grams, age: 8 weeks). The animal grouping was classified as detailed in table 1. Before treatment, each mouse was given 0.3 ml of 0.2 M sodium bicarbonate solution. All of these treatments were given orally.

The dose (250 µg/injection) for subunit administration was obtained from previous research²⁶ (based on their protein adhesion inhibition test). Immunization was given 4 times a week (on days 0, 7, and 14). The mice were sacrificed in the 4th week. The mucus was taken for SIgA measurement, and the spleen was taken for CD4⁺ and CD8⁺ analysis.

Table 1. Animal grouping classification

	OMP (250 μg/0.3 ml PBS)	Fimbriae (250 μg/0.3 ml PBS)	Flagellin (250 μg/0.3 ml PBS)	ISCOM (adjuvant, 7 μg/0.3 ml PBS)
Control	-	-	-	-
ISCOM	-	-	-	✓
OMP	✓	-	-	✓
Fimbriae	-	✓	-	✓
Flagellin	-	-	✓	✓

Salmonella typhi:

Salmonella typhi was collected from typhoid fever patients' clinical specimens at Dr. Saiful Anwar Hospital, Malang. For 18 to 24 hours, the bacteria were cultured at 37 °C on a biphasic medium (MH agar ⁺ BHI broth). Gram-staining was performed to identify colonies that had grown, and the colonies were then cultivated on MacConkey differential media with BSA selective medium for 18 to 24 hours at 37 °C²⁷. Oxidase levels in the developing colonies on MacConkey media were measured. The Microbact system also carried out biochemical identification to identify the bacterial species.

Once S. typhi was properly defined, the bacterial isolates were grown on MacConkey medium and kept at 37 °C for 18 to 24 hours. After that, cultures from MacConkey's medium were moved to a biphasic medium composed of TCG slanted agar medium and BHI liquid medium, which was incubated at 37 °C for 24 hours^27,28.

Subunits preparation:

Omp:

It was carried out using the previous method²⁶. The bacterial cells were separated from the pili parts (using a pili cutter), then OMP was extracted using Chaps detergent. After dialysis using PBS pH 7.4 OMP crude electrophoresis (SDS-PAGE). The visible band at a position of about 36 kDa was cut and collected. The gel pieces were then subjected to an electroelution process to obtain pure protein AdhO36.

Fimbriae:

The liquid culture from the biphasic medium (BHI) was transferred into a 100 cc centrifuge tube, added TCA so that the concentration was 3%, then rotated in a cold centrifuge (Biofuge 28 RS, Heraeus Sepatech) at 4 °C 6000 rpm for 15 minutes. The fimbriae were cut using a modified omni-mixer at 4 °C after the precipitate was suspended with enough PBS pH 7.4²⁹. A centrifugation in cold conditions was carried out at 12,000 rpm for 15 min. The filtrate is separated (containing fimbriae). The precipitate is sufficiently suspended with PBS pH 7.4, and the fimbriae are then cut once more. This process was repeated several times and was stopped after a clear difference in protein pattern was seen between the fimbriae fraction and the cell fraction (electrophoresis was performed). From this procedure, a filtrate containing fimbriae protein and a precipitate that is part of bacterial cells was obtained²⁸.

The fimbriae fraction was collected. The dialysis process was carried out using PBS pH 7.4 solution to remove the remaining TCA. Furthermore, the dialysate was precipitated with 35% ammonium sulfate, rotated at 4 °C 6000 rpm, the filtrate was removed, the precipitate was suspended with sufficient PBS, and dialysis was carried out again. The result of dialysate is the fimbriae fraction.

For the purification of protein subunits, a gel chromatography method was used using Sephacryl HR-100. TEA is used as a washing solution and TEAN as an eluent. The results of the SDS-PAGE electrophoresis of the eluate chromatography according to the previous method³⁰. Then the protein band at the position of the molecular weight of about 36 kDa was cut and collected. The pure protein AdhF36 was harvested by an electroelution method.

Flagellin:

On LB agar, Salmonella typhi was cultured. A single colony was subcultured once again after being transferred to 10 mL of LB broth (constantly shaken). The cultures were inoculated into 1 L of LB broth while being shaken continuously (at 37 °C).

Centrifugation at 5000xg for 10 minutes was performed to precipitate the bacterial culture. All pellets were centrifuged once more under similar circumstances after being suspended in sterile PBS (200 mL, pH 7.4). After being washed twice, the pellets were resuspended in sterile PBS (50 mL, pH 2). For 30 minutes, the bacterial suspension was stirred with a magnetic stirrer in cold conditions (4 °C). This step is performed to separate the bacterial filament monomerization and the flagellin. The remaining bacteria were subsequently precipitated by centrifuging for 20 minutes at 10,000xg. The supernatant—which included bacterial flagellin—was collected. To precipitate the flagellin protein from the aqueous phase, the supernatant was slowly saturated with 70% ammonium sulfate for 45 minutes in a cold environment. Next, 10 minutes of 10,000xg centrifugation at 4 °C was performed. To remove any remaining residue, the suspended pellet was collected and dialyzed. SDS-polyacrylamide gel electrophoresis (PAGE) analysis was also carried out to ascertain the degree of purity³¹.

Profiling:

This method was carried out based on the previous method and modified by Dulley and Grieve³². Protein samples were dissolved for five minutes at 100 °C in sample buffer (0.625 M Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, and 5% mercaptoethanol). 10% buffered gel separation was used for SDS-PAGE. The gel was stained with 0.1% (wt/vol) Coomassie brilliant blue R-250 (Merck, India) at 45.4% and 9.2% glacial acetic acid and destained with 5% and 7% glacial acetic acid in methanol, respectively. The electrophoresis was performed for 90 minutes with a current of 20 mA (120 V).

ELISA:

Parameters observed in this antibody examination were SIgA levels in mucus. The mucus was collected as pooled mucus from the small intestine of both groups (the control and treatments). The results were noted in antilog units of 100. The preparation was as follows: intestinal sections were washed with cold PBS (containing 1.0 mM EDTA and a protease inhibitor (25 g/mL inhibitor cocktail)). The small intestine's mucosa was revealed once the gut was dissected. By scraping longitudinally with a spatula, the mucus layer was removed and placed in a tube containing a protease inhibitor in sterile PBS. The suspension was shaken before being centrifuged for ten minutes at 12,000 rpm and 4 °C. The supernatant was taken, purified as in serum purification, and used as a mucus sample for S-IgA (secretory IgA) examination by ELISA reader (Biorad Model 550)^33,34,35.

Flow cytometry:

The expression of CD8⁺ T cells was measured using Flow Cytometry with the following procedure: all reagents to be used were placed at room temperature. A total of 50 L of cell suspension derived from the spleen homogenate was taken and put into a 1.5 mL centrifuge tube. Subsequently, the surface antibody was diluted by adding 5 L of PE (phycoerythrin) Anti-mouse CD8⁺ per sample for every 50 L of cell Staining Buffer. Cell surface antigen staining was performed, then incubated at room temperature for 20 minutes under dark conditions. After incubation, 500 L of Cell Staining Buffer was added, mixed evenly, then transferred to a round bottom tube and analysed using Flow cytometry.

Data analysis:

The data were statistically evaluated with SPSS 25 using the independent-T test and ANOVA at a 95% confidence level (α= 0.05). The ability of the proteins OMP, Fimbriae, and flagellin to elicit cellular and mucosal immune responses were compared statistically using the independent-T test. In addition to the results of statistical analysis, a descriptive analysis will also be presented in the form of a histogram. If there is a significant difference, then the Post Hoc analysis is continued with the Duncan Multiple Range Test (DMRT) to find out which groups show significant differences.

RESULT AND DISCUSSION:

Profiling

Figure 1: SDS-PAGE result confirmed the protein profile, (1) whole cell (fimbriae included), (2) Fimbriae 1, (3) Fimbriae 2, (4) whole-cell (fimbriae excluded), (5) marker, (6) omp, and (7) flagellin.

Profiling was performed to determine the protein subunit profile based on its molecular weight by the SDS-PAGE method. In figure 1 (number 1), the whole cell that the smear shows a variety of proteins with various molecular weights. It contains all parts of the cell, including OMP, fimbriae, flagellin, and more.

Lane numbers 2 and 3 show fimbriae (AdhF36) 1 and fimbriae 2. Fimbriae 1 was obtained by cutting the fimbriae from the first whole cell. Fimbriae 2 is the resuspension of fimbriae 1. The resuspension was performed until a clear result is yielded. Hypothetically, fimbriae 2 should be "cleaner" from residue and have a clearer band result rather than fimbriae 1. The result supports the hypothesis. Lane 3 shows a slightly better result than lane 2. Type-1 fimbriae also known as AdhF36 is an essential virulence factor composed of alpha-D-mannoside-specific Lectin also called FimH. FimH has the main role to determine adhesiveness and bacterial pathogenesis³⁶. A previous study has shown that type-1 fimbriae can induce a strong immune response and promote adherence to gut mucosa. Type-1 fimbriae demonstrated its advantage in self-limiting gastrointestinal infections³⁶. AdhF36 is identified as a stable protein because this protein is unimpacted by alteration of temperature. Thus, it will be a potential subunit to inhibit bacterial adhesion as a typhoid vaccine candidate³⁸.

Lane 4 contains whole cells without fimbriae. However, the results showed that there was still a visible band around 36 kDa that should have had the fimbriae removed. This band is probably another protein weighted 36 kDa such as outer membrane protein (OMP)³⁹.

OMP or AdhO36 is on lane 6 and the previous study have shown that the molecular weight of AdhO36 is approximately 36 kDa³⁹. A protein band is visible around 50 kDa and below 29 kDa (but no 36 kDa). Another study exclaimed that the OMP protein of Salmonella typhi was found around 15-100 kDa. Among those ranges, 49 kDa protein was also detected³⁹. This setup matches our SDS-PAGE result. Although this gel failed to perform the protein confirmation of AdhO36, in fact, for the production of AdhO36 to be injected into the mice, the result is fair and clear (not shown).

While for flagellin, the protein was detected in the range of 43-50 kDa. This result is in accordance with a recent study that explains the flagellin protein has a molecular weight of 47 kDa. Flagellin has the main role in bacterial flagella formation and mediator of epithelial activation as well as systemic inflammation caused by Salmonella⁴¹. Flagellin from Salmonella typhi has a molecular mass of about 50 kDa⁴². Toll-like receptor 5 (TLR5) mediates the effects of a flagellin-stimulated immune response and initiates a signaling cascade that causes macrophages and dendritic cells to express co-stimulatory molecules and produce cytokines⁴³. Previous studies have also shown that recombinant flagellin from Salmonella typhimurium induces Th2 response in animal models⁴⁴. Our results proved that this subunit isolated from Salmonella typhi potential as a typhoid vaccine candidate.

CD4⁺ and CD8⁺:

In order to evaluate the immune response against the bacteria invasion and subunit vaccination, T-cell subsets (CD4⁺ and CD8⁺) were studied. The CD4⁺ and CD8⁺ concentration data were obtained from T lymphocytes that were isolated from the spleen of each animal group treatment. The total CD8⁺ and CD4⁺ T Cell production levels can be depicted in Figures 2 and 3.

On CD4⁺ T cell expression, all treatment groups showed an increase in CD4⁺ production compared with the control (p<0.004), except for OMP. OMP has a lower production of CD4⁺ T cells. This was also confirmed after Duncan's test where all treatment groups other than OMP were inhomogeneous compared with the control. For CD8⁺ T cell concentration, all groups demonstrated an increase in CD8+ T cell production following the 4 weeks of treatments. however, the Duncan test confirmed the statistically different data are only flagellins and fimbriae. as can be seen in Figure 3, it is obvious that fimbriae and flagellin have higher numbers than control and OMP. Overall, based on CD4⁺ and CD8⁺ T cell production results, subunit flagellin and fimbriae both are more effective to induce the immunological response of S. typhi incursion. While OMP is the less effective one. This ability of fimbriae and flagellin subunits indicates that both subunits are suitable candidates to be used as a typhoid vaccine since they can play a role in the mechanism of cellular immunity. Recently, a study has demonstrated that FimH as a component of type 1 fimbriae stimulated the proinflammatory cytokines in macrophages infected with S. enterica serovar Typhimurium⁴⁵. Furthermore, flagellin also stimulated the cytokines production by T and B cells recruitment and activate the T cells in different way⁴⁶.

An infectious disease occurs when pathogens invade host cells and initiate their replication process^47,48,49,50. Infection of S. typhimurium greatly increases the expression of macrophage cell surface receptors, which stimulate macrophages to secrete cytokines. This cytokine increase induces the differentiation of B cells into immunoglobulin-producing plasma cells. Conversely, increased cytokines stimulate B cell differentiation and T cell activation. More B cells circulating in the body means more antibody production in the circulation⁵¹.

According to clinical research was found that patients infected with S. typhi showed an immune response by elevating the CD4⁺ and CD8⁺ expression significantly during typhoid fever⁵². S. typhi will activate CD₄ T cells by specific immune system mechanisms through primary histocompatibility complex class 2 (MHC-II) and facilitate macrophages to carry out phagocytosis⁵³. By presenting infected cells to the MHC class I groups, APC allows the CD8⁺ T lymphocytes to be directly activated by these cells. In this scenario, CD8⁺ T cells will generate pro-inflammatory cytokines that are in charge of transporting proteins from the blood to the infection site as well as mediators that act as the host's cell defense⁵⁴.

Figure 2. Representative diagram of CD4⁺ and CD8⁺ T Cell, each group shows a distinctive result, (a) control, (b) ISCOM, (c) OMP (d) fimbriae, and (e) flagellin.

Figure 3. Mean Concentration of T Cell CD4⁺ and CD8⁺ after the injection of ISCOM adjuvant and protein subunit OMP, fimbriae, and flagellin in the 4^th week.

The administration of the ISCOM adjuvant demonstrated a considerable increase in CD4+ T cell production (p>0.05) and a significant decrease in CD8+ T cell production (insignificant, failed Duncan's test). Nevertheless, it is still safe to represent that ISCOM can be used as an adjuvant to boost the action of a subunit against a given subunit. ISCOM has been shown to increase MHC class II expression in APCs, leading to increased antigen presentation⁵⁵. Strong antibody and cellular immunological responses can be provoked by ISCOM-based adjuvants in a wide range of animal species. ISCOM can induce the balancing of Th1 and Th2 cells and CMI including the cytotoxic T cells response⁵⁶. ISCOM is active after mucosal administration and induces local and systemic immune responses and is considered the most potent adjuvant for the induction of cell-mediated immunity⁵⁷.

Secretory IgA (SIgA):

After the fourth week, SIgA levels in the small intestine's mucus showed a significant difference between the control, ISCOM, and treatment groups (p 0.001). The Duncan test indicated the mice that were injected with fimbriae and flagellin subunits have inhomogeneous data. Both group treatments prevail over an increase in the concentration of SIgA. Whilst for the OMP subunit, there was a decrease in the concentration of SIgA.

Figure 4: Mean Concentration of SIgA after the injection of ISCOM adjuvant and protein subunit OMP, fimbriae, and flagellin in the 4^th week.

The ISCOM adjuvant administration showed an increase in the concentration of SIgA compared to the control. Statistical analysis with Duncan's test also stated that the data was not homogeneous. Thus, it can be declared that the ISCOM adjuvant is effective to be used as an inducer of the immune response in the mucosa.

Similar to the results of the analysis of CD4⁺ and CD8⁺, the fimbriae and flagellin subunit proteins are suitable subunits proposed as vaccine candidates against typhoid fever. Whereas CD4⁺ and CD8⁺ are indicators of the cellular immunity response, SIgA is an indicator of the mucosal immune response. Thus, when the fimbriae and flagellin protein subunits are administered, it is expected that a more specific immune response can occur by intervening in the adhesion mechanism of S. typhi during its invasion. Bacterial infection of the gastrointestinal tract is the common stimulation that normally produces a secretory response (SIgA)^22,58. Secretory IgA (SIgA) is the predominant antibody class in mucosal secretions. The mucosal membranes that line the digestive, respiratory, and genitourinary tracts are constantly in contact with a wide range of microorganisms. The gastrointestinal tract (GIT) is inhabited by a wide range of microbial populations. The mucosal immune system is induced to synthesize SIgA, which controls interactions between the host and bacteria and anticipates the invasion of harmful microorganisms⁵⁹.

CONCLUSION:

Whilst the OMP protein subunit failed to show the expected result of this study, the fimbriae and flagellin protein subunits showed promising results. Both subunits showed their ability to increase the cellular immune response, which was confirmed by an increase in the production of CD4⁺ and CD8⁺ T cells after four weeks of treatment. In addition, it has been demonstrated that these two subunits can boost the mucosal immune response by increasing SIgA synthesis. Accordingly, it can be declared that fimbriae and flagellin are suitable protein subunits and have more potential to be conducted as vaccine candidates against typhoid fever.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank the Faculty of Medicine, Universitas Brawijaya for funding this research and Laboratory of Microbiology, Faculty of Medicine, Universitas Brawijaya for facilitating the research process.

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Received on 29.09.2022 Modified on 18.11.2022

Research J. Pharm. and Tech 2023; 16(8):3891-3898.

DOI: 10.52711/0974-360X.2023.00641