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 ~  Abstract
 ~ Introduction
 ~ Subjects and Methods
 ~ Results
 ~ Discussion
 ~ Conclusions
 ~  References
 ~  Article Figures

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 1  |  Page : 37-45
 

Generating mucosal and systemic immune response following vaccination of vibrio cholerae adhesion molecule against shigella flexneri infection


1 Department of Clinical Microbiology, Faculty of Medicine, University of Brawijaya, Malang, Indonesia
2 Department of Clinical Parasitology, Faculty of Medicine, University of Brawijaya, Malang, Indonesia
3 Master Program in Biomedical Sciences, Faculty of Medicine, University of Brawijaya, Malang, Indonesia
4 Bachelor Medical Program, Faculty of Medicine, University of Brawijaya, Malang, Indonesia

Date of Submission27-Oct-2019
Date of Decision01-Mar-2020
Date of Acceptance08-Jul-2020
Date of Web Publication25-Jul-2020

Correspondence Address:
Dr. Aisyah Amalia
Medical Faculty, Brawijaya University Jalan Veteran, Malang, 65145, East Java
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_19_411

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 ~ Abstract 


Introduction: Previous studies have shown 37.8 kDa pili subunit protein of Vibrio cholerae and 49.8 kDa pili subunit protein of Shigella flexneri can act as an adhesion molecule to initiate infection. These molecules also have the ability to agglutinate blood. The present study assessed mucosal and systemic immunity following vaccination using 37.8 kDa V. cholerae and protection against S. flexneri. Subjects and Methods: Haemagglutination test was performed after purification of V. cholerae protein, followed by an anti-haemagglutination test. The intestinal weight and colony count were used to validate the protective effect on balb/c mice which were divided into the naive group, Shigella-positive control group, Vibrio-positive control group, V. cholerae infected-Vibrio-vaccinated group and S. flexneri-infected-Vibrio-vaccinated group. Th17, Treg, interleukin (IL) IL-17A, β-defensin and secretory-immunoglobulin A (s-IgA) were also measured to determine the systemic and mucosal immunity after vaccination. Results: The haemagglutination and anti-haemagglutination tests showed that the 37.8 kDa protein could inhibit 49.8 kDa of the S. flexneri pili subunit. Decreased intestinal weight and colony count of vaccinated group compared to naive group also support cross reaction findings. Vaccination also generates higher level of Th17, Treg, IL-17A, β-defensin and s-IgA significantly. Conclusions: 37.8 kDa subunit pili can act as a homologous vaccine candidate to prevent V. cholerae and S. flexneri infection.


Keywords: 37.8 kDa Vibrio cholerae, 49.8 kDa Shigella flexneri, adhesion molecule, vaccine


How to cite this article:
Prawiro SR, Poeranto S, Amalia A, Widyani EL, Indraswari G, Soraya M, Dwi Pradipto SR, Prasetya A, Hidayat GR, Alitha Putri SN. Generating mucosal and systemic immune response following vaccination of vibrio cholerae adhesion molecule against shigella flexneri infection. Indian J Med Microbiol 2020;38:37-45

How to cite this URL:
Prawiro SR, Poeranto S, Amalia A, Widyani EL, Indraswari G, Soraya M, Dwi Pradipto SR, Prasetya A, Hidayat GR, Alitha Putri SN. Generating mucosal and systemic immune response following vaccination of vibrio cholerae adhesion molecule against shigella flexneri infection. Indian J Med Microbiol [serial online] 2020 [cited 2020 Aug 14];38:37-45. Available from: http://www.ijmm.org/text.asp?2020/38/1/37/290675





 ~ Introduction Top


Diarrhoea is the second leading cause of morbidity and mortality worldwide. Shigellosis and cholera are still a common cause of acute diarrhoea in developing countries.[1],[2] In Indonesia, the prevalence of diarrhoea reaches 8% of all infectious diseases, with 18.5% of cases found in infants.[3] Of all other subtypes of Shigella, Shigella flexneri is the major cause of bacterial shigellosis in developing country.[4] Meanwhile, cholera in Indonesia was likely caused by Vibrio cholerae O1.[5]

V. cholerae and S. flexneri have a different pathomechanism to cause signs and symptoms. After adhesion, V. cholerae produces and secretes toxins that increase the levels of cyclic nucleotides (e.g., cAMP and cGMP), thereby inducing massive fluid loss observed during secretory diarrhoea. Meanwhile, shigellosis is characterised by neutrophil accumulation in the intestinal mucosa followed by transmigration of these cells into the intestinal lumen that causes mucosal damage of the intestine, loss of protein-rich fluids and decrease in the ability to reabsorb them.[6]

Nowadays, vaccination becomes the most effective way to eradicate infectious diseases. Several studies have found that haemagglutinin pili subunit protein with molecular weight (MW) 49.8 kDa and anti-haemagglutinin pili protein with MW 7.9 kDa were suspected to become adhesion molecules.[7],[8],[9] We have also found that Shigella dysenteriae pili subunit protein with MW 49.8 kDa and MW 7.9 kDa can cross-react with pili subunit proteins of other Shigella species, including S. flexneri.[10] Haemagglutinin molecules derived from the pili subunit weighing 37.8 kDa on V. cholerae O1 MO94V (Indonesian strain) were discovered by Sumarno; can act as adhesion molecule and initiate infection process.[11]

The purpose of this study is to find out whether there is a cross-reaction of the immune response between the adhesion molecules in S. flexneri and V. cholerae through the measurement of β-defensin and secretory-immunoglobulin A (s-IgA) levels as markers of the mucosal immune response and interleukin (IL) IL-17A levels, Th17 cells and Treg cells as markers of the systemic immune responses. Protectivity test was also carried out by measuring the intestinal weight and number of colonies from the culture of mucous intestinal secretions after vaccination. Cross-protection between Shigella and Cholera infection hopefully will make a potential homologue vaccine.


 ~ Subjects and Methods Top


This is an experimental study is using in vivo and in vitro approaches. The subject used in this study was Balb/c male mice. Ethical clearance was obtained from the Ethical Committee of Medical Faculty of Brawijaya University, Malang, Indonesia. The mice were divided into negative control group, control-positive Shigella group (infected with Shigella only), control-positive Vibrio group (infected with Vibrio only) and Vibrio-vaccinated group (subsequently infected with Vibrio and Shigella separately). We used four mice in each group.

Culture and pili protein isolation of Vibrio cholerae and Shigella flexneri

The bacteria V. cholerae O1 classical M094V and S. flexneri 2a were obtained from patients admitted in the General Hospital Saiful Anwar and cultured in Microbiology Laboratory of Faculty of Medicine Brawijaya Malang. The medium used to breed the bacteria to enhance pili production was TCG medium which was incubated for 2 × 24 h. The TCG medium contains 0.02% thioproline, 0.3% NaHCO3, 0.1% mono sodium l-glutamat, 1% bactotryptone, 0.2% yeast extract, 0.5% NaCl, 2% bacto agar and 1 mM β amino-ethyl ether-N, N, N 'n'. S. flexneri and V. cholerae bacterias then harvested from the medium, incubated, and subsequently re-suspended by adding tri chlor acetic solution until the concentration reached 3%. The suspension was then shaken for 30 s and stored at room temperature for 1 h and then the pellet was made by centrifugation at a speed of 6000 rpm for 30 min at 4°C. The centrifuged pellet was then resuspended with phosphate-buffered saline (PBS) at a pH of 7.4. The pili were sliced with pilus bacterial cutter at a speed of 5000 rpm for 30 s at a temperature of 4°C. The pili were isolated by centrifugation at six cycles. The first cycle was run at 12,000 rpm for 30 min at 4°C temperature. After centrifuging, the supernatant and the pellet were separated, and the second and third cycles were continued in a manner similar to that of the first cycle. The fourth cycle was centrifugation of the pili at 10,000 rpm for 60 s at the pilus bacteria cutter. Similar to the previous step, the supernatant constituting rich protein pili was collected, and the pellet was continued to the fifth and the sixth cycles, similar to that of the fourth cycle.

Sodium dodecyl sulphate-polyacrylamide electrophoresis of Vibrio cholerae and Shigella flexneri

Sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) was used to measure the MW of the pili. The SDS-PAGE was done according to the Sumarno et al.'s[12] method. The protein sample was heated at 100°C for 5 min in a buffer solution containing 62.5 mMTris with pH 6.8, 10% glycerol, bromophenol blue with 5% (v/v)-mercaptoethanol prior to electrophoresis through 5% stacking and 15% separating gels. Coomassie brilliant blue was used to stain the gels. The desired protein of each bacteria (V. cholerae at MW 37, 8 kDa; Shigella flexneri at MW 49, 8 kDa) was purified and amplified according to the next method.

Pili protein purification of Vibrio cholerae with molecular weight 37.8 kDa and Shigella flexneri with molecular weight 49.8 kDa

The gel from electrophoresis was cut at the protein of interest perpendicularly according to the method suggested by Agustina et al.,[7] so each piece contains one protein band. The cut band was collected into a piece of membrane tape containing electrophoresis running buffer. Electroelution was done in a horizontal apparatus of electrophoresis using electric voltage 120 mV for 90 min. PBS (pH 7.4) fluid buffer was used to dialyse the membrane tape for 28 h. The dialysis fluid was replaced four times. The result of electroelution of SDS-PAGE band dialysis fluid was used in the haemagglutination test.

Immunisation of mice with Vibrio cholerae adhesion molecule with MW 37.8 kDa

Before immunisation, the mice were given 0.3 ml of 0.2 M natrium bicarbonate. The dose of immunisation was 250 μg of protein via gastric gavage for Vibrio-vaccinated group with subunit B of cholera toxin (CTB) as an adjuvant. Immunisation was given weekly at days 7, 14, 21, 28 and 35. Immunisation was also given intraperitoneally in different mice with CFA (complete Freud adjuvant) as an adjuvant for anti-haemagglutination assay.

Haemagglutinin and anti-haemagglutination assays

Haemagglutinin assay was conduct after S. flexneri 2a pili protein purification method[13]. The assay was conducted using a microplate that has 96 V bottom hole-wells in which each well had 100 μL of volume. Double dilution of the sample was prepared at several concentrations. 50 ml of red blood cells from the mice balb/c with a concentration of 0.5% and S. flexneri's pili was added to each well and shaken on a rotator plate for a minute and then stored at room temperature for an hour. The same procedure was done with the V. cholerae's pili. The agglutination of red blood cells on the highest dilution was observed to determine the titre. Anti-haemagglutinin assay was conduct by adding V. cholerae's polyclonal antibody containing serum into the result of haemagglutination assay at several concentrations. The sedimentation of erythrocytes was observed.

Protectivity test and infection of the intestine with Shigella flexneri and Vibrio cholerae bacteria

Protectivity test was conducted using mice ligated ileal loop (MLIL) according to the previously reported method.[14] The mice intestine was cut from 4 cm proximal and 4 cm distal, and then injected with S. flexneri and V. cholerae bacteria to stimulate infection process. In control-positive Shigella group, the intestine was infected with S. flexneri, meanwhile control-positive Vibrio group infected with V. cholerae. The intestine from mice vaccinated with 37, 8 kDa V. cholerae (further called Vibrio-vaccinated mice) then infected with S. flexneri and V. cholerae separatedly. The weight fluctuation of the section of the ileal loop was then observed serially.

Calculation of bacterial colonisation

The colony taken from the intestine of the mice was placed on SSA medium and then incubated at 37°C for 18––24 h. The S. flexneri were then identified from the grown colony and calculated by using a colony counter.

Secretory-immunoglobulin A, β defensin and interleukin-17A examination using enzyme-linked immunosorbent assay method

Enzyme-linked immunosorbent assay (ELISA) was done for measuring s-IgA, β-defensin and IL-17. The ileum was cut, and the mucous was collected and suspended with the same volume of PBS. Then, it was centrifuged at 6000 rpm at a temperature of 4°C for 30 min and the supernatant was stored at 4°C. Then, s-IgA and β-defensin were measured, respectively, using s-IgA ELISA kit from Elabscience (Donghu Hi-Tech Development Area, China) (E-EL-M1040) and β-defensin ELISA kit from MyBioSource (MyBioSource, Inc. San Diego, USA) (MBS2886605) with standard method. Blood was collected from the mice's heart and stored for 10 min at 70°C. IL-17 was measured from blood plasma using IL-17A ELISA kit from BioLegend (San Diego, CA, USA) with standard method.

Th17 and Treg cell count

The absolute counts of Th17 and Treg cells were assessed by flowcytometry, respectively, using anti-human CD4+ IL-17APE antibodies (BioLegend, San Diego, CA, USA) and anti-human CD4+ CD25+ Foxp3-PE antibodies (BioLegend, San Diego, CA, USA) with standard method. Peripheral blood mononuclear cells s were adjusted to the concentrations of 1 × 106 cells/L and incubated with various antibodies. All the samples were analysed by BD Cell- Quest™ Pro software (BD Biosciences).

Data analysis

All statistical analyses were performed using IBM® SPSS® version 23.0 (International Business Machines Corporation, Armonk, New York, USA) software. The research was considered statistically significant when P < 0.05 was achieved.


 ~ Results Top


Identification of pili Vibrio cholerae and Shigella flexneri proteins using sodium dodecyl sulphate-polyacrylamide electrophoresis

From TCG media, V. cholerae were obtained and bacteria pili were isolated with pili bacteria cutter. After isolation completed, SDS-PAGE was used to identify the proteins in V. cholerae pili. The results were consistent from the 5th until 7th band with MW 37.8 kDa.

Vibrio cholerae polyclonal antibodies inhibit adhesion molecule ligand Shigella flexneri assessed using haemagglutination and anti-haemagglutination assays

The adhesion molecule act as the first step of the pathogenesis of infection in many bacteria, as well as V. cholerae. The adhesion molecule allowing adherence of the bacteria to our cell which can caused the bacteria colonization then caused infection. With its haemagglutinin characteristic, adhesion molecule is known to have high virulence factor that is responsible for the attachment of the bacteria to host cells. Haemagglutination assay was performed to verify whether purified subunit pili have the ability to agglutinate.

In order to assess V. cholerae pili subunit's ability to agglutinate erythrocyte, haemagglutination assay was performed [Figure 1]. Twelve microplates were used. Serial dilution was done in the first until tenth microplate which has 1/512 dilution with a concentration of 0.65 mg/ml. Positive results indicated with absence of 'dot' (indicating the erythrocyte's agglutination) at the base of microplate. From haemagglutination test [Figure 1], the highest haemagglutination titre was found to be 1/8. Hence, this titre was utilised in the anti-haemagglutinin assay. V. cholerae pili subunits were then added to microplates and then instilled with polyclonal antibody of S. flexneri. Anti-haemagglutination reaction was then marked by the presence of erythrocyte sediment at the base of the microplate. From [Figure 2], it can be concluded that the highest titre that possesses the ability of anti-haemagglutinin assay is 1/64.
Figure 1: Haemagglutinin assay. Following Vibrio cholerae purification, haemagglutinin assay was performed in a serially diluted started from 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128 and 1/256, until 1/512 that filled the second until the tenth microplate. The first microplate contained Vibrio cholerae 37, 8 kDa protein only, 11th microplate was empty and the last microplate contained erythrocyte suspension and phosphate-buffered saline (negative control group)

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Figure 2: Anti-haemagglutinin assay. Using the highest titre showed a positive result during haemagglutinin assay (1/8), 37,8 kDa Vibrio cholerae protein purification then diluted with the serum containing 49, 8kDa Shigella flexneri polyclonal antibody produced from Balb/c mice that had been vaccinated with 49, 8kDa Shigella flexneri. The first microplate was empty, the 11th microplate was filled with Shigella flexneri serum and mice's erythrocyte suspension (negative control [N]), while the last microplate is filled with erythrocyte suspension and Vibrio cholerae without polyclonal antibody (positive control [P])

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To evaluate the effect of vaccination, the mouse intestine were inoculated with S. flexneri and V. cholerae according to MLIL method. After inoculation, the weight of the intestine was measured at 0, 5, 15 30, 45 and 60 minutes [Figure 3]. Macroscopic findings can be found in [Figure 4]. It was noted that escalation of intestinal weight reached its peak at 15 min, and then decreased at 45 min and became stable at 60 min. It is also noted that the average of the infection control group, either Shigella-positive control group or Vibrio-positive control group, at first was lower than that of the naive group, but when it reached 15 min, the average increased and became stable until 60 min. Meanwhile, the vaccinated group, either Shigella-vaccinated or Vibrio-vaccinated group, showed a different pattern; even though there was an increase in intestinal weight at 15 min, at 60 min, the intestinal weight decreased below that of the naive group.
Figure 3: Difference mean of intestinal weight in each group over time with MLIL method. Intestinal weight measured at 0, 5, 15, 30 and 60 min, while infection is on progress with MLIL method. Data are represented as means. N: Naïve group, SPC: Shigella-positive control group, VPC: Vibrio-positive control group, SVv: Shigella infection + Vibrio vaccination, VVv: Vibrio infection + Vibrio vaccination

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Figure 4: Macroscopic findings mice ligated ileal loop tie test in each treatment group after 60 minutes. (a) Naive group, (b) Shigella-positive control group, (c) Vibrio-positive control group, (d) Shigella-infected + Vibrio-vaccinated group, (e) Vibrio-infected + Vibrio-vaccinated group

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Count of Vibrio cholerae and Shigella flexneri colony-forming unit after vaccination

In this research, the mice were given adhesion molecule subunit pili of V. cholerae vaccination at days 7, 14, 21 and 28. At day 35, abdominal incision and intestinal isolation were performed with MLIL method. The intestines were then inoculated with S. flexneri and V. cholerae. The colony was taken, cultured in TCBS or SS agar and counted using colony counter. The result showed a significant difference average of colony numbers between groups that vaccinated with 37,8 kDa V. cholerae infected with V. cholerae(VVv) compared with positive control groups (VPC). V. cholerae adhesion molecule vaccination groups leave no colony-forming unit after V. cholerae inoculation. The number of colonies in the vaccinated group (both Shigella and Vibrio-infected) was statistically lower than the positive control group. With significant difference of colony count and intestinal weight, the mucosal and systemic parameters were taken to seek the cause.

Per-oral immunisation increases β-defensin and secretory-immunoglobulin A as response of mucosal immunity

To see whether vaccination caused alteration to mucosal immunity, several parameters were examined. Several defensive factors were found in the mucus layer. After series of vaccination, intestinal secretory was obtained immediately to measure β-defensin level and s-IgA titres using ELISA. β-defensin1 is one of the antimicrobial peptides expressed in epithelial cells of gut and is also an essential component of intestinal immune system as part of innate immunity. To determine whether there is difference of antimicrobial peptide concentration, β-defensin1 level from control group and vaccinated group was compared. The result showed that the β-defensin level of vaccinated group (3316 ± 0.39 ng/mL) was significantly higher than that of the control group (2655 ± 0.48 ng/mL) (P < 0.05) [Figure 5]b. Meanwhile, IgA, known as antigen-specific immunoglobulin, is an important component of adaptive mucosal protection. Mice that were fed with 37.8 kDa adhesion molecule exhibited statistically significantly higher sIgA titres (3392 ± 0.39 μg/mL) compared to control mice (2.95 ± 0.48 μg/mL) (P < 0.05) [Figure 5]a.
Figure 5: Intestinal secretory-immunoglobulin A titres and β-defensin1 in mice from control group and Vibrio-vaccinated. C. Serum interleukin-17A level in each group. After series of vaccination, Balb/c mouse blood and mucus were collected immediately and analysed with enzyme-linked immunosorbent assay. Data are represented as means ± standard errors of the means

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Generation of systemic Th17 and Treg response following vaccination on systemic immune response

IL-17A is known as Th17 cell's product that plays protective roles in host immunity. To investigate the effect of vaccination on systemic immune response, the level of IL-17A from blood was measured at the end of immunisation. Higher serum IL17 levels were noted in Vibriovaccinated group (1685.73 ± 204.53 pg/mL) compared to control group (1685.73 ± 154.74 pg/mL) (P < 0.05) [Figure 6].{Figure 6}

To examine whether administering V. cholerae adhesion molecule would induced antigen-specific immunity, Th17 and Treg were measured. After immunisation, the lymphocyte was collected from spleen and examined using flowcytometry. Some representative figure Treg and Th17 can be seen in [Figure 5]. After vaccination with 37,8 kDa V. cholerae, the levels of both Treg and Th17 cells were significantly higher than those of control group (P < 0.05) [Figure 7]. However, in proportion, even vaccinated group reached higher Treg/Th17 proportion, but there was no statistically significant difference with control group (P > 0.05) [Figure 7]c.
Figure 6:Representative figures of Treg flowcytometry from control group (a), Vibrio-vaccinated group (b), Th17 from control group (c) and Vibrio-vaccinated group (d). After isolating CD4+ T cells from peripheral blood mononuclear cells, staining of surface antibodies anti-CD25 and FOXP3 and interleukin- 17 intracellular was done and measured with flowcytometry

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 ~ Discussion Top


Sumarno et al. revealed that pili proteins 50.3 kDa, 37.8 kDa, 35.9 kDa, 21.3 kDa and outer membrane protein (Omp) 37.8 kDa are adhesion molecules of V. cholerae.[11] From these molecules, the 37.8 kDa pili protein can induce s-IgA production and inhibit liquid secretion in the small intestine of mice if combined with CTB.[14] Hence, we used 37.8 kDa pili protein of V. cholerae in our study to reveal the immune response of this molecule. Our previous study also revealed the cross-reactivity of 49.8 kDa pili subunit haemagglutinin proteins and 7.9 kDa pili subunit anti-haemagglutinin proteins of Shigella spp.[10] There is also cross-reactivity among S. flexneri pili and Omp subunit haemagglutinin.[15] After knowing that both of them have the same hemagutinin characteristics, this study wanted to assess whether there was cross-reactivity between V. cholerae with MW 37.8 kDa + CTB and pili protein of S. flexneri 49.8 kDa.

SDS-PAGE analysis is a fundamental procedure in many protein analysis applications. The protein profile of the V. cholerae pili was cut using a pili cutter. The result of our study is similar to that of a preliminary study previously conducted by Sumarno et al.[11],[14] The authors in their study used a local strain of V. cholerae O1 classical M094V. The extraction method using 0.5% N-octyl-beta-D-glycopyranoside (NOG) successfully isolated the 37.8 kDa haemagglutinin protein and showed that this protein has protectivity characteristic by inducing immune response.

Haemagglutination test aims to determine whether there is a bond between antigens (pili) and receptors found on the surface of erythrocyte cells because there is a similarity of receptors on erythrocyte cell membranes with receptors on the mucosal surface of the host cell.[16],[17] From the results of the anti-haemagglutination test, it is known that 37.8 kDa V. cholerae protein which was previously used in the production of polyclonal antibodies can inhibit the activity of 49.8 kDa of S. flexneri protein, thus reflecting the cross-reaction between the two proteins.

To verify the ability of vaccination from V. cholerae's 37.8 kDa adhesion molecule + CTB against infection of S. flexneri, protectivity test using MLIL method were conducted. This method was chosen and performed according to previous study because it is an easy less harmful method.[14] The result from this study shows that the vaccinated group develops resolution at 60 min, which is almost the same compared to naive group, and indicates that the 37.8 kDa has the ability of diminishing liquid secretion[14] and decreasing intestinal weight. In addition to the protectivity test, we also count the colony after MLIL method. We found that there is an inhibition of S. flexneri growth in Vibrio-vaccinated group compared to the unvaccinated group. This also confirmed the cross-reactivity between them.

We also examined the immune effector cells that were affected when vaccination was carried out into the body of mice. In this study, we focused on Th17, Treg immune effector cells and IL-17A pro-inflammatory cytokines. Th17, a subset of CD4+ T-cells, often called pro-inflammatory T cells, produces not only IL-17 but also IL-23, IL-21, tumour necrosis factor-alpha and granulocyte macrophage-colony-stimulating factor.[18] Higher splenic Th17 was exhibited after 37.8 kDa vaccination compared to control group (P = 0.005). Th17 has plasticity towards the Tfh phenotype in the intestine, especially Peyer's patch environment. In Peyer's patch, Th17 not only shows Tfh phenotype, but also upregulated IL-21 and Bcl-6 and mimics Tfh cells.[19] Several evidences support Th17's role in vaccine-induced protection against Bordetella pertussis, Streptococcus pneumonia and BCG for Mycobacterium tuberculosis infection.[20] Another study shows that after 4 weeks' challenge of Helicobacter pylori, mice that were vaccinated with UreB subcutaneously had higher splenic Th17, but low expression of homing receptor expression,[21] which means that Th17 could not migrate back into gastric tissue. These suggest that splenic Th17 might play different roles in protection that has not yet been fully understood.

In this study, the level of splenic Treg in the vaccinated group was higher than that in the control group (P < 0.05) [Figure 7]a. Treg, mostly known with Foxp3+ T cells, has been known as a major regulator of immune homeostasis through its immunosuppressive function. Instead of eliminating bacteria, Treg in the gut keeps microbiota in balance and maintains diversity. Foxp3+ T cells are also required for T follicular regulatory (Tfr) for gut microbiota regulation. However, splenic Foxp3+ cells need to migrate to germinal centres of Peyer's patches and convert into Tfr to regulate microbiota. The migration receptor expression is not measured in this study.[22]

Th17 cells and Treg cells are present in large amounts of intestinal lamina propria. In stable conditions, the number of Treg cells will be more dominant, whereas Th17 cells will continue to produce IL-17A in small amounts to maintain the mucosal barrier. However, when an infection occurs, the suppression function of Treg cells will be controlled so that Th17 cells can secrete IL-17A in higher amounts.[23],[24] However, to maintain the condition of homeostasis, the role of FoxP3-Treg is needed to maintain immune tolerance through IL-10 and TGF-β.[25],[26],[27] However, the proportion of Treg/Th17 shows that an increase in Th17 cell count was not followed significantly by an increase in Treg (P = 0.441). This might be caused by differences in measurement time during the study. In infection, the Th17 response is suppressed by Treg cells. However, immunisation enhances Th17 cell's response, which can overcome Treg cell suppression.

The higher level of Th17 is followed by a statistically significantly higher level of IL-17A in the blood of vaccinated mice (P = 0.029). However, the role of IL-17 in secreting T cells is a considerable controversy. IL-17-secreting T cell is activated in the early stage of inflammation and plays a role in maintaining neutrophil homeostasis. Upon bacterial infection, the IL-23/IL17 pathway is activated which recruits neutrophils to the infection site, leading to the extracellular clearance of bacteria.[28] IL-17 also induces expression of an anti-microbial peptide, including β-defensin,[18] and this could be the reason why β-defensin levels were increased in this study.

In this study, the levels of β-defensin were measured as a humoral innate immune response to the vaccination of adhesion molecule of V. cholerae with MW 37.8 kDa + CTB. This results in significantly increased β-defensin production (P = 0.028) compared to control. Defensins as cationic effectors interact with negatively charged membranes of pathogens. The electrostatics changes in membrane cause altered metabolism production and caused the death of the microbes.[29] Knowing that the act is not specific, the high level of defensin could be the cause why 37.8 MW kDa vaccination gives protective effect not only to V. cholerae infection but also to S. flexneri infection. Interestingly, besides producing innate immune response, several evidences have suggested that defensins act as modulators and activators of the adaptive immune system.[30] Defensin links innate and adaptive immunity mostly by stimulating immune cell migration, promoting the release of pro-inflammatory cytokines and recruiting antigen-presenting cells and activating them to induce a Th1-skewed immune response.[29]

IL-17 was needed in lamina propria to transport IgA via polymeric Ig receptor (pIgR) across epithelium to the mucus layers after produced by plasma cell in the mucosa.[31] IgA itself functions as neutralisation and clearance of pathogens play a crucial role as mucosal homeostasis. The results showed that s-IgA levels from the vaccinated group are statistically significantly higher when compared to that of the control group (P = 0.029). The administration of cholera toxin as an adjuvant in those studies can induce the production of s-IgA in the intestinal mucosa.[24] In this study, the mechanism of s-IgA production is still unclear and needs further investigations, but there is some suspected pathway how s-IgA is produced. IgA is a sign which indicates that an adaptive immune system was activated in this study. IgA can be produced from T-cell-dependent or -independent mechanism.[32] IgA is generated in germinal centres of Peyer's Patches with the help of T follicular helper (Tfh) cells. After APC presents antigen, Tfh will generate antigen-activated B cell and produce IgA. After class switching recombination, plasmablast will also be generated with memory B cell. Unlike T-dependent mechanism, T-independent are generated without involvement of T cells causing low affinity immunoglobulin and not developing memory B cell.[33] In this study, both mechanisms are known to be the possible cause of the increase of s-IgA levels. Therefore, the affinity of IgA and the existence of B cell memory need to be investigated. For vaccine development, it is important to develop high-affinity and B cell memory for long life immunity.


 ~ Conclusions Top


The vaccination of 37.8 MW kDa of V. cholerae pili has protectivity not only for V. cholerae itself but also for S. flexneri. The cause of this cross-reactivity is unclear, but several mechanisms are suggested from the induction of β-defensin and/or production of IgA – independent or dependent mechanism. Furthermore, to prove the cross-reactivity among them, the immunoblotting procedure must be done. Thus, further research is needed to identify the characteristic of an immune response against the adhesion molecule of V. cholerae 37.8 MW kDa and the expansion of the cross-protection.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 7], [Figure 7]



 

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2004 - Indian Journal of Medical Microbiology
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