Acessibilidade / Reportar erro

A CssA, CssB and LTB chimeric protein induces protection against Enterotoxigenic Escherichia coli

Abstract

OBJECTIVES:

Enterotoxigenic Escherichia coli (ETEC), a major cause of diarrhea in children under 5, is an important agent for traveler's diarrhea. Heat-labile enterotoxin (LT) and colonization factors (CFs) are two main virulence mechanisms in ETEC. CS6 is one of the most prevalent CFs consisting of two structural subunits viz., CssA, CssB, necessary for attachment to the intestinal cells.

METHODS:

In the present research, a chimeric trivalent protein composed of CssB, CssA and LTB was constructed. The chimeric gene was synthesized with codon bias of E. coli for enhanced expression of the protein. Recombinant proteins were expressed and purified. Mice were immunized with the recombinant protein. The antibody titer and specificity of the immune sera were analyzed by ELISA and Western blotting. Efficiency of the immune sera against ETEC was evaluated.

RESULTS:

Antibody induction was followed by immunization of mice with the chimeric protein. Pretreatment of the ETEC cells with immunized animal antisera remarkably decreased their adhesion to Caco-2 cells.

DISCUSSION:

The results indicate efficacy of the recombinant chimeric protein as an effective immunogen, which induces strong humoral response as well as protection against ETEC adherence and toxicity.

Enterotoxigenic Escherichia coli ; CS6; Enterotoxin; Chimeric protein


Introduction

Enterotoxigenic Escherichia coli (ETEC) is one of the important agents causing diarrhea in children under five years old in developing countries and is a major cause of diarrhea in travelers to those regions.11. Wajima T, Sabui S, Fukumoto M, et al. Enterotoxigenic Escherichia coli CS6 gene products and their roles in CS6 structural protein assembly and cellular adherence. Microb Pathog. 2011;51:243-9. and 22. Byrd W, de Lorimier A, Zheng ZR, Cassels FJ. Microencapsulated subunit vaccine approach to enterotoxigenic Escherichia coli and other mucosal pathogens. Adv Drug Deliv Rev. 2005;57:1362-80. This microorganism is responsible for 400,000 to 800,000 deaths per year.33. Yu J, Cassels F, Scharton- Kersten T, et al. Transcutaneous immunization using colonization factor and heat- labile enterotoxin induces correlates of protective immunity for enterotoxigenic Escherichia coli. Infect Immun. 2002;70:1056-68. ETEC strains have several virulence factors like heat-labile enterotoxin (LT), heat-stable enterotoxin (ST) and colonization factors (CFs).11. Wajima T, Sabui S, Fukumoto M, et al. Enterotoxigenic Escherichia coli CS6 gene products and their roles in CS6 structural protein assembly and cellular adherence. Microb Pathog. 2011;51:243-9. CFs are surface proteins that mediate adherence to intestinal mucosa, the first step to begin pathogenesis.44. Ghosal A, Bhowmick R, Banerjee R, et al. Characterization and studies of the cellular interaction of native colonization factor CS6 purified from a clinical isolate of enterotoxigenic Escherichia coli. Infect Immun. 2009;77:2125-35. Following adherence, either or both LT or ST are expressed, resulting in diarrheal disease.55. Lapa JA, Sincock SA, Ananthakrishnan M, et al. Randomized clinical trial assessing the safety and immunogenicity of oral microencapsulated enterotoxigenic Escherichia coli surface antigen 6 with or without heat- labile enterotoxin with mutation R192G. Clin Vacc Immunol. 2008;15: 1222.

Of more than 25 identified CFs,66. Walker RI, Steele D, Aguado T. Ad Hoc E. Analysis of strategies to successfully vaccinate infants in developing countries against enterotoxigenic E.coli (ETEC) disease. Vaccine. 2007;25:2545-66. CS6 is one of the most epidemiologically prevalent CF in many countries, often accounting for up to 20-30% of all clinical isolates.11. Wajima T, Sabui S, Fukumoto M, et al. Enterotoxigenic Escherichia coli CS6 gene products and their roles in CS6 structural protein assembly and cellular adherence. Microb Pathog. 2011;51:243-9. and 44. Ghosal A, Bhowmick R, Banerjee R, et al. Characterization and studies of the cellular interaction of native colonization factor CS6 purified from a clinical isolate of enterotoxigenic Escherichia coli. Infect Immun. 2009;77:2125-35. This has led to interest to try to use CS6 alone or in combination with other antigens in an ETEC vaccine.11. Wajima T, Sabui S, Fukumoto M, et al. Enterotoxigenic Escherichia coli CS6 gene products and their roles in CS6 structural protein assembly and cellular adherence. Microb Pathog. 2011;51:243-9. and 77. Tobias J, Lebens M, Kallgard S, Nicklasson M, Svennerholm AM. Role of different genes in the CS6 operon for surface expression of enterotoxigenic Escherichia coli colonization factor CS6. Vaccine. 2008;26:5373-80. The morphology of CS6 is non-fimbrial and is expressed either alone or with CS5 or less commonly with CS4 on ETEC strains.77. Tobias J, Lebens M, Kallgard S, Nicklasson M, Svennerholm AM. Role of different genes in the CS6 operon for surface expression of enterotoxigenic Escherichia coli colonization factor CS6. Vaccine. 2008;26:5373-80. and 88. Tobias J, Svennerholm AM, Carlin NIA, Lebens M, Holmgren J. Construction of a non- toxigenic Escherichia coli oral vaccine strain expressing large amounts of CS6 and inducing strong intestinal and serum anti-CS6 antibody responses in mice. Vaccine. 2011;29:8863-9.

The genes associated with CS6 are expressed as a typical bacterial operon, consisting of four genes viz., cssA, cssB, cssC, and cssD.77. Tobias J, Lebens M, Kallgard S, Nicklasson M, Svennerholm AM. Role of different genes in the CS6 operon for surface expression of enterotoxigenic Escherichia coli colonization factor CS6. Vaccine. 2008;26:5373-80. CssA and CssB are structural subunits, whereas CssC and CssD are recognized to be chaperone and usher respectively.99. Wolf MK, Haan LAM, Cassels FJ, et al. The CS6 colonization factor of human enterotoxigenic Escherichia coli contains two heterologous major subunits. FEMS Microbiol Lett. 1997;148:35-42. CssC protein assists in the folding of the CssA and CssB structural subunits, and CssD has been ascribed an usher function responsible for transport of CssA and CssB to the cell surface.77. Tobias J, Lebens M, Kallgard S, Nicklasson M, Svennerholm AM. Role of different genes in the CS6 operon for surface expression of enterotoxigenic Escherichia coli colonization factor CS6. Vaccine. 2008;26:5373-80. 40 kDa CssA and 15.5 kDa CssB are necessary for attachment to intestinal cells.44. Ghosal A, Bhowmick R, Banerjee R, et al. Characterization and studies of the cellular interaction of native colonization factor CS6 purified from a clinical isolate of enterotoxigenic Escherichia coli. Infect Immun. 2009;77:2125-35. and 77. Tobias J, Lebens M, Kallgard S, Nicklasson M, Svennerholm AM. Role of different genes in the CS6 operon for surface expression of enterotoxigenic Escherichia coli colonization factor CS6. Vaccine. 2008;26:5373-80. The CssA interacted with the host cell fibronectin while CssB binds to cell surface sulfatide.44. Ghosal A, Bhowmick R, Banerjee R, et al. Characterization and studies of the cellular interaction of native colonization factor CS6 purified from a clinical isolate of enterotoxigenic Escherichia coli. Infect Immun. 2009;77:2125-35. and 1010. Jansson L, Tobias J, Jarefjäll C, Lebens M, Svennerholm AM, Teneberg S. Sulfatide recognition by colonization factor antigen CS6 from enterotoxigenic Escherichia coli. PloS one. 2009;4:e4487. Studies have indicated that antibodies against both structural subunits are essential to inhibit the adhesion.11. Wajima T, Sabui S, Fukumoto M, et al. Enterotoxigenic Escherichia coli CS6 gene products and their roles in CS6 structural protein assembly and cellular adherence. Microb Pathog. 2011;51:243-9. and 44. Ghosal A, Bhowmick R, Banerjee R, et al. Characterization and studies of the cellular interaction of native colonization factor CS6 purified from a clinical isolate of enterotoxigenic Escherichia coli. Infect Immun. 2009;77:2125-35.

LT toxin consists of a pentameric ring of five identical binding (LTB) subunits of 11.5 kDa surrounding an active subunit (LTA) and bind strongly to the intestinal receptor GM1 on the surface of eukaryotic cells.1111. Nicklasson M. Studies on the expression and regulation of enterotoxins and colonization factors in Enterotoxigenic Escherichia coli (ETEC): Inst of Biomedicine. Dept. of Medical Microbiology and Immunology ; 2008. and 1212. Hardy S, Holmgren J, Johansson S, Sanchez J, Hirst TR. Coordinated assembly of multisubunit proteins: oligomerization of bacterial enterotoxins in vivo and in vitro. Proc Natl Acad Sci USA. 1988;85:7109. The B subunit mostly associates with immunity against LT and induces both systemic and mucosal antibody responses.1313. Svennerholm AM, Steele D. Progress in enteric vaccine development. Best Pract Res Clin Gastroenterol. 2004;18:421-45. Since most clinical ETEC isolates can produce LT and has adjuvant properties, therefore it is a suitable candidate antigen to provide anti-LT immunity.1414. Salimian J, Salmanian A, Khalesi R, Mohseni M, Moazzeni S. Antibody against recombinant heat labile enterotoxin B subunit (rLTB) could block LT binding to ganglioside M1 receptor. Iran J Microbiol. 2010;2:120. and 1515. Millar DG, Hirst TR, Snider DP. Escherichia coli heat- labile enterotoxin B subunit is a more potent mucosal adjuvant than its closely related homologue, the B subunit of cholera toxin. Infect Immun. 2001;69:3476.

In order to provide protection, an ETEC vaccine should enclose the most prevalent CF and a LT toxoid or B subunit of LT toxin until it blocks adherence and toxin activity.55. Lapa JA, Sincock SA, Ananthakrishnan M, et al. Randomized clinical trial assessing the safety and immunogenicity of oral microencapsulated enterotoxigenic Escherichia coli surface antigen 6 with or without heat- labile enterotoxin with mutation R192G. Clin Vacc Immunol. 2008;15: 1222. and 1616. Svennerholm AM, Tobias J. Vaccines against enterotoxigenic Escherichia coli. Expert Rev Vacc. 2008;7:795-804.

Because live attenuated or killed bacterial cell vaccines are potentially associated with transient side effects, use of subunit vaccines has been preferred. The subunit vaccines are considerably safe immune system stimulators with the ability to target the site where immunity is required.1717. Nazarian S, Mousavi Gargari SL, Rasooli I, Amani J, Bagheri S, Alerasol M. An in silicochimeric multi subunit vaccine targeting virulence factors of enterotoxigenic Escherichia coli (ETEC) with its bacterial inbuilt adjuvant. J Microbiol Methods. 2012;90:36-45.

In this study a trivalent recombinant protein was designed against ETEC CS6 strains. A construction containing structural subunits of CS6 (CssB, and 100 amino acids from the C-terminal of the protein CssA) and B subunit of LT toxin was designed. The immunogenicity of a trivalent recombinant protein coded by a synthetic gene is reported.

Materials and methods

Bacterial strains, plasmids and media

ETEC strains were isolated from children with diarrhea. The strains were previously characterized for the presence of CFs and enteroxins and used for challenge purpose. Plasmids pET-28a and pET-32a were from Novagen (USA). E. coli BL21 (DE3), DH5a and Top10 were procured from Pasteur Institute of Iran. The E. coli strains were grown in Luria-Bertani (LB) broth at 37 °C.

In silico predictions and construction of chimeric gene

Chimeric gene was designed based on the nucleotide sequence of ltb, cssA and cssB genes (Gen Bank accession numbers; M17874.1, GQ241328.1, UO4844.1, respectively). The sequences were used to generate a chimeric trivalent protein fused together by (EAAAK)4 hydrophobic linkers in order to find the best epitope exposing chimeric antigen. The in silico gene analysis and multi parameter gene optimization of the synthetic chimeric gene was performed using OPTIMIZER server. Itasser and phyre software were used in order to estimate tertiary structure prediction. The chimeric proteins were analyzed for B-cell epitopes using Bcepred server. The gene encoding protein was synthesized by Shine Gene Molecular Biotech, Inc. (Shanghai, China).

Expression and purification of chimeric protein

The synthetic gene (cssA- cssB -ltB) subcloned into pET28a was transformed to E. coli BL21 (DE3) (Novagen). The transformants were grown in Luria Bertani (LB) broth supplemented with 30 µg of kanamycin/ml. Expression of the chimeric protein was induced at OD600 of 0.5 by addition of 1 mM isopropyl-ß-D-galactopyranoside (IPTG) and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Batch purification of His-tagged proteins from E. coli under denaturing conditions (Qiagen) was carried out and validated by SDS-PAGE. The denaturant (8 M urea) was removed by stepwise dialysis.

Western blot analysis

The proteins separated by SDS-PAGE were blotted onto nitrocellulose membrane using transfer buffer (39 mM glycine, 48 mM Tris-base, 0.037% SDS, and 20% methanol). The membrane was incubated in 10 ml blocking buffer (5% skim milk in PBST [137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4 and 0.05% (v/v) Tween-20]) at 4 ° C overnight. The membrane was then incubated in a 1:10,000 dilution of mice anti-His-tag -conjugated HRP and anti-CTX (Sigma) in the PBST, with mild shaking for 1 h at 37 °C. Detection was carried out using HRP staining solution containing 3,3'-diaminobenzidine (DAB) (Sigma) and H2O2.

Animal immunization

Five-week old female BALB/C mice and New Zealand White female rabbits procured from Pasteur Institute of Iran were randomly divided into test and control groups. Animals were immunized four times. Each mouse received 20 µg recombinant CS6 protein along with complete Freund's adjuvant (SIGMA) subcutaneously. Booster doses of 15 and 10 µg recombinant proteins with incomplete Freund's adjuvant were injected after 20 and 35 days, respectively. PBS served as control in parallel to the aforementioned procedure. The sera taken from animal blood after the second and third injections were stored at -70 °C for further analyses.

Determination of serum IgG responses to recombinant proteins

Indirect ELISA was used for antibody titration. A 96-well ELISA plates (Nunc) were coated with 5 µg of CssA, CssB and LTB proteins in coating buffer (64 mM Na2CO3, 136 mM NaHCO3, pH 9.8) at 4 °C overnight. The plates were washed three times with PBST and the non-specific sites were blocked with skim milk solution 5% (w/v) in PBST. The serum samples serving as primary antibodies were serially diluted to 1:500 in PBST and added to the ELISA plates with incubation at 37 °C for 45 min. The plates were washed thrice in PBST and Goat Anti-Mouse IgG Antibody (12,000 in PBST) was added to the ELISA plates as the secondary antibody. The plates were incubated for 30 min at 37 °C and were then washed. The wells added with 100 µl of citrate buffer containing 0.06% (W/V) of O-phenylene diamine dihydrochloride (OPD) (SIGMA) and 0.06% (V/V) hydrogen peroxide were incubated at room temperature for 15 min. The reaction was stopped with 100 µl of 2 M H2SO4 and the OD492 was read on a microplate reader (Bio-Rad).

ETEC binding inhibition assay

Caco-2 cells (104/well) were added and grown to confluence in 96-well plates with DMEM supplemented with 10% (v/v) FBS at 37 °C in a culture flask. ETEC strain was grown in LB broth. The bacterial suspension (105/well) was pretreated with 40 µl of immunized mice antisera for 15 min at room temperature. The bacterial mixture was added to Caco-2 cells and incubated for 2 h at room temperature. The supernatant was decanted, and the cells were washed with PBS. After digestion, the suspended bacteria were serially diluted and incubated in LB agar at 37 °C for 16 h. Subsequently, the number of bacteria adhered to the Caco-2 cells was counted. The sera of non-immunized mice were used as control.

Anti-LT antibody neutralization

The production of LT toxin neutralizing antibodies was performed with rabbit ileal loop, cAMP immunoassay and Y-1 cell rounding assay. The rabbit ileal loop test was carried out according to the methods described by De and Chatterjee.1818. De SN, Chatterje D. An experimental study of the mechanism of action of Vibrio cholerae on the intestinal mucous membrane. J Pathol Bacteriol. 1953;66:559-62. New Zealand White rabbits (1.5 kg) were fasted for 24 h before surgery. The rabbits were anesthetized by the intramuscular injection of fentanyl (SIGMA) (0.3 ml/kg of body weight) and zolazepam chloridrate (0.4 ml/kg of body weight). 10-cm ligated ileal loops were constructed. ETEC at 1 × 108 concentration were incubated with mouse serum raised against the chimeric protein, for 30 min. ETEC bacteria incubated with non-immune mouse serum served as negative control. The above bacterial mixture and PBS were inoculated into each ligated loop using a 25-gauge needle. After 18 h, the animals were sacrificed with intravenous 3% pentobarbital, and zolazepam chloridrate (0.4 ml/kg). For each ileal loop, the volume of secretion was measured after excision. Experimental infection of the rabbits was performed at the Faculty of Veterinary Medicine, University of Tehran. Research was conducted in compliance with the Animal Welfare Act and regulations related to experiments involving animals. The production of antibodies that neutralized LT toxin was also performed with a cAMP immunoassay kit (Abnova) and CHO cells. CHO cells were seeded in 24-well sterile culture plates at a density of 5 × 104 cells per well and were grown overnight to approximately 80% confluence. 0.1 µg of LT was incubated with 50 µl mice serum at room temperature for 1 h, and the mixture was added to each well for incubation at 37 °C in 5% CO2 for 2 h After washes, the cells were lysed with 0.1 M HCl and the supernatant was used in cAMP assay following the manufacturer's protocol.

For cell rounding assay, Y-1 mouse adrenal cells (Pasteur Institute of Iran) were seeded in cell culture plates at a concentration of 5 × 104 cells per well and grown to confluence at 37 °C/5% CO2 atmosphere. Y-1 cells were mixed with LT pretreated with serum samples and incubated under 5% CO2 incubator at 37 °C for 10 h. The morphological changes were observed under the compound microscope.

Statistical analysis

All statistical analyses were conducted using a SPSS 12.0 statistical program. Student's t-test was used to analyze the data for antibody responses between immunized and non-immunized groups; t-test was also used to evaluate the significance of differences in inhibition of ETEC binding to Caco-2 cells generated by test sera. A value of p < 0.05 was considered statistically significant.

Results

Codon optimization and tertiary structure prediction

A chimeric trivalent protein fused together by (EAAAK)4 hydrophobic linkers in order to find the best epitope exposing chimeric antigen (Fig. 1A and B). Codon optimization was performed to improve the transcription efficiency and transcript stability. Percentage of codon having a frequency distribution of 91-100 in the native chimeric CS6 gene was 66%, which was significantly improved to 91% in the optimized gene sequence. The overall GC content, which is a measure of transcriptional and translational efficiency, was improved from 38.96% to 48.75% upon codon optimization. There were five negative cis elements in the native CS6 gene sequence, which were removed after optimization.

Fig. 1
Design and modeled tertiary structure of chimeric protein: (A) schematic construction of recombinant chimeric protein containing CssB, CssA and LTB bound together by appropriate linkers, (B) amino acid sequences of chimeric protein and (C) modeled tertiary structure of chimeric protein by I-TASSER software. This model showed a protein with three main domains linked together with linkers.

Ab initio modeling of the synthetic sequence was exploited to produce 3D models of the chimeric protein. The result of tertiary structure of the chimeric protein construction using I-TASSER showed a protein with three main domains linked together with linker (Fig. 1C). B-cell epitope was predicted and the epitopes having cutoff values >0.8 were selected (Table 1).

Table 1
The predicted B-cell epitopes from full-length chimeric protein.

Expression and purification of recombinant protein

The genes were subcloned into pET28a and pET32a vectors respectively and over expressed in E. coli (BL21DE3) (Fig. 2A). The SDS-PAGE analysis showed the presence of a 47 KD recombinant chimeric protein. The purification of recombinant proteins was carried out under denaturation condition by Ni-NTA affinity (Fig. 2B). The expression of the recombinant protein was confirmed by the detection of the protein by Western blot using anti-His-tag and anti CTX antibodies (Fig. 3).

Fig. 2
Expression and purification of recombinant chimeric protein: (A) SDS-PAGE analysis of the rCS6 expression after IPTG induction. Lane 1: protein weight marker. Lanes 2, 4: non induced cells. Lane 3: the induced bacteria. (B) Purification of rCS6 protein by Ni-NTA column. Lane 1: protein weight marker. Lane 2: expression of chimeric protein. Lane 3: flow-through. Lane 4: column washed with buffer D (pH = 5.9). Lane 5: column washed with elution buffer (pH = 4.5). Lane 6: column washed with mes Buffer.

Fig. 3
Western blot analysis rCS6: (A) using anti 6X-His-tag antibodies. Lane 1: protein weight marker. Lane 2: rCS6 and (B) using anti-CTXB. Lane 1: protein weight marker, Lane 2: rCS6, Lane 3: antibody. Negative control (bacterial lysate without IPTG induction).

Response to immunization with recombinant protein

Animals were immunized with the recombinant chimeric protein. All immunized animals had anti-CssA, anti-CssB, and anti-LT IgG antibodies detected in serum (Fig. 4).

Fig. 4
Serum anti-LTB, -CssB, and -CssA antibody titers of mice immunized with chimeric protein. Titers calculated as log 10 of last reciprocal dilution above cut-off. Error bars represent mean ± SEM (n = 3).

Inhibition of ETEC binding to Caco-2 cells

Assessment of Caco-2 cell monolayers revealed that ETEC cells pretreated with serum from non-immunized mice were distributed on the Caco-2 cells. However, pretreatment of the ETEC cells with immunized mice antisera considerably blocked their binding to Caco-2 cells. The immune sera could inhibit 68 ± 3.5% bacterial adhesion onto the target cells. Comparison of pretreatment of the bacteria with non-immune antisera with that of immunized mice serum, showed a considerable reduction in their adhesion to Caco-2cells followed by pretreatment of the ETEC cells with immunized mice antisera.

Anti-LT toxin antibody neutralization assay

For each ileal loop, the volume of secretion measured after excision was as follows: ETEC as a positive control: 2.59 ± 0.3 g/cm, ETEC+ antisera: 0.51 ± 0.01 g/cm, PBS: 0.31 ± 0.12 g/cm. The fluid accumulation was not observed in rabbit ileal loops 18 h post-infection with ETEC treated with the immunized mice serum. In contrast, infection with standard ETEC was accompanied by an increase of fluid secretion. Data from the cAMP immunoassay kit showed that antibodies in serum and fecal samples of the immunized mice neutralized LT and prevented the toxin from stimulating intracellular cyclic AMP levels in CHO cells (Fig. 5). No morphological changes were observed in Y1 cells incubated with LT pretreated with anti-chimeric antibody, whereas normal serum had no inhibitory effects on rounding (Fig. 6).

Fig. 5
Anti-LT antibodies neutralization assay with cAMP immunoassay. Serum samples from immunized or from the control mice were tested to neutralize LT toxin. Cyclic AMP concentrations of treated CHO cells were measured using a cAMP kit. Mean values are shown, and error bars represent standard deviations.

Fig. 6
Inhibitory effect of anti-L2C3 antibody on the rounding of Y-1 cells. Y-1 cells were co-cultured with LT pretreated with serum of immunized mice (A) or normal serum (B).

Discussion

A vaccine against ETEC could have a significant impact on morbidity and mortality caused by diarrheal diseases in children living in developing countries.1919. Byrd W, Mog SR, Cassels FJ. Pathogenicity and immune response measured in mice following intranasal challenge with enterotoxigenic Escherichia coli strains H10407 and B7A. Infect Immun. 2003;71:13-21. ETEC vaccine development has been based on two strategies of blocking adherence and/or toxin activity.55. Lapa JA, Sincock SA, Ananthakrishnan M, et al. Randomized clinical trial assessing the safety and immunogenicity of oral microencapsulated enterotoxigenic Escherichia coli surface antigen 6 with or without heat- labile enterotoxin with mutation R192G. Clin Vacc Immunol. 2008;15: 1222. A trivalent recombinant protein was designed consisting of CssB, CssA, LTB subunits.

The production of chimeras consisting of ETEC adhesion and enterotoxin B subunit may be capable of eliciting both antitoxin and anti-adhesion immune responses.1616. Svennerholm AM, Tobias J. Vaccines against enterotoxigenic Escherichia coli. Expert Rev Vacc. 2008;7:795-804. Chimeric method reduces the need for producing three separate antigens, which would make it more cost-effective and would ease the manufacturing process.

In the present study LTB reported to be expressed by over half of LT expressing ETEC isolates was used, the presence of antibody against LTB can block the binding of the toxin to epithelial cells.1414. Salimian J, Salmanian A, Khalesi R, Mohseni M, Moazzeni S. Antibody against recombinant heat labile enterotoxin B subunit (rLTB) could block LT binding to ganglioside M1 receptor. Iran J Microbiol. 2010;2:120. and 2020. Mudrak B, Kuehn MJ. Heat- labile enterotoxin beyond GM1 binding. Toxins. 2010;2:1445-70. LTB shows adjuvant effect that elicited strong humoral and cellular immune response against the vaccine components.1515. Millar DG, Hirst TR, Snider DP. Escherichia coli heat- labile enterotoxin B subunit is a more potent mucosal adjuvant than its closely related homologue, the B subunit of cholera toxin. Infect Immun. 2001;69:3476. and 2121. da Hora VP, Conceição FR, Dellagostin OA, Doolan DL. Non- toxic derivatives of LT as potent adjuvants. Vaccine. 2011;29:1538-44. On the other hand, although ST antigens are prevalent in ETEC isolates, they cannot be used as vaccine components for their poor immunogenicity.1111. Nicklasson M. Studies on the expression and regulation of enterotoxins and colonization factors in Enterotoxigenic Escherichia coli (ETEC): Inst of Biomedicine. Dept. of Medical Microbiology and Immunology ; 2008. CssB have been reported as a key factor for binding to host cells77. Tobias J, Lebens M, Kallgard S, Nicklasson M, Svennerholm AM. Role of different genes in the CS6 operon for surface expression of enterotoxigenic Escherichia coli colonization factor CS6. Vaccine. 2008;26:5373-80. and a hundred amino acids from the C-terminal region of CssA have been reported to interact with the host cell fibronectin.44. Ghosal A, Bhowmick R, Banerjee R, et al. Characterization and studies of the cellular interaction of native colonization factor CS6 purified from a clinical isolate of enterotoxigenic Escherichia coli. Infect Immun. 2009;77:2125-35. We combined three fragments with linkers (EAAAK)4. Chimeric proteins with a helical linker are thought to have a more elongated conformation compared to those with a flexible linker. (EAAAK)4 sequences were introduced between different domains for more flexibility and efficient separation. The successful experience of using (EAAAK)4 sequences in chimeric gene has paved the way for it to bring about rationally acceptable results.1717. Nazarian S, Mousavi Gargari SL, Rasooli I, Amani J, Bagheri S, Alerasol M. An in silicochimeric multi subunit vaccine targeting virulence factors of enterotoxigenic Escherichia coli (ETEC) with its bacterial inbuilt adjuvant. J Microbiol Methods. 2012;90:36-45. The recombinant protein was confirmed by western blot using anti-His-tag -conjugated HRP and anti-CTXB antiserum as LTB is structurally very similar to B subunit of cholera toxin (CTXB). The purified rCS6 was used for immunization of mice and rabbit. In order to achieve an optimal induction and powerful response, we applied booster doses at least three weeks after the first injection. The doses were gradually decreased to increase affinity and to fully activate the B-cell immune cascade. The ELISA test results indicated that the immunized animals produced higher titer of anti-chimer antibody than each individual gene (to each individual gene alone) and immunization with chimeric rCs6 strongly protected animals from ETEC challenges. Accordingly a significant difference in antibody titer was observed between sera of the immunize animals and control groups. The finding further indicates that B-cell epitopes of this chimeric protein might be located in three distinct parts, selected as the CssA, CssB, and LTB.

The adhesion inhibition capacity of the antibodies raised against the recombinant chimer protein assayed with Caco-2 cells revealed that pretreatment of ETEC with immunized mice antisera decreased their adhesion properties and blocked their binding to Caco-2 cells. Attachment to epithelial cell culture of ETEC was effectively reduced in immunized mice compared to the control. This is consistent with the reports indicating that CssA, CssB were necessary for attachment to intestinal cells.44. Ghosal A, Bhowmick R, Banerjee R, et al. Characterization and studies of the cellular interaction of native colonization factor CS6 purified from a clinical isolate of enterotoxigenic Escherichia coli. Infect Immun. 2009;77:2125-35. and 77. Tobias J, Lebens M, Kallgard S, Nicklasson M, Svennerholm AM. Role of different genes in the CS6 operon for surface expression of enterotoxigenic Escherichia coli colonization factor CS6. Vaccine. 2008;26:5373-80.

The ability of the antibodies against rCS6 to block fluid accumulation was tested in rabbit intestinal loops. The results indicate that rCS6 antibody is capable of binding to the toxin and neutralizing its effect. Vaccination with a recombinant chimeric trivalent immunogen could protect the host against ETEC. Pretreatment of the ETEC cells with immunized mice antisera remarkably decreased their adhesion properties and blocked their binding to Caco-2 cells.

Since mucosal immunity, especially the production of IgA antibodies, is thought to be important for blocking the attachment of E. coli to epithelial cells, the latter aspect, such as the application of the chimeric recombinant protein in oral administration, should be considered in further immunization studies. However, it has been demonstrated with Citrobacter rodentium that secretory IgA is not necessary for preventing bacterial colonization in mice using intimin as a vaccine antigen.2222. Ghaem- Maghami M, Simmons CP, et al. Intimin-specific immune responses prevent bacterial colonization by the attaching-effacing pathogen Citrobacter rodentium. Infect Immun. 2001;69:5597-605.

In conclusion, our chimeric protein not only induced anti-toxins neutralizing antibodies, but also induced anti-adhesion antibodies for protecting against ETEC. The findings are in support of its use as an immunogen protein toward taking advantage of its components in vaccine strategies against ETEC.

Acknowledgments

The authors wish to thank Shahed University for the sanction of grants to conduct the present study.

REFERENCES

  • 1
    Wajima T, Sabui S, Fukumoto M, et al. Enterotoxigenic Escherichia coli CS6 gene products and their roles in CS6 structural protein assembly and cellular adherence. Microb Pathog. 2011;51:243-9.
  • 2
    Byrd W, de Lorimier A, Zheng ZR, Cassels FJ. Microencapsulated subunit vaccine approach to enterotoxigenic Escherichia coli and other mucosal pathogens. Adv Drug Deliv Rev. 2005;57:1362-80.
  • 3
    Yu J, Cassels F, Scharton- Kersten T, et al. Transcutaneous immunization using colonization factor and heat- labile enterotoxin induces correlates of protective immunity for enterotoxigenic Escherichia coli. Infect Immun. 2002;70:1056-68.
  • 4
    Ghosal A, Bhowmick R, Banerjee R, et al. Characterization and studies of the cellular interaction of native colonization factor CS6 purified from a clinical isolate of enterotoxigenic Escherichia coli. Infect Immun. 2009;77:2125-35.
  • 5
    Lapa JA, Sincock SA, Ananthakrishnan M, et al. Randomized clinical trial assessing the safety and immunogenicity of oral microencapsulated enterotoxigenic Escherichia coli surface antigen 6 with or without heat- labile enterotoxin with mutation R192G. Clin Vacc Immunol. 2008;15: 1222.
  • 6
    Walker RI, Steele D, Aguado T. Ad Hoc E. Analysis of strategies to successfully vaccinate infants in developing countries against enterotoxigenic E.coli (ETEC) disease. Vaccine. 2007;25:2545-66.
  • 7
    Tobias J, Lebens M, Kallgard S, Nicklasson M, Svennerholm AM. Role of different genes in the CS6 operon for surface expression of enterotoxigenic Escherichia coli colonization factor CS6. Vaccine. 2008;26:5373-80.
  • 8
    Tobias J, Svennerholm AM, Carlin NIA, Lebens M, Holmgren J. Construction of a non- toxigenic Escherichia coli oral vaccine strain expressing large amounts of CS6 and inducing strong intestinal and serum anti-CS6 antibody responses in mice. Vaccine. 2011;29:8863-9.
  • 9
    Wolf MK, Haan LAM, Cassels FJ, et al. The CS6 colonization factor of human enterotoxigenic Escherichia coli contains two heterologous major subunits. FEMS Microbiol Lett. 1997;148:35-42.
  • 10
    Jansson L, Tobias J, Jarefjäll C, Lebens M, Svennerholm AM, Teneberg S. Sulfatide recognition by colonization factor antigen CS6 from enterotoxigenic Escherichia coli. PloS one. 2009;4:e4487.
  • 11
    Nicklasson M. Studies on the expression and regulation of enterotoxins and colonization factors in Enterotoxigenic Escherichia coli (ETEC): Inst of Biomedicine. Dept. of Medical Microbiology and Immunology ; 2008.
  • 12
    Hardy S, Holmgren J, Johansson S, Sanchez J, Hirst TR. Coordinated assembly of multisubunit proteins: oligomerization of bacterial enterotoxins in vivo and in vitro. Proc Natl Acad Sci USA. 1988;85:7109.
  • 13
    Svennerholm AM, Steele D. Progress in enteric vaccine development. Best Pract Res Clin Gastroenterol. 2004;18:421-45.
  • 14
    Salimian J, Salmanian A, Khalesi R, Mohseni M, Moazzeni S. Antibody against recombinant heat labile enterotoxin B subunit (rLTB) could block LT binding to ganglioside M1 receptor. Iran J Microbiol. 2010;2:120.
  • 15
    Millar DG, Hirst TR, Snider DP. Escherichia coli heat- labile enterotoxin B subunit is a more potent mucosal adjuvant than its closely related homologue, the B subunit of cholera toxin. Infect Immun. 2001;69:3476.
  • 16
    Svennerholm AM, Tobias J. Vaccines against enterotoxigenic Escherichia coli. Expert Rev Vacc. 2008;7:795-804.
  • 17
    Nazarian S, Mousavi Gargari SL, Rasooli I, Amani J, Bagheri S, Alerasol M. An in silicochimeric multi subunit vaccine targeting virulence factors of enterotoxigenic Escherichia coli (ETEC) with its bacterial inbuilt adjuvant. J Microbiol Methods. 2012;90:36-45.
  • 18
    De SN, Chatterje D. An experimental study of the mechanism of action of Vibrio cholerae on the intestinal mucous membrane. J Pathol Bacteriol. 1953;66:559-62.
  • 19
    Byrd W, Mog SR, Cassels FJ. Pathogenicity and immune response measured in mice following intranasal challenge with enterotoxigenic Escherichia coli strains H10407 and B7A. Infect Immun. 2003;71:13-21.
  • 20
    Mudrak B, Kuehn MJ. Heat- labile enterotoxin beyond GM1 binding. Toxins. 2010;2:1445-70.
  • 21
    da Hora VP, Conceição FR, Dellagostin OA, Doolan DL. Non- toxic derivatives of LT as potent adjuvants. Vaccine. 2011;29:1538-44.
  • 22
    Ghaem- Maghami M, Simmons CP, et al. Intimin-specific immune responses prevent bacterial colonization by the attaching-effacing pathogen Citrobacter rodentium. Infect Immun. 2001;69:5597-605.

Publication Dates

  • Publication in this collection
    May-June 2014

History

  • Received
    02 June 2013
  • Accepted
    16 July 2013
Brazilian Society of Infectious Diseases Rua Augusto Viana, SN, 6º., 40110-060 Salvador - Bahia - Brazil, Telefax: (55 71) 3283-8172, Fax: (55 71) 3247-2756 - Salvador - BA - Brazil
E-mail: bjid@bjid.org.br