Enterotoxigenic Escherichia coli Adhesin-Toxoid Multiepitope Fusion Antigen CFA/I/II/IV-3xSTa_N12S-mnLT_G192G/L211A-Derived Antibodies Inhibit Adherence of Seven Adhesins, Neutralize Enterotoxicity of LT and STa Toxins, and Protect Piglets against Diarrhea

Bacterial strains and plasmids.E. coli strains and plasmids used for this study are included in Table 1. ETEC field isolates producing CFA/I, CS3, CS4/CS6, CS5/CS6, or CS6 (provided by Johns Hopkins University, Washington University, and the University of Gothenburg E. coli Reference Strain Center, Sweden) and E. coli recombinant strains expressing CS1 or CS2 fimbrial adhesin (35, 36) were used for bacterial fimbria extraction (for coating antigens of antibody titration enzyme-linked immunosorbent assays [ELISAs]) and for in vitro antibody adherence inhibition assays. Vector pET28α (Novagen, Madison, WI) and E. coli BL21 (GE Healthcare, Piscataway, NJ) were used to express the adhesin-toxoid MEFA recombinant protein.

Construction of the tagless adhesin-toxoid MEFA gene.PCRs with designed primers (Table 3) were used to amplify the CFA/I/II/IV MEFA gene from strain 9175 (18) and the toxoid fusion 3xSTa_N12S-mnLT_R192G/L211A gene from strain 9331 (21). Toxoid fusion 3xSTa_N12S-mnLT_R192G/L211A, previously named 3xSTa_N12S-dmLT, carried three copies of STa toxoid STa_N12S and a monomeric LT peptide, which consists of one LT_A subunit domain mutated at the 192th and the 211th residues and one LT_B subunit domain. As described previously (19, 22), splicing overlap extension (SOE) PCR was used to overlap three PCR products into a single chimeric gene (Fig. 1A). The first PCR, using primers STaNcoI-F and LT30CFA-R and toxoid fusion 3xSTa_N12S-mnLT_R192G/L211A DNA template, amplified the first copy of STa_N12S and the first 30 amino acids of the LT_A domain. The second PCR, using primers LT30CFA-F and LT160CFA-R and CFA/I/II/IV MEFA DNA template, generated the CFA/I/II/IV fragment. The third PCR, using primers LT160CFA-F and T7-R and 3xSTa_N12S-mnLT_R192G/L211A DNA template, yielded the C-terminal fragment of 3xSTa_N12S-mnLT_R192G/L211A, starting from the 160th amino acid of LT_A. Three PCR products generated with Pfu DNA polymerase were purified and then overlapped in a final SOE PCR. The overlapped chimeric gene was further amplified with primers STaNcoI-F and T7-R, digested with NcoI and EagI restriction enzymes (New England BioLabs, Ipswich, MA), and ligated into expression vector pET28α. Since the NcoI restriction site in pET28α expression vector is located upstream of the 6×His tag region and the third copy of the STa_N12S toxoid gene (at the 3′ end) has a stop codon, this adhesin-toxoid MEFA chimeric gene did not carry 6×His tag nucleotides. The cloned tagless chimeric gene was verified initially by DNA sequencing.

Expression, extraction, and characterization of the tagless adhesin-toxoid MEFA protein.Plasmids (pET28α) carrying the chimeric adhesin-toxoid MEFA gene were introduced into E. coli BL21 bacteria to express tagless adhesin-toxoid MEFA protein. Protein expression, extraction, and refolding followed protocols described previously (37, 38). Briefly, a single colony of the recombinant strain was cultured in 5 ml lysogeny broth (LB) supplemented with kanamycin (30 μg/ml) at 37°C on a shaker (150 rpm). Overnight-grown bacteria were transferred to 500 ml 2× YT (2× yeast extract tryptone) medium broth and incubated continuously until culture optical density at 600 nm (OD₆₀₀) reached 0.5 and then induced with isopropyl-1-thio-β-d-galactoside (IPTG; 0.5 mM) and cultured for 4 h. Bacteria were collected, lysed in 10 ml bacterial protein extraction reagent (B-PER; Pierce, Rockford, IL) after freeze-thaw, and incubated at room temperature for 30 min. Bacterial lysates were centrifuged, suspended in 10 to 20 ml B-PER with vigorous vortexing and pipetting, mixed with 200 to 400 μl freshly prepared lysozyme (10 mg/ml in B-PER), and incubated at room temperature for 20 min. Suspensions were centrifuged, and pellets were suspended in B-PER and incubated with lysozyme. After centrifugation, inclusion body proteins were suspended in 100 ml 1:10 diluted B-PER, vortexed, and centrifuged. Protein pellets were washed with 100 ml phosphate-buffered saline (PBS) three times, vortexed vigorously, centrifuged at 12,000 rpm for 15 min at 4°C, and finally dissolved in 10 to 20 ml PBS.

Extracted proteins were further solubilized with solubilization buffer and refolded with a protein refolding kit by following the manufacturer's protocols (Novagen). Proteins were refolded in molecular porous membrane tubing (Spectrum Laboratories, Inc., Rancho Dominquez, CA) with refolding buffer (20 mM Tris-HCl supplemented with 0.1 mM dithiothreitol [DTT]) and then dialyzed with refolding buffer without DTT. Refolded and solubilized proteins were centrifuged and collected (in supernatant). Proteins were measured for concentration (milligrams per milliliter) and stored at −80°C.

Refolded protein (10 μg) was examined by 12% SDS-PAGE and immune blot assays with monoclonal anti-CFA/I, -CS1, -CS2, -CS3, -CS4, -CS5, and -CS6 antibody hybridoma supernatant (provided by A. M. Svennerholm at the University of Gothenburg, Sweden), rabbit anti-CT (1:5,000; Sigma, St. Louis, MO), and anti-STa sera (provided by D. C. Robertson at Kansas State University). IRDye-labeled goat anti-mouse or anti-rabbit IgG (1:5,000 dilution; LI-COR, Lincoln, NE) and a LI-COR Odyssey premium infrared gel imaging system (LI-COR) were used for protein detection.

Mouse i.p. immunization with the tagless adhesin-toxoid MEFA protein.Three groups of 8-week-old female BALB/c mice (Charles River Laboratories International, Inc., Wilmington, MA), 10 mice per group, were used in the i.p. immunization study. Each mouse in the first group was i.p. injected with 200 μg tagless adhesin-toxoid MEFA protein CFA/I/II/IV-3xSTa_N12S-mnLT_R192G/L211A (in 200 μl protein buffer, 20 mM Tris-HCl) and 200 μl Freund's incomplete adjuvant (Sigma). The second group was immunized with the same amount of CFA/I/II/IV-3xSTa_N12S-mnLT_R192G/L211A MEFA protein but with 2 μg dmLT adjuvant (in 200 μl protein buffer; provided by Walter Reed Army Institute of Research, Silver Spring, MD). The third group was injected with 200 μl protein buffer as the negative control. Immunized mice received two booster injections with the same dose of the primary at an interval of 2 weeks. Mice were anesthetized with CO₂ and exsanguinated 2 weeks after the second booster. Blood samples were collected from each mouse before the primary immunization and 14 days after the final booster. Mouse serum samples were stored at −80°C until use. Mouse immunization complied with the Animal Welfare Act by following the 1996 National Research Council guidelines and was approved by the Kansas State University Institutional Animal Care and Use Committee (IACUC).

Mouse serum antiadhesin and antitoxin antibody titration.IgG antibodies to CFA/I, CS1, CS2, CS3, CS4, and CS5 fimbriae and also to STa and LT toxins from mouse serum samples were titrated in ELISAs as described previously (18, 19, 21, 22, 25). Briefly, wells of 2HB plates (Thermo Scientific, Rochester, NY) were coated with heat-extracted CFA/I, CS1, CS2, CS3, CS4, or CS5 fimbriae (200 ng per well) and LT (List Biological Laboratories, Inc., Campbell, CA; 100 ng per well) to titrate antibodies to each CFA fimbrial adhesin and to LT, whereas STa-ovalbumin conjugates (10 ng per well) and Costar plates (Corning Inc., Corning, NY) were used to titrate anti-STa IgG antibodies. Serum samples of each mouse were 2-fold diluted (from 1:200 to 1:51,200) and were examined in triplicate. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:3,300; Sigma) and the 3,3′,5,5′-tetramethylbenzidine (TMB) microwell peroxidase substrate system (2-C) (KPL, Gaithersburg, MD) were used to measure optical absorbance (OD₄₀₅). The highest serum dilution that produced OD readings of >0.3 above the background was calculated for antibody titers. Antibody titers were presented in a log₁₀ scale as previously described (20, 21, 23, 25, 37).

Mouse serum antibody adherence inhibition assay.Mouse serum samples from each group were examined for in vitro antibody inhibition activities against adherence of E. coli bacteria expressing CFA/I, CS1, CS2, CS3, CS4/CS6, CS5/CS6, or CS6 to Caco-2 cells (ATCC HTB-37). As described previously (18, 25), overnight-grown ETEC or E. coli bacteria scraped off from sheep blood agar plates were gently suspended in sterile PBS (1.05 × 10⁷ bacteria in 100 μl PBS, with a multiplicity-of-infection ratio set at 15 to 1). Bacteria were pretreated with 4% mannose (to prevent type 1 fimbriae from attachment), mixed with 20 μl mouse serum sample from each group, brought to 300 μl with PBS, and then transferred to 90 to 95% confluent monolayer Caco-2 cells (7 × 10⁵ per well in 700 μl cell culture medium). After incubation in a CO₂ incubator (5% CO₂) for 1 h at 37°C, Caco-2 cells were gently washed with PBS to remove nonadherent ETEC or E. coli bacteria and then dislodged with 0.25% trypsin (200 μl per well). Bacteria adherent to the Caco-2 cells were collected by centrifugation, suspended in 1 ml PBS, serially diluted, and plated on LB plates. Bacteria were counted for CFU after overnight growth at 37°C. Antibody adherence inhibition activities were reflected by reduction of the numbers of bacteria adherent to Caco-2 cells (in CFU or percentages, with the control set to 100%).

Mouse serum antibody neutralization against CT and STa enterotoxicity.Enzyme immunoassay (EIA) cAMP and cGMP kits (Enzo Life Sciences, Farmingdale, NY) and T-84 cells were used to measure mouse serum antibody neutralization activities against enterotoxicity of STa and CT (cholera toxin; a homologue of LT commonly used in anti-LT antibody neutralization assays). As described previously (19–23, 25), mouse serum samples were examined for prevention of STa toxin from stimulating intracellular cGMP levels or CT from elevating intracellular cAMP levels in T-84 cells. Mouse serum sample (30 μl) from each group was mixed with STa toxin (2 ng or 5 ng) or CT (10 ng). Each serum-toxin mixture was incubated at room temperature for 30 min and then was transferred to 90 to 95% confluent T-84 cells and incubated in a CO₂ incubator (1 h for STa to measure cGMP or 3 h for CT to measure cAMP). Intracellular cGMP or cAMP levels (in picomoles per milliliter) of T-84 cells were measured with an EIA cGMP or cAMP kit by following the manufacturer's protocol (Enzo Life Sciences).

Pig i.m. immunization with the tagless adhesin-toxoid MEFA protein.A total of eight pregnant gilts from the Kansas State University Swine Unit and a local producer, which had no record of diarrhea and were verified not to have preexisting anti-STa and anti-LT antibodies, were raised at a university large-animal research center. As described previously (24), four pregnant gilts each were i.m. immunized with 500 μg tagless adhesin-toxoid MEFA protein and 5 μg dmLT adjuvant 6 to 8 weeks before farrowing, followed by a booster injection at the same dose of the primary 4 weeks later. Four pregnant gilts without immunization were used as the control.

Blood samples were collected from gilts before immunization and after farrowing, and colostrum samples were collected before farrowing.

Pig serum and colostrum antiadhesin and antitoxin antibody titration.Serum samples from each immunized or control gilt were examined for IgG antibodies to STa, LT, CFA/I, and CS1 to CS6, and pig colostrum samples were titrated for IgG and IgA antibodies to each toxin and adhesin. HRP-conjugated goat-anti-porcine IgG and IgA (1:3,000 dilution; Bethyl Laboratories, Montgomery, TX) were used as the secondary antibodies (24). Serum and colostrum samples from each immunized or control pig were examined for antibody neutralization activities against STa and CT, as well as antibody adherence against CFA/I and CS1 to CS6 adhesins as described above.

Piglet ETEC challenge study.Piglets born to immunized and control mothers were used to assess protective efficacy of anti-STa and anti-LT antibodies against ETEC diarrhea. As described previously (24), piglets born to three immunized mothers and three control mothers, after 24 h of suckling, were orally inoculated with 5 × 10⁹ CFU STa⁺ ETEC strain 8823 (G58/987P/STa) (24). Suckling piglets born to the fourth immunized sow and the fourth control sow were inoculated with 5 × 10⁹ CFU LT⁺ ETEC strain 8819 (G58/987P/LT). Challenged piglets were monitored every 2 to 4 h until 24 h postinoculation and were weighed before challenge and 24 h postchallenge. Clinical symptoms, including diarrhea, dehydration, and lethargy, were recorded from each challenged piglet.

Serum samples were collected from each piglet at necropsy and were titrated for IgG antibodies to each CFA adhesin and toxin as described above. Pig immunization and piglet challenge studies complied with the USDA Animal Welfare Act Regulations and were approved by the Kansas State University IACUC.

Statistical analysis.Mouse and pig antibody titration and antibody neutralization data are presented as means ± standard deviations. Differences in antibody titers (in log₁₀ scale) between the immunized group and the control group were measured using Student's t test with SAS for Windows, version 8 (SAS Institute, Cary, NC). Differences of antibody neutralization activities (cAMP and cGMP, in picomoles per milliliter), antibody adherence inhibition activities from serum or colostrum samples between the immunized group and the control group, and piglet challenge outcome data between the piglets born to immunized mothers and piglets born to control mothers were compared with the nonparametric Wilcoxon matched-pairs test. A P value of <0.05 indicated significance of differences when two treatments were compared using two-tailed distribution and two-sample unequal variance.