A Self-Assembling Whole-Cell Vaccine Antigen Presentation Platform

Bacterial strains and culture conditions.Vibrio cholerae strains were cultured in Luria-Bertani (LB) broth supplemented with 100 μg/ml streptomycin at 27°C, with shaking at 200 rpm. Escherichia coli was grown in LB broth at 37°C with shaking. Where necessary, plasmids were maintained with 100 μg/ml of ampicillin in the culture medium. Protein production from plasmid-borne P_tac was induced with 0.5 mM IPTG (isopropyl-β-d-1-thiogalactopyranoside). Frozen stocks were maintained in 15% glycerol at −80°C. The strains used in this study are listed in Table S1 in the supplemental material.

(i) Construction of prototype vaccines expressing plasmid-encoded R-CTB.The previously described pFLAG-CTC derivative carrying the gene encoding the B subunit of cholera toxin (CTB) fused to the gene encoding RbmA (R-CTB) (5) or CTB alone under the control of an IPTG-inducible promoter was introduced into wild-type MO10, MO10ΔctxA, or MO10ΔvpsA by electroporation. Protein production in positive transformants was verified by Western blotting using an anti-CTB antibody as described below.

(ii) Electroporation.Vibrio cholerae strains were inoculated into 30 ml of LB broth and were incubated at 22°C until an optical density of 0.3 was reached. Cultures were chilled on ice for 20 min and were then collected by centrifugation at 8,000 × g for 10 min at 4°C. Cells were washed twice in 13 ml of cold 2 mM CaCl₂ and once in 2 ml of cold 10% glycerol and were then collected by centrifugation at 6,000 × g for 10 min. The cells were finally resuspended in 10% glycerol and were electroporated in 50-μl aliquots by applying 1.8 kV to a 0.1-cm-gap cuvette. Cells were immediately recovered by adding 250 μl of LB medium and incubating for 1 h at 37°C with shaking at 200 rpm. Transformants were selected on LB agar containing 100 μg/ml of ampicillin and were confirmed by PCR and Western blotting.

Vaccine preparation. (i) Protein induction.Frozen stocks were inoculated into 3 ml of LB medium supplemented with streptomycin and ampicillin (LB-Sm/Amp) and were cultured at 27°C overnight. This starter culture was collected by centrifugation, washed once with LB-Sm/Amp, and subcultured in 25 ml of fresh LB-Sm/Amp in a 250-ml flask. After incubation for 6 to 8 h at 27°C with shaking, IPTG was added to a final concentration of 0.5 mM, and the culture was incubated for an additional 10 h at 27°C with shaking.

(ii) TCA precipitation of secreted proteins.Proteins secreted into the supernatant were precipitated with trichloroacetic acid (TCA) and were washed with acetone. Briefly, spent supernatant was passed through a 0.2-μm filter to remove bacterial cells. TCA was added to the cell-free supernatant to a final concentration of 10%, and the supernatant was incubated overnight at 4°C with gentle mixing. Precipitated proteins were collected by centrifugation and were washed three times with ice-cold acetone. Residual acetone was evaporated by brief incubation at 95°C. The protein pellet was resuspended in 4× Laemmli buffer containing β-mercaptoethanol and was prepared for Western blotting as described below.

(iii) Preparation of whole-cell vaccines.After protein induction, the bacterial culture was centrifuged at 5,000 × g for 15 min at 4°C to collect cells. The supernatant was passed through a 0.2-μm filter, and the resulting cell-free supernatant was used for Western blot analysis. The remaining bacterial pellet was washed three times with 12 ml of sterile PBS and was finally resuspended in 1 ml of PBS. This constituted the live whole-cell vaccine. For each vaccine preparation, 10 μl was removed to quantify CFU, and 20 μl was reserved for Western blot analysis. For each immunization, the vaccine was prepared and used within 2 h.

(iv) Western blot analysis.Supernatants and cell pellet samples were separated by centrifugation. TCA precipitation of the supernatant was performed prior to the detection of CTB. These samples were combined with 4× Laemmli buffer containing β-mercaptoethanol, sonicated in an ice bath, boiled for 5 min, and finally briefly centrifuged to remove particulates. Proteins were resolved on a denaturing 4-to-20% gradient Tris-HCl gel and were then transferred to a polyvinylidene difluoride membrane by semidry transfer (Bio-Rad). The membrane was blocked in Tris-buffered saline with 0.1% Tween (TBS-T) and 5% skim milk for 2 h at room temperature with gentle shaking. Fresh blocking solution containing a primary antibody was added at a 1:1,000 dilution. A polyclonal anti-CTB antibody conjugated to horseradish peroxidase (HRP) (PA1-85293; Pierce) was used to detect RbmA-CTB in cell pellets and native CTB in supernatants. After overnight incubation with primary antibodies, the membrane was washed three times with TBS-T. All membranes were developed using an ECL Western blotting substrate (Pierce).

(v) Quantification of R-CTB by densitometry.Known concentrations of purified CTB (List Laboratories) were resolved by SDS-PAGE alongside R-CTB samples and were used as standards for quantification. ImageJ was used to generate a standard curve fitted to the intensities of bands corresponding to the CTB standards. The concentration of R-CTB was calculated using the linear portion of the standard curve.

(vi) Coomassie staining.To ensure equal loading for Western blots, a duplicate gel was run in parallel, stained with Imperial Protein Stain (Thermo Fisher) according to the manufacturer's instructions, and destained with Nanopure water.

Immunization and sample collection. (i) Animals.Female 6- to 8-week old BALB/c mice were used in all immunization experiments. For sublingual immunizations, mice were purchased from Charles River Laboratories and were housed in a biosafety level 2 facility at Boston Children's Hospital with food and water ad libitum. Mice were acclimatized for 5 days. All procedures had been approved by the Institutional Animal Care and Use Committee previously.

(ii) Sublingual administration of a live-attenuated vaccine.Mice were first anesthetized by intraperitoneal injection with a cocktail of ketamine (100 mg/kg) and xylazine (10 mg/kg) and were then held upright while 10 μl of the vaccine was delivered under the tongue by a micropipette directed toward the floor of the mouth. Mice were maintained in the upright position for 2 min before resting, ventral side down, for at least 30 min until regaining consciousness.

(iii) Collection of blood and stool samples.Blood and stool samples were collected 1 day before vaccination and at designated time points throughout the study period. Fresh stool pellets were frozen at −80°C until use. Blood was collected from the tail vein using capillary tubes with a clot activator (Sarstedt). Sera were obtained by clearing the clotted blood by centrifugation at 10,000 × g for 5 min at room temperature and were stored at −20°C. Stool samples were prepared as described previously (29). Briefly, pellets were thawed on ice, transferred to 15-ml conical tubes containing 3 ml of chilled resuspension solution (0.1 mg/ml soybean trypsin inhibitor, a 3:1 mixture of PBS and 0.1 M EDTA), thoroughly homogenized, and centrifuged at 650 × g for 10 min at room temperature. The supernatant was collected and was centrifuged once more at 15,300 × g for 10 min at 4°C. PMSF (phenylmethane sulfonyl fluoride) was added to the supernatant to a final concentration of 2 mM. Stool samples were kept at −20°C or, for long-term storage, at −80°C.

(iv) Enumeration of V. cholerae CFU in fecal pellets.A fresh stool pellet was collected from each mouse 24 h and 48 h after sublingual immunization. The pellet was weighed, homogenized in 1 ml sterile PBS, and serially diluted. Portions (100 μl) of the undiluted and diluted stool suspensions were plated onto LB agar containing 100 μg/ml streptomycin and were incubated at 37°C overnight. The number of CFU was recorded and normalized to the weight of the pellet in order to calculate CFU per gram of stool. One-tenth of the total stool suspension, which contained approximately 22.7 mg stool/ml, was plated. Therefore, we estimate the lower limit of detection to be approximately 440 CFU/g.

Enzyme-linked immunosorbent assays (ELISA). (i) Quantification of CTB-specific antibodies by ELISA.(a) Standard curve. CTB-specific IgA was not available for use in a standard curve. Therefore, to assess linearity, standard curves were generated by capturing IgA and IgG in a reference mouse serum sample with goat anti-mouse IgG or IgA. Microtiter plate wells were incubated overnight with 100 ng of goat anti-mouse IgG or IgA diluted in sodium carbonate buffer. The wells were washed in PBS with 0.05% Tween 20 (PBS-T) and were blocked with PBS–bovine serum albumin (BSA). The reference mouse serum sample (Bethyl Laboratories) was diluted to 1 μg/ml of total IgG or IgA and was then applied to the wells. The wells were washed after overnight incubation, probed with HRP-conjugated goat anti-mouse antibodies, and developed with 3,3′,5,5′-tetramethylbenzidine substrate (TMB; Thermo Fisher).

(b) Test samples. Microtiter plates were coated with GM1 followed by 100 ng of purified CTB. The plates were blocked in PBS-BSA and were washed in PBS-T. Serially diluted serum or stool samples were applied to the wells and were incubated overnight. Serum dilutions ranged from 1:50 to 1:6,400, and stool dilutions ranged from 1:2 to 1:128. The plates were probed and developed as described above.

(ii) Quantification of total stool IgA by ELISA.Total fecal IgA was used to normalize antigen-specific IgA in the stool. Each well of a microtiter plate was coated with 100 ng of a goat anti-mouse IgA antibody in sodium bicarbonate buffer and was incubated overnight. Plates were washed in PBS-T and were blocked in PBS-BSA. Stool samples were serially diluted from 1:200 to 1:25,600 in PBS-T–BSA and were added to the plates. The plates were incubated overnight and were then probed and developed as described above. Standard curves were generated as described for CTB-specific antibodies above.

(iii) LPS extraction and measurement of O-antigen-specific antibodies.Lipopolysaccharide (LPS) was extracted from 50 ml of Vibrio cholerae MO10 (serotype O139) and N16961 (serotype O1) overnight cultures using a commercial kit (Bulldog Bio). Serum and stool antibodies recognizing the O1 or O139 serotype were quantified as described previously (30). A 1:1,000 dilution of LPS in sodium carbonate buffer was applied to microtiter plates and was incubated overnight. The plates were washed in PBS-T and were blocked for 40 min at 37°C in PBS-BSA. Serum and stool samples were applied to the plates in dilutions similar to those used to measure CTB-specific antibodies. The plates were first incubated for 90 min at 37°C and then washed in PBS-T. Plates were incubated for 90 min at 37°C after the addition of 100 ng of HRP-conjugated goat anti-mouse antibodies per well. Plates were developed using the same protocol described for the quantification of CTB-specific antibodies above.

Serum vibriocidal titers.Serum vibriocidal antibody titers were determined as described previously with the following modifications (31). Immunized mouse sera were incubated at 56°C for 1 h to inactivate endogenous complement, serially diluted 2-fold in PBS in 0.5-μl tubes, and kept on ice. Wild-type MO10 was grown to mid-logarithmic phase in brain heart infusion broth containing 100 μg/ml streptomycin and was diluted in PBS containing 20% guinea pig complement to 4 × 10⁶ CFU/ml. An equal volume of this suspension was added to the serum dilutions to obtain a final concentration of 10% complement and 2 × 10⁶ CFU/ml V. cholerae; the mixture was incubated for 1 h at 37°C with shaking at 200 rpm; and viable cells were enumerated by plating. The bactericidal titer was determined as the reciprocal of the serum dilution capable of killing 50% or more of the indicator strain compared with a control containing preimmune or PBS-immunized serum. Sera from mice that received the inactivated vaccine were randomly pooled into groups of three for the determination of vibriocidal titers.

Infant mouse challenge model. (i) Orogastric challenge of infant mice.At the end of the study period (between 60 and 70 days after the initial immunization), vaccinated female mice were mated with age-matched males. Nonvaccinated, nontimed pregnant mice were purchased from Charles River Laboratories and were housed in the same facility as the vaccinated mice. When pups were born, wild-type MO10 was grown overnight in LB broth with 100 μg/ml streptomycin at 27°C. The cell density was adjusted to 5 × 10⁹ CFU/ml. Bacteria were collected by centrifugation at 6,000 × g for 5 min and were resuspended in 2.5% sodium bicarbonate (0.29 M). Four- to 5-day-old pups were challenged with 2.5 × 10⁷ CFU of wild-type MO10 delivered in 50 μl of sodium bicarbonate solution by oral gavage. Pups were monitored for immediate signs of distress and were then returned to the dam. All pups were sacrificed 24 h after infection, and signs of disease were documented.

(ii) Quantification of bacterial colonization.The small and large intestines were weighed, added to sterile conical tubes containing 1 ml of PBS, and homogenized. The homogenates were serially spread plated on LB agar supplemented with 100 μg/ml of streptomycin. Plates were incubated overnight at 37°C. The limit of detection for each spread plate is 10 CFU per intestine.

Ethics statement.Animal experiments were performed in accordance with the standards outlined in the National Research Council's Guide for the Care and Use of Laboratory Animals (32) and Boston Children's Hospital's Public Health Service Animal Welfare Assurance. The protocol was approved by the Boston Children's Hospital Institutional Animal Care and Use Committee (IACUC), appointed to review proposals for research involving vertebrate animals.

Statistical analysis.Statistical analyses were performed in GraphPad Prism, version 7. One-way ordinary analysis of variance (ANOVA) with Tukey's test was used for multiple comparisons. A two-tailed, unpaired Mann-Whitney test was used for pairwise comparisons. All vaccine groups consisted of 10 mice. Error bars indicate standard deviations unless otherwise noted. Western blot images are representative of experimental triplicates.