Immunization with gold nanoparticle-delivered EHEC antigens reduces in vivo colonization.

To test the immunogenicity and protective capacity of these four antigens when delivered as conjugates to gold nanoparticles, we immunized BALB/c mice intranasally with 5-μg doses in 2-week intervals (Fig. 1). Following the administration of a prime and two boosts of each vaccine formulation, we assess the in vivo protection against EHEC colonization in a mouse model of intestinal colonization. Three weeks after receiving the last immunization (Fig. 1), animals were challenged via gavage with 9×10⁹ CFU/mouse of EHEC O157:H7 strain 86-24. Three and five days after challenge, individual segments along the gastrointestinal tract were collected, processed, and plated to enumerate bacterial colonization. Animals immunized with individual formulations, including AuNP-LomW, AuNP-EscC, AuNP-LpfA1, and AuNP-LpfA2, showed significantly lower bacterial colonization along the large intestines (LI) and ceca by days 3 and 5 postchallenge (Fig. 2A to ​toD).D). However, no significant differences were observed in the small intestine (SI) at any of the time points evaluated (Fig. 2E and ​andF).F). Taken together, these results demonstrate the ability of the gold nanoparticle EHEC-specific vaccine candidates to reduce colonization when administered intranasally.

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FIG 1

Timeline depicting the intranasal vaccination strategy. Illustration highlights the timeline of the experiments used to test the efficacy and mechanism of protection of the different AuNP protein candidates. BALB/c (n=10) mice were intranasally immunized with three doses of 50-μl formulations in 2-week intervals. Two weeks after receiving the last immunization, serum and fecal samples were obtained to evaluate humoral responses. Seven days after sample collection, animals were challenged with 9×10⁹ cells of EHEC O157:H7 via gavage. Individual intestinal segments were collected 3 or 5 days after infection and processed for CFU enumeration. Two weeks after receiving the last immunization, the intestinal epithelial layer (IEL) was isolated and stimulated in the presence of each antigen for 5days. After stimulation, samples were analyzed by a multiplex cytokine array. Small-intestine lavage samples from immunized and naive animals were obtained 2 weeks after receiving the last immunization to assess secretory IgA levels.

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FIG 2

Animals immunized with individual AuNP-coupled vaccines exhibited reduced EHEC colonization. The cecum, small intestine (SI), and large intestine (LI) from vaccinated animals (n=5) were collected three (A, C, and E) and five (B, D, and F) days after EHEC challenge, processed, and plated to assess colonization. Numbers of adhered bacteria were determined per gram of tissue, and adhesion is shown on a log₁₀ scale. The bacterial limit of detection (LOD) was 10 CFU/organ. All colonization data are shown as means ± standard errors of the means (SEM) from results determined for each group. Significant differences in organ colonization were determined via one-way ANOVA followed by Tukey’s post hoc test (*, P<0.05; **, P<0.01).

Gold nanovaccine single formulations elicit robust humoral and cell-mediated responses, both systemically and tissue specific, following intranasal immunization.

To evaluate the immunogenicity of the different single-vaccine formulations, we evaluated the antigen-specific humoral responses 2 weeks after receiving the last dose. To assess differences in systemic versus local humoral responses, we collected the serum and intestinal lavage of immunized mice to assess IgG and sIgA antibody responses (Fig. 3). We detected significantly higher antigen-specific IgG and sIgA responses from single-formulation-immunized animals than naive mice (Fig. 3A and ​andB).B). In addition, we evaluated the cell-mediated tissue-specific responses elicited at the intestinal epithelial layer (IEL). Intraepithelial lymphocytes were isolated from the intestine of immunized animals, and the cell suspensions were restimulated for 5 days in the presence of each antigen to assess antigen recall responses by assessing cytokine secretion. Intraepithelial lymphocytes from AuNP-EscC or AuNP-LpfA2 did not show enhanced cytokine secretion upon restimulation, whereas AuNP-LpfA1-immunized animals in the presence of LpfA1 produced significantly higher levels of interleukin-2 (IL-2), gamma interferon (IFN-γ), IL-17, MCP-1, and MIP-1β than naive controls (Fig. 3C, ​,D,D, and ​andFF to ​toH).H). Furthermore, animals immunized with the AuNP-LomW formulation elicited higher IFN-γ and IL-4 levels in response to antigen restimulation (Fig. 3D and ​andE).E). Taken together, these results highlight the ability of single AuNP-protein formulations to elicit robust mixed humoral and tissue-specific cellular responses associated with reduced colonization and protection against in vivo infection.

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FIG 3

AuNP vaccine intranasal immunization elicits robust antigen-specific humoral and cell-mediated responses associated with reduced bacterial adherence. Systemic and mucosa-specific humoral responses were analyzed using sera (A) and intestinal lavage (B) from immunized animals. The intestinal epithelial layer containing intraepithelial lymphocytes was isolated from immunized animals and stimulated in the presence of each antigen for 5days. Medium-only and anti-CD3/CD28-coated beads served as negative and positive controls, respectively. (C to H) Supernatants from cells stimulated in the presence or absence of antigen were collected and analyzed for cytokine release using a mouse cytokine Bio-Plex. Only the antigens (LpfA1 and LomW) that showed a cytokine response are shown. All humoral and cytokine data are shown as ±SEM from results determined for each group. Significant differences in humoral or cytokine levels were determined via one-way ANOVA followed by Tukey’s post hoc test (*, P<0.05; **, P<0.01; ***, P<0.001).

Immune sera from animals receiving individual AuNP vaccine formulations inhibited bacterial adherence on primary IECs as well as pedestal formation.

To further understand the mechanism of vaccine-induced protection, we evaluated whether serum from immunized mice bind and neutralize bacteria, preventing adherence onto IECs. We observed a complete ablation of EHEC-mediated A/E lesions, as measured by the reduction on pedestal formation, as well as a reduction on adherence onto IECs, of bacteria in contact with immune sera from vaccinated mice compared to untreated or naive sera (Fig. 4A). To quantify the percentage of bacterial adherence onto IECs, a serum inhibition of adherence assay was performed. We detected significantly lower bacterial adherence in the presence of AuNP-LomW, AuNP-EscC, AuNP-LpfA1, and LpfA2 compared to naive or untreated bacteria (Fig. 4B). To assess if the inhibition of adherence was due to the antibody recognition properties from immune serum, immunostaining with the serum from immune animals was used. The sera from immunized animals recognized the outer membrane of EHEC O157:H7 (Fig. 4C), suggesting that serum recognition of surface-exposed antigens is responsible for the inhibition on adherence. Together, these data indicate that AuNP-delivered EHEC-specific vaccine antigens elicit a robust humoral response associated with inhibition of adherence and pedestal formation, a phenomenon mediated by surface-expose antigen recognition.

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FIG 4

Sera from immunized animals with individual vaccine formulations reduced adherence and pedestal formation on mouse primary intestinal epithelial cells (IECs). (A) Mouse primary IECs were infected at an MOI of 10 with EHEC O157:H7 previously incubated in the presence or absence of naive (adjuvant-only) or immune sera for 1 h at 37°C. After infection, cells were washed to remove nonadherent bacteria and fixed and stained with rhodamine phalloidin and DAPI for IECs and an EHEC-specific primary antibody conjugated to FITC. (B) The percentage of adhered bacteria was calculated 1 h after infection, where cells were washed, detached, and processed for CFU enumeration. (C) Recognition of EHEC O157:H7 by immune sera was visualized using immune or naive sera from vaccinated animals, followed by a secondary goat anti-mouse IgG antibody conjugated to Alexa488. All adherence data are expressed as means ± SEM of results from at least three independent experiments using sera from n=8 mice per group. Significant differences were determined via one-way ANOVA followed by Tukey’s post hoc test (**, P<0.01; ns, not significant).

Cloning.

EHEC (EDL933) genomic DNA was isolated via a Qiagen DNeasy blood and tissue kit according to the manufacturer’s directions. Sequences encoding LomW (GI:12514345), EscC (GI:12518466), LpfA1 (GI:12518278), and LpfA2 (GI:12518581) were amplified via Phusion polymerase (New England BioLabs) and cloned into a pET30a(+) expression vector using NdeI and XhoI or HindIII-HF (New England BioLabs) restriction sites. The open reading frame encoding each protein was inserted in-frame with a 6×-histidine (His) tag on the C terminus. Ligation, transformation, and expression were performed according to the manufacturer’s directions (pET System; Novagen), with some modifications. Upon confirmation of successful gene insertion via gel electrophoresis and directional sequencing (UTMB Genomics Core), plasmids were transformed into BL21(DE3) competent E. coli (New England BioLabs) via heat shock treatment.

Protein purification.

To induce protein expression, overnight cultures were diluted 1:20 in 1 liter of LB broth, grown to an optical density at 600 nm between 0.6 and 0.8, and induced with 1mM final concentration of isopropyl β-d-1-thiogalactopyranoside (IPTG). At 3 h postinduction, cultures were centrifuged (4,000×g for 15min), and the resulting bacterial pellet was frozen at −20°C. The bacterial pellets were then resuspended in phosphate-buffered saline containing 10% glycerol and 25 mM sucrose with 1-mg/ml final concentration of lysozyme, 0.2% sodium deoxycholate, and a tablet of cOmplete EDTA-free protease inhibitor cocktail (Roche, Germany). This lysate was then sonicated and centrifuged, and the pellet was used for subsequent washes to maximize soluble protein extraction. After spindown, the supernatant was filtered sterilized (0.2mm). Soluble protein extracts were then bound to Talon nickel columns (GE Healthcare, USA) and washed with a PBS buffer containing 50mM imidazole. Proteins were eluted from affinity columns by applying a PBS buffer with 10% glycerol, 25mM sucrose, and 250mM imidazole. Fractions were collected and pooled before dialyzing overnight at 4°C. The purified proteins and protein standards were subjected to a colorimetric bicinchoninic acid (BCA) assay according to the manufacturer’s protocol and then stored at −80°C until use. For protein visualization, 2mg of protein was run on SDS-PAGE gel. Protein bands were visualized by staining with Coomassie blue stain (Bio-Rad), or gels were transferred to a nitrocellulose membrane for Western blot analysis. A mouse antihistidine antibody (1:5,000) was incubated overnight at 4°C, and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG was used as a secondary antibody. Protein bands were visualized by adding ECL substrate (ThermoFisher Scientific, USA) and imaged on a film.

Coupling of protein candidates onto AuNPs.

AuNPs, 15nm in diameter, were synthesized, conjugated to individual proteins, and characterized as previously described (25,–27). Briefly, 1mM gold(III) chloride trihydrate underwent a reduction reaction with 90mM sodium citrate dihydrate. Particle size and shape were established by transmission electron microscopy (TEM). To stabilize the conjugation of soluble antigens onto the AuNP surface, 0.1mM 16-mercaptohexadecanoic acid (16-MHDA) and 0.1% Triton X-100 were added to AuNPs. After 2 h of incubation, this solution was filtered with centrifugation (EMB Millipore Amicon Ultra-15, 30-kDa molecular size cutoff) and repeated to ensure coverage. To covalently link soluble protein, 20mg/ml nanoparticles was added in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate (DMTMM). The reactions were carried out in a 100mM borate buffer to allow for conjugation. Attachment of 16-MHDA and protein was confirmed by measuring plasmon resonance via UV-Vis spectroscopy as well as by SDS-PAGE.

Animal studies.

Female, 6- to 8-week-old BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and maintained at animal biosafety level 2 (ABSL2). Animals were housed in microisolator cages under pathogen-free conditions with food and water ad libitum and maintained on a 12-h light cycle. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch (protocol no. 1007037C). To allow adequate acclimation, mice were housed within the animal facility for 1week prior to experimentation.

Immunization and challenge study.

To evaluate the protective immunogenicity of LomW, EscC, LpfA1, or LpfA2 coupled to AuNPs (AuNP-LomW, AuNP-EscC, AuNP-LpfA1, or AuNP-LpfA2), anesthetized BALB/c mice (n=10 per group) were intranasally (i.n.) vaccinated three times in 2-week intervals. Animals received either the AuNP-LomW, AuNP-EscC, AuNP-LpfA1, or AuNP-LpfA2 formulation. Each vaccine formulation contained 5μg of protein along with 10μg of detoxified cholera toxin (CT) (Sigma). Control groups received equivalent amounts of adjuvant alone. To evaluate antibody titers, blood was drawn retroorbitally 2weeks following the last boost. To isolate sera, blood was incubated for 30min at room temperature to allow for clotting and centrifuged (10,000×g for 10min). Serum was removed and stored at −80°C until use. For assays requiring serum, the sera from all immunized animals (n=10) were pooled and stored.

Infection and bacterial colonization.

Two weeks after administering the last immunization, all mice were challenged with a dose of ~9×10⁹ CFU of E. coli O157:H7 strain 86-24 via gavage (200μl). Food was restricted 12 h before infection but was administered ad libitum throughout the remainder of the study. Two hours prior to the challenge, mice were injected intraperitoneally with cimetidine (50mg/kg; Sigma) to reduce stomach acidity. To enumerate bacterial colonization in the gastrointestinal tract, mice were euthanized and ceca, large intestines, and small intestines were removed. Sections from the gastrointestinal (GI) tract were homogenized in 1ml PBS, serially diluted, and plated on MacConkey agar and incubated at 37°C to enumerate bacterial colonization.

Detection of protein-specific antibodies.

Baseline serum and fecal samples from adjuvant-only, AuNP-LomW-, AuNP-EscC-, AuNP-LpfA1-, or AuNP-LpfA2-vaccinated mice were collected 7days before prime (day 0) and 14days after the second boost. Whole blood was collected via retroorbital bleeding and stored in microvette tubes without anticoagulant. The serum was separated by centrifugation and stored at −80°C until use. Intestinal lavage was collected 14days after the last immunization by washing the small intestine with 1ml of PBS using a 3-gauge needle and centrifuged to remove fecal debris, and the supernatants were stored at −80°C until use. The protein-specific total IgG and IgA titers were determined by indirect enzyme-linked immunosorbent assay. A microplate (Costar, Cambridge, MA) was coated at 4°C overnight with 1μg/well of each protein (LomW, EscC, LpfA1, LpfA2) in 1× Dulbecco’s phosphate-buffered saline (DPBS) (Corning, USA). Wells were washed twice with washing buffer (0.05% Tween 20 in 1× DPBS) and then blocked with blocking buffer (0.05% Tween 20, 2% BSA, 1× DPBS) at room temperature (RT) for 2 h. Blocked wells were washed twice before adding sample diluent (1% BSA, 0.05% Tween 20 in 1× DPBS). The sera or intestinal lavage were added to each top dilution well in triplicate, and 2-fold dilutions were performed following incubation at RT for 2 h. Diluted goat anti-mouse IgG or IgA (Southern Biotech, USA) (1:5,000) was added into each well and then incubated for 3 h after washing. Plates were washed four times prior to addition of tetramethylbenzidine (TMB) substrate solution (Invitrogen, USA). The stop solution (2N H₂SO₄) was added, and the samples were immediately read at 450 and 570nm using a microplate reader (BioTek, USA). The results were reported as the reciprocal of the highest titer giving an optical density (OD) reading of at least the means ± 2 standard deviations from baseline sera or intestinal lavage. All assays were performed in triplicate, and results were shown as the mean reciprocal endpoint titer.

Cell-mediated response from intraepithelial lymphocytes.

Small intestine epithelial cells from AuNP-LomW-, AuNP-EscC-, AuNP-LpfA1-, or AuNP-LpfA2-immunized mice were obtained 2weeks after the last immunization. IEL were isolated from the small intestine from control (adjuvant-only) and different formulation-vaccinated mice according to an established protocol (36). Single-cell suspensions of isolated IELs were cultured in 12-well cell culture-grade plates (Corning, USA) in duplicate at 1×10⁶ cells/ml in RPMI 1640 (Gibco, Life Technologies) supplemented with 10% fetal calf serum (Invitrogen Life Technologies), 1mM sodium pyruvate, 2mM l-glutamine, 100 U penicillin/ml, and 100mg streptomycin/ml (complete medium), with protein antigens as stimuli. The IEL cell suspensions were stimulated with different stimuli for 5days, including LomW (10μg/ml), EscC (10μg/ml), LpfA1 (10μg/ml), LpfA2 (10μg/ml), and αCD3/αCD28 magnetic antibody-coupled beads with 30 U/ml mouse recombinant IL-2 and complete medium alone. After 5days of incubation at 37°C in a humidified atmosphere (5% CO₂ and 95% air), cell culture supernatants were collected and immediately stored at −80°C until further analysis. Cytokine production was analyzed using a BioPlex kit (Bio-Rad, USA) according to the manufacturer’s instructions. Cytokine analysis of restimulated mouse IEL cell supernatants was done using BioPlex kit (Bio-Rad, USA) analysis according to the manufacturer’s instructions.

Macrophage survival assay.

C57BL/6 murine bone marrow-derived primary macrophages (BMDM) (no. C57-6030; Cell Biologics Inc., Chicago, IL) were routinely grown in complete primary cell culture medium by following the manufacturer’s instructions (no. M3368; Cell Biologics). Cells were incubated at 37°C and 5% CO₂. For infection and microscopic analysis, 5×10⁵ cells/well were grown in 12-well cell culture-grade plates in round coverslips and incubated overnight prior to treatment. Bacterial inoculum used at a multiplicity of infection of 10 (5×10⁶ CFU) was incubated in the presence or absence of immune serum from immune groups (AuNP-LomW, AuNP-EscC, AuNP-LpfA1, and AuNP-LpfA2) or naive sera (final concentration of 10%) for 1 h at 37°C, with slight agitation. After incubation in the presence or absence of sera, bacteria were collected in 1ml of fresh medium and used to infect cell culture plates containing 5×10⁵ cells. After 2 h of infection at 37°C with 5% CO₂, cells were washed and fixed with 4% paraformaldehyde–PBS for 30min. Following that step, cells were slightly permeabilized with 0.1% saponin in PBS for 10min at room temperature. Cells were then stained with a LIVE/DEAD BacLight (Molecular Probes, Life Technologies) containing propidium iodide (PI) or SYTO 9 by following the manufacturer’s instructions. Cells were washed three times with PBS, fixed with 4% paraformaldehyde for 20min, and then directly mounted using ProLong gold antifade (Molecular Probes, Life Technologies). Cells were visualized using an Olympus BX51 upright fluorescence microscope and analyzed using ImageJ software from the National Institutes of Health (37).

Serum-mediated bacterial adherence inhibition assay.

C57BL/6 mouse primary small intestinal epithelial cells (catalog no. C57-6051; Cell Biologics Inc., Chicago, IL) were routinely grown in complete primary cell culture medium by following the manufacturer’s instructions (catalog no. M6621; Cell Biologics Inc.). For adhesion assays, 12-well plates were seeded with 1×10⁶ cells/per well. Approximately 1 h prior to infection, the monolayer was washed twice with 1ml PBS prior to addition of 1ml medium containing no supplements. Overnight cultures of EHEC O157:H7 strain 86-24 were diluted in DMEM (1:20) without supplements and incubated at 37°C under static growth conditions for 2 h. Bacterial inoculum was adjusted to a multiplicity of infection (MOI) of 10 (1×10⁷ CFU) and incubated in the presence of AuNP-LomW, AuNP-EscC, AuNP-LpfA1, or AuNP-LpfA2 immune serum, adjuvant-only serum, or naive serum (10%) for 1 h at 37°C with slight agitation. After incubation in the presence of sera, bacteria were collected in 1ml of fresh medium and used to infect cell culture plates containing 1×10⁶ cells. Monolayers were incubated for 2 h at 37°C with 5% CO₂. After incubation, cells were washed three times with PBS prior to addition of 100μl of 0.1% Triton X-100 in PBS. After detachment, the cells were serially diluted in PBS and plated on LB agar plates to enumerate adhered bacteria (output). The percentage of adhered bacteria was determined as the output/input×100. Data were representative of two independent experiments using pooled sera from n=10 mice. For microscopy analysis, primary intestinal epithelial cells were cultured in round coverslips placed at the bottom of 12-well plates and incubated for 24 h at 37°C with 5% CO₂. Bacterial inoculum using an MOI of 10 (1×10⁶ CFU) was incubated in the presence of AuNP-LomW, AuNP-EscC, AuNP-LpfA1, AuNP-LpfA2, or naive sera (10%) for 1 h at 37°C with slight agitation. After incubation in the presence or absence of sera, bacteria were collected in 1ml of fresh medium and used to infect cell culture plates containing 1×10⁵ cells. Additionally, evaluation of bacterial viability was performed by using the LIVE/DEAD staining (LIVE/DEAD BacLight bacterial viability kit; Invitrogen, USA) by using the manufacturer’s instructions. For cell infection, monolayers were incubated for an additional 2 h at 37°C with 5% CO₂. After infection, cells were washed and fixed with 4% paraformaldehyde-PBS.

Fluorescence microscopy and bacterial serum recognition.

For fluorescence microscopy analysis, fixed and permeabilized primary intestinal epithelial cells or primary macrophages were used. Polymerized actin was detected by staining with tetramethyl rhodamine isothiocyanate-phalloidin (Molecular Probes-Invitrogen, USA). DNA from cell nuclei and bacteria were mounted using Fluoroshield and detected with 4′,6-diamidino-2-phenylindole (DAPI). EHEC (86-24) was detected by immunofluorescence with anti-E. coli O plus E. coli K antibody coupled to fluorescein isothiocyanate (FITC) (Abcam, USA). Evaluation of bacteria serum recognition was performed by interaction of EHEC O157:H7 strain 86-24 with sera (1:500) from different immune groups (AuNP-LomW, AuNP-EscC, AuNP-LpfA1, or AuNP-LpfA2) during 1 h at 37°C while shaking. After incubation, cells were washed, fixed, and stained with a goat anti-mouse conjugated to Alexa-488 secondary antibody (1:10,000). Bacterial DNA was visualized using DAPI (Molecular Probes, Invitrogen) and mounted using ProLong Gold antifade (Molecular Probes, Invitrogen). Images were taken using an Olympus BX51 upright fluorescence microscope and analyzed by Image J software (37).

This work was supported by NIH NIAID grant AI148913 and partially by NIH NIAID AI126601 and UTMB seed funds awarded to A.G.T. D.T. was funded by an NIH NIAID Research Supplement for Underrepresented Minorities. J.I.S.-V. was supported by the Conacyt ConTex Postdoctoral fellowship.

The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIAID or NIH.