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Original Articles: Gastroenterology

Neonatal Sublingual Vaccination with Salmonella Proteins and Adjuvant Cholera Toxin or CpG Oligodeoxynucleotides Induces Mucosal and Systemic Immunity in Mice

Huang, Ching-Feng*,†; Wang, Chih-Chien; Wu, Tzee-Chung; Wu, Keh-Gong; Lee, Chin-Cheng§; Peng, Ho-Jen*,‡,¶

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Journal of Pediatric Gastroenterology and Nutrition: March 2008 - Volume 46 - Issue 3 - p 262–271
doi: 10.1097/MPG.0b013e318156050d
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The development of neonatal vaccines has been hindered by poor efficacy because of immature immunity (1,2). Experimental bacterial vaccines against mucosal pathogens are usually administered by injection (3,4). However, injected vaccines cause discomfort in newborns and are unable to induce mucosal immunity. Although Salmonella enteritidis is one of the most common enteric pathogens in children with acute gastroenteritis (5) there has been limited study of mucosal Salmonella vaccines.

Secretory IgA (SIgA) antibodies are crucial for mucosal immunity, and they represent the first-line defense against mucosal pathogens (6). A recent study showed that neonatal immune responses can be induced by intranasal administration of Salmonella live vector vaccines to newborn mice (7). Both CpG-oligodeoxynucleotides (CpG) and cholera toxin (CT) have been identified as effective adjuvants for activating adult systemic immune responses (8,9). They have been also shown as mucosal adjuvants to preferentially induce SIgA antibody production in adult animals (10,11). However, their adjuvant effects in neonatal Salmonella vaccines on both mucosal and systemic immunity have not been investigated.

Sublingual immunotherapy has recently been used for the treatment of allergic diseases such as asthma and allergic rhinitis (12,13), but its mechanisms remain unclear (14,15). There is still no established neonatal animal model for sublingual vaccination. In the present study, we first evaluated the effect of neonatal sublingual administration with sonicated Salmonella proteins (SSP) plus CpG or CT on specific mucosal and systemic immunity in mice. We showed for the first time, as far as we are aware, that neonatal sublingual vaccination with sonicated bacterial proteins SSP combined with either CpG or CT could successfully stimulate specific mucosal SIgA and systemic IgG antibody production. Furthermore, neonatal sublingual vaccination increased protection against oral Salmonella challenge.


Adult (10–12 weeks) BALB/c mice were obtained from the National Animal Center of Taiwan (Taipei). They were given a standard laboratory rodent maintenance diet (Lab Diet, PMI Feeds, St Louis, MO) in the Animal House of Taipei Veterans General Hospital and National Defense Medical Center (Taipei). Nineteen days after mating, each pregnant mouse was confined in a cage and was checked twice daily from that time on. Newborn mice received the first vaccination within 24 h of birth. The offspring were weaned when they were 3 weeks old. All of the animal experiments were approved by the Institutional Animal Care and Use Committee of Taipei Veterans General Hospital (VGHTPE-94-43) and were performed at least twice.

Preparation of Sonicated Salmonella Proteins (SSP)

Salmonella enteritidis (ATCC 13076) was obtained from the Bioresource Collection and Research Center (Chunan, Taiwan). A suspension of bacteria was prepared by overnight growth at 37°C in a shaking incubator in nutrient broth. The bacterial suspension was sterilized by gamma radiation (2.4 Mrad). Thereafter, the solution was centrifuged and washed in phosphate-buffered saline (PBS) containing protease inhibitor (Calbiochem, San Diego, CA). Salmonella enteritidis was disrupted by sonicator (Misonix, Farmingdale, NY) on ice for 20 min. The sonicated debris was centrifuged at 1800g for 20 min at 4°C, and the membrane fraction was then sedimented by ultracentrifugation (L8-80M, Beckman, Fullerton, CA) at 100 000 g for 30 min at 4°C. The supernatant of the sonicated extract of S enteritidis was filtered with a 0.22-μm filter. The protein concentration was assessed by the bicinchronic acid protein assay (Pierce, Rockford, IL).


A total of 20 base phosphorothioate-modified oligonucleotides were synthesized with the embedded CpG motifs (CpG ODN 1826; Sigma): 5′-TCCATGACGTTCCTGACGTT-3′. Both CpG and CT (Calbiochem) were used as adjuvants.


Six newborn BALB/c offspring in each group were given a sublingual vaccination of 6 μg SSP only or mixed with 1 μg CpG or 0.2 μg CT in 1.2 μL PBS once daily for the first 3 days of life. A booster vaccination of 40 μg SSP only or combined with 10 μg CpG or 1 μg CT was given 7 weeks after the last treatment. Control mice were similarly treated with PBS only. Mice were sedated with 1 mg Nembutal sodium (Abbott Laboratories, North Chicago, IL) before the booster vaccination. The mice were bled from the tail just before the booster vaccination and weekly thereafter. Saliva was collected on the same day of bleeding after intraperitoneal injection of the salivation-stimulating drug carbamylcholine chloride (10 μg/mouse; Sigma).


Experiment I: Groups of neonatally vaccinated mice were given a booster dose on day 49, and their immune responses were assessed without challenge. Experiment II: Groups of mice vaccinated as described above were challenged with intragastric administration of S enteritidis 2 weeks after the booster vaccination. Their intestinal histopathology and survival rate were investigated after challenge. The experimental groups and protocol are summarized in Fig. 1A and B, respectively.

FIG. 1
FIG. 1:
A, Experimental groups. B, Flowchart. SSP = sonicatedSalmonella proteins; CT = cholera toxin; B & S = blood and saliva sampling.

Enzyme-linked Immunosorbent Assay

Antibody levels were assessed by enzyme-linked immunosorbent assays (ELISA) as previously described (16). Briefly, 96-well plates (Nunc, Kamstrup, Roskilde, Denmark) were coated with 50 μg/mL SSP, 10 μg/mL goat anti-mouse IgA (Sigma), 10 μg/mL goat antimouse IgG1 or IgG2a (Sigma) in 0.05 mol/L carbonate buffer, pH 9.6 (100 μL/well) overnight at 4°C. Free sites were blocked with 3% skim milk in PBS-Tween 20 for 1 h. Saliva or serum samples (1/50–200) and standards (pooled hyperimmune sera collected after monthly immunization with sonicated SSP/IFA for 3 months) were added in duplicate, and the plates were incubated for 5 h at room temperature. After washing, 100 μL horseradish peroxidase conjugated goat antimouse IgA (1/4000; Sigma), IgG (1/4000; Jackson, West Grove, PA), IgG2a, or IgG1 (1/4000 respectively; Southern Biotechnology, Birmingham, AL) were added, and the plates were incubated overnight at 4°C. After washing, o-phenylenediamine (0.5 mg/mL; Sigma) in citrate-carbonate buffer containing 0.015% H2O2 was added, and the plates were incubated in the dark. Color development was stopped by 4 N H2SO4. Absorbance at 492 nm was read by use of a microplate spectrophotometer (SPECTRAmax 250, Molecular Devices, Sunnyvale, CA), and unknowns were interpolated.

Immunodot Assay

Saliva-specific IgA antibody responses were examined by immunodot assays performed by the application of 10 μg SSP onto a polyvinylidene fluoride membrane (Millipore Corp, Bedford, MA). After blocking with 3% skim milk for 2 h, blots were immersed in pooled saliva (1/4) overnight at 4°C. The blots were then immersed in horseradish peroxidase conjugated goat antimouse IgA (1/4000) at room temperature for 5 h. Blots were washed with Tris-buffered saline after each treatment. Last, blots were immersed in enhanced chemiluminescence reagent (Amersham, Buckinghamshire, UK) for 2 min and exposed to x-ray films (Kodak, Rochester, NY) for 5 to 30 s at room temperature. The film was quantified by densitometer (Personal Densitometer SI, Amersham Biosciences, Piscataway, NJ).

Cytokine Assay

One week after the booster vaccination, spleens were collected, and the spleen cells were cocultured in duplicate in complete RPMI-1640 medium with SSP (1 mg/mL), 10% fetal calf serum, and antibiotics in 24-well flat-bottomed microtiter plates (1 mL/well; Costar, Cambridge, MA). The supernatants were harvested after 24 and 72 h of culture, and cytokines interleukin (IL)-4, IL-5, IL-6, and IFN-γ were measured by use of sandwich ELISA kits (e-bioscience, San Diego, CA).

Challenge Tests and Survival Rate

All of the experimental mice were challenged 2 weeks after the booster vaccination with intragastric administration of 1 × 109 colony forming units S enteritidis. The mice were starved overnight, but water intake was stopped for only 2 h. They then underwent gavage with antacid (0.5 mL/mouse, Amphojel, Whitehall, Australia) 1 h before challenge. The mice were then monitored for 28 days.


For assessment of the microscopic morphology of intestinal inflammation, the mice were killed by inhalation of carbon dioxide 3 days after intragastric bacteria challenge, and their intestines were resected immediately. The intraluminal contents were first irrigated gently with PBS. They were then fixed in 10% formalin (Sigma) for 48 h. A 2-μm section of each sample was placed on a glass slide and stained with hematoxylin and eosin (Vector, Burlingame, CA) for histopathological examination with a light microscope.

Statistical Analysis

Antibody and cytokines titers were expressed as means + 1 SD. Two-tailed Wilcoxon tests were used for comparison of antibody responses between groups. For challenge experiments, the Kaplan-Meier method was applied to evaluate the survival rate, and the significance of differences was determined by log rank tests. The χ2 test and the Fisher exact test were used for comparison of survival rates between groups with different specific antibody levels. A value of P < 0.05 was considered statistically significant.


Effect of Neonatal Sublingual Vaccination on Systemic and Mucosal-specific Antibody Responses

Each newborn BALB/c mouse was sublingually vaccinated with SSP alone or with adjuvant CT or CpG daily for the first 3 days of life. The control newborn mice were treated with PBS only. A booster vaccination was given 7 weeks after the last treatment. Systemic and mucosal-specific immune responses were measured just before the booster vaccination and weekly thereafter for 3 weeks (Fig. 2). In comparison with control mice, the mice vaccinated with SSP sublingually had enhanced IgG, IgG2a (P < 0.05, respectively; Fig. 2A, C), and SIgA (Fig. 2D) antibody responses 2 weeks after the booster vaccination. Compared with the mice vaccinated with SSP or PBS only, the group that received SSP plus CT had markedly increased IgG, IgG1, IgG2a (all P < 0.005; Fig. 2A, B, C) and strong SIgA (Fig. 2D) antibody responses, and the group that received SSP in combination with adjuvant CpG also had significantly enhanced IgG, IgG2a (P < 0.005, respectively; Fig. 2A, C), and SIgA (Fig. 2D) antibody responses.

FIG. 2
FIG. 2:
Effect of neonatal sublingual vaccination on systemic and mucosal antibody responses. Newborn BALB/c mice (n = 6) were given a sublingual vaccination of SSP only or SSP mixed with CpG or CT once daily for the first 3 days of life. A booster vaccination was given 7 weeks after the last treatment. Control mice were similarly treated with PBS only. Their systemic and mucosal-specific antibody responses were examined just before boosting and weekly after it. A, IgG. B, IgG1. C, IgG2a. D, SIgA. SSP = sonicatedSalmonella proteins; CT = cholera toxin.

Effect of Neonatal Sublingual Vaccination on Cytokine Secretion of Spleen Cells

The cytokine profiles after neonatal sublingual vaccination were further studied. Newborn BALB/c mice were sublingually vaccinated as described in Fig. 2. One week after the booster vaccination, spleens were collected and spleen cells were cocultured with SSP. The cytokine profiles are shown in Fig. 3. Mice receiving SSP combined with adjuvant CpG or CT had enhanced spleen cell production of IFN-γ compared with mice that received SSP or PBS only (Fig. 3A). Moreover, only mice receiving SSP and CT had markedly increased spleen cell production of IL-4, IL-5, and IL-6 (Fig. 3). Not only were the cytokine profiles without SSP stimulation low but also there were no significant differences between any experimental group and the control group (data not shown).

FIG. 3
FIG. 3:
Effect of neonatal sublingual vaccination on cytokine secretion of spleen cells. Groups of newborn mice (n = 6) were vaccinated as described inFig. 2. One week after boosting, their spleens were collected, and the spleen cells were co-cultured with SSP for 1 to 3 days. The cytokines in the supernatant were measured in duplicate using enzyme-linked immunosorbent assay kits. A, Interferon-γ (IFN-γ). B, Interleukin (IL)-4. C, IL-5. D, IL-6. SSP = sonicated Salmonella proteins; CT = cholera toxin.

No Influence of Neonatal Sublingual Vaccination on Total Mucosal and Systemic Immunity

The effect of neonatal sublingual vaccination on total mucosal and systemic immunity was also evaluated. Newborn BALB/c mice were sublingually vaccinated as described above. Total saliva, serum antibody levels, and serum cytokine levels were measured 1 week after the booster vaccination (Fig. 4).

FIG. 4
FIG. 4:
Effect of neonatal sublingual vaccination on saliva total SIgA and serum total IgG1, IgG2a antibody titers and serum cytokine levels. Newborn BALB/c mice (n = 6) were treated as described inFig. 2. Their saliva and serum samples were collected 1 week after boosting. A, saliva SigA. B, serum IgG1/IgG2a ratio. C, serum IFN-γ. D, serum IL-4. SSP = sonicated Salmonella proteins; CT = cholera toxin.

The total SIgA antibody levels, the ratio of total IgG1 to IgG2a, and serum cytokine profiles of IFN-γ and IL-4 were not significantly different between any experimental group and the control group (Fig. 4).

Survival Rate and Intestinal Histopathological Characteristics After Enteric Challenge

Groups of newborn BALB/c mice were vaccinated as described in Fig. 2. Two weeks after a booster vaccination on day 63, they were challenged with live S enteritidis. The mice vaccinated with SSP plus CpG or CT had a higher mean survival rate (78% and 80%, respectively) when compared with the survival rate (46%) in PBS-treated control mice (P < 0.05, respectively; Fig. 5A). The survival rate in those vaccinated with SSP alone (58%), however, showed no significant difference from that in the control mice (Fig. 5A).

FIG. 5
FIG. 5:
Survival rate (A) and correlation of survival rate with specific antibody levels after bacterial challenge (B). Groups of newborn mice (n = 6) were treated as described inFig. 2. Two weeks after boosting, they underwent intragastric challenge respectively with 1 × 109 colony forming units Salmonella enteritidis, and the survival rate was recorded for 4 weeks. Saliva and serum samples were collected just before challenge. The survival rate was categorized according to different levels of SSP-specific IgG and SIgA antibodies. The challenge experiments were repeated 3 times, and the survival data from 18 mice for the same grouping were analyzed. SSP = sonicated Salmonella proteins; CT = cholera toxin.

Saliva SIgA and serum IgG antibody levels were assessed just before challenge. The correlation between specific antibody responses and survival rate is shown in Fig. 5B. A prominent difference was found between the mice with high (>132) and low (<132) IgG antibody titers (P < 0.001). Whereas the mice with high IgG antibody titers survived, only 31% of those with low IgG antibody titers survived. Moreover, all of the mice with low IgG antibody titers (<132) and low SIgA antibody levels (<13) died (P < 0.001 and P < 0.05, respectively, in comparison with the mice with high or low IgG antibody titers).

The small intestines of the mice vaccinated with SSP plus CT or CpG showed no apparent swelling or necrotic inflammation (Fig. 6B and C, respectively). By contrast, the small intestines of PBS-treated control mice were severely damaged and lost the normal texture of villi (Fig. 6D). In addition, the small intestines of the mice treated with SSP alone showed marked swelling and vacuolar degeneration (Fig. 6A).

FIG. 6
FIG. 6:
Histopathological appearance of intestinal tissue after challenge with liveSalmonella enteritidis. Groups of newborn BALB/c mice (n = 4) were treated as described in Fig. 5. Their intestines were collected 3 days after challenge and fixed in 10% formalin. A, SSP only. B, SSP + CT. C, SSP + CpG. D, controls. SSP = sonicated Salmonella proteins; CT = cholera toxin. (Hematoxylin & eosin, original magnification ×200.)


To our knowledge, this is the first study to demonstrate that neonatal sublingual vaccination of BALB/c mice with enteric bacterial antigens SSP and mucosal adjuvant CpG or CT can preferentially activate systemic and mucosal-specific immune responses that provide protection against lethal enteric challenge with live S enteritidis. This study also shows that SSP combined with adjuvant CT or CpG provides much more effective systemic and mucosal-specific immune responses than does vaccination with bacterial antigens SSP alone.

Our previous study showed that injection vaccination with ovalbumin (OVA) and CpG can effectively induce Th1-predominant immunity in adult mice (9). Another study found that intranasal vaccination with sonicated Helicobacter antigens and adjuvant CpG can induce systemic Th1-predominant and mucosal immunity in adult mice (17). Induction of systemic immune responses to vaccines has been considered to be less efficient in newborns than in adults because of the immature immunity of newborns (1,2,18). As is consistent with a recent study of neonatal injection vaccination (19), however, we found that neonatal sublingual vaccination with bacterial antigens SSP and mucosal adjuvant CT or CpG could simultaneously induce systemic and mucosal immune responses.

This study showed that neonatal sublingual vaccination with SSP and adjuvant CpG exclusively induced systemic Th1-predominant and mucosal immune responses in mice. The systemic Th1 responses were characterized by serum IgG2a-dominant antibody responses and high levels of spleen cell production of the Th1 cytokine IFN-γ. CT was shown in early studies to preferentially induce Th2-dominant immune responses (20), but some recent studies have shown that CT can stimulate both Th1 and Th2 responses (21,22). As is consistent with the latter, we found that mice sublingually treated with SSP plus CT had strong IgG1 and moderate IgG2a antibody responses. The spleen cells of these mice generated high Th2 cytokine IL-4, IL-5, and IL-6 levels and also showed enhanced production of Th1 cytokine IFN-γ. By contrast, there were no differences in total saliva SIgA and total serum IgG1 or IgG2a antibody levels, serum circulating cytokines IL-4, or IFN-γ between mice receiving adjuvant CpG or CT and the control mice. These findings suggest that neonatal sublingual vaccination with SSP and adjuvant CpG or CT modulate only SSP-specific mucosal and systemic immunity, but not the entire mucosal and systemic immunity.

The side effects of injection vaccines include soreness at the injection site and the possibility of lethal anaphylaxis. In addition, injection vaccination is not able to effectively induce mucosal immunity (23). Therefore, vaccination through mucosal routes is a better choice not only to avoid the side effects of injection but also to induce mucosal immunity. Recent animal studies have repeatedly shown that oral or intranasal vaccination is effective for the development of bacterial vaccines against mucosal infection (24,25). However, the use of sublingual vaccination against mucosal pathogens in adults or newborns has not been reported. Sonicated bacterial components have been conventionally applied as potent vaccines (26). Whereas recombinant proteins are monovalent, sonicated bacterial protein antigens possess multivalent potency and can be easily prepared. Jiang et al (17) demonstrated that H felis infection was prevented in mice by intranasal immunization with sonicated H felis plus adjuvant CpG and CT. In this study, we further demonstrated that sublingual vaccination with sonicated S enteritidis plus adjuvant induced powerful protective mucosal and systemic immunity.

SIgA antibodies are crucial to defend against mucosal pathogens (6,27). This study showed for the first time, as far as we are aware, that neonatal sublingual vaccination could effectively induce mucosal immunity. The induction of specific SIgA antibodies has been shown to be beneficial to mice undergoing subsequent oral challenge with live bacteria (28). Similar protective effects were demonstrated in this study after challenge with S enteritidis. Most of the mice vaccinated with SSP together with CpG or CT survived, but more than half of the control mice died. Intestinal necrotic inflammation was found in control mice but not in mice vaccinated with SSP and CpG or CT. By contrast, systemic IgG antibodies may also play some roles in the defense against enteric pathogens. In this study, all of the mice with high serum-specific IgG antibody titers survived, compared with only 31% of the mice with low IgG antibody levels. Moreover, all of the mice with poor specific IgG and SIgA antibody responses died. Specific IgG antibody responses were better correlated with the survival rate than were SIgA antibodies. Whereas SIgA antibodies are the first line of defense against local invasion of mucosal pathogens, serum IgG antibodies are the second line of defense and play a more important role against disseminated systemic infection. Therefore, mucosal SIgA and systemic IgG reactions are both crucial against enteric pathogens.

A prime-boost vaccine regimen confers better systemic antibody responses and better bacteria clearance than does a prime vaccine regimen alone (29,30). Similarly, a mucosal prime-boost vaccine regimen also provides better mucosal and systemic immune responses (31). In addition to their influence on maternal antibodies, early prime-boost strategies in young BALB/c mice obtained effective systemic antibody responses in a previous study (32). In this study, we showed that early prime-boost strategies beginning in the neonatal stage provided effective mucosal and systemic antibody responses. Whereas the mice neonatally vaccinated on days 1 through 3 and boosted on day 49 had prominent secondary mucosal and systemic antibody responses, the adult mice vaccinated only once on day 49 had very weak primary antibody responses (data not shown). However, our recent study in adult mice showed that multiple sublingual vaccines with SSP and adjuvant CpG or CT can more effectively activate systemic and mucosal-specific immune responses, and they can confer protection from lethal enteric challenge with live S enteritidis (unpublished data).

To examine whether SSP has an adjuvant effect, sublingual administration of OVA combined with SSP in newborn mice was also studied. The results showed that mice treated with OVA plus SSP did not have significantly enhanced OVA-specific IgG antibody responses compared with mice treated with OVA alone (23.99 ± 2.97 and 26.12 ± 3.93 units, respectively). This finding indicates that the SSP used in this study does not provide any adjuvant effect. Among the variety of preparations of Salmonella vaccines that have been studied (28,33–36), recombinant proteins have the advantage of avoiding risk of systemic dissemination, but they are more expensive and cannot be easily prepared. By contrast, whole bacterial vaccines are readily prepared and inexpensive. Although the present study verified the neonatal immunizing effect of sublingual SSP, further study of the effect of sublingually administered recombinant Salmonella protein antigens is needed.

In conclusion, this study demonstrated that neonatal sublingual vaccination with sonicated bacterial proteins and mucosal adjuvant CpG or CT can efficaciously prevent S enteritidis infection in the gut of mice. The sublingual pathway provides a more convenient route for the administration of neonatal vaccines. Inasmuch as both specific mucosal and systemic immune responses could be simultaneously activated in this neonatal mouse model, the method seems to offer promise as a feasible strategy for the development of adequate immune protection against mucosal pathogens. Its potential clinical application deserves further investigation.


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Adjuvant; Mucosal vaccine; Newborn; Salmonella enteritidis; Sublingual

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