M-cell targeting of whole killed bacteria induces protective immunity against gastrointestinal pathogens - PubMed (original) (raw)
M-cell targeting of whole killed bacteria induces protective immunity against gastrointestinal pathogens
Yok-Teng Chionh et al. Infect Immun. 2009 Jul.
Abstract
As the majority of human pathogens infect via a mucosal surface, delivery of killed vaccines by mucosal routes could potentially improve protection against many such organisms. Our ability to develop effective killed mucosal vaccines is inhibited by a lack of adjuvants that are safe and effective in humans. The Ulex europaeus agglutinin I (UEA-I) lectin specifically binds M cells lining the murine gastrointestinal tract. We explored the potential for M-cell-targeted vaccination of whole, killed Helicobacter pylori, the main causative agent of peptic ulcer disease and gastric cancer, and Campylobacter jejuni, the most common cause of diarrhea. Oral delivery of UEA-I-agglutinated H. pylori or C. jejuni induced a significant increase in both serum and intestinal antibody levels. This elevated response (i) required the use of whole bacteria, as it did not occur with lysate; (ii) was not mediated by formation of particulate clumps, as agglutination with a lectin with a different glycan specificity had no effect; and (iii) was not due to lectin-mediated, nonspecific immunostimulatory activity, as UEA-I codelivery with nonagglutinated bacteria did not enhance the response. Vaccination with UEA-I-agglutinated, killed whole H. pylori induced a protective response against subsequent live challenge that was as effective as that induced by cholera toxin adjuvant. Moreover, vaccination against C. jejuni by this approach resulted in complete protection against challenge in almost all animals. We believe that this is the first demonstration that targeting of whole killed bacteria to mucosal M cells can induce protective immunity without the addition of an immunostimulatory adjuvant.
Figures
FIG. 1.
Lectin agglutination of H. pylori. Photographs portray typical agglutination of H. pylori strain SS1 (108 cells/ml) observed under light microscopy after mixing bacteria for 5 s at room temperature with PBS (a), UEA-I at 20 μg/ml (b), or BS-I at 50 μg/ml (c). At these concentrations, the lectins produced aggregates of similar size. Bar, 100 μm.
FIG. 2.
Mucosal and systemic antibody responses induced by oral delivery of UEA-I-agglutinated live H. pylori. C57BL/6 mice (n = 8) were orally dosed twice with live H. pylori (SS1 or 11637) mixed with either PBS or the lectin BS-I or UEA-I. One week after the second dose, sera and intestinal scrapings were collected and specific anti-H. pylori antibody end point titers determined by ELISA. Box plots present the median antibody titers (horizontal bar), the interquartile range (boxed region), and the 10th and 90th percentile values (error bars). All treated groups had significantly elevated levels of anti-H. pylori IgG and IgA in their sera and intestines compared with the naïve group (#, P < 0.05 by ANOVA). Oral delivery of live H. pylori agglutinated with UEA-I lectin significantly increased IgG and IgA antibody titers compared with all other groups (*, P < 0.005 by ANOVA).
FIG. 3.
Mucosal and systemic antibody responses induced by oral delivery of UEA-I-agglutinated, formalin-fixed H. pylori. BALB/c mice (n = 5) were orally dosed twice with UEA-I-agglutinated live H. pylori (SS1 UEA-I), formalin-fixed H. pylori (F-SS1 UEA-I), or H. pylori lysate (HpL). Control groups received the same bacteria without lectin (SS1 and F-SS1, respectively) or were left untreated (naïve). One week after the second dose, sera and intestinal scrapings were collected and specific anti-H. pylori antibody end point titers determined by ELISA. Box plots present the median antibody titers (horizontal bar), the interquartile range (boxed region), and the 10th and 90th percentile values (error bars). *, oral delivery of UEA-I-agglutinated, fixed bacteria induced mucosal and systemic antibody levels equivalent to those for UEA-I-agglutinated live bacteria but significantly greater than those for all other groups (P < 0.01 by ANOVA). #, significantly greater than untreated control (P < 0.04 by ANOVA).
FIG. 4.
Protective immunity against H. pylori induced by oral delivery of UEA-I-agglutinated bacteria. BALB/c mice (n = 8) were orally dosed twice with formalin-fixed H. pylori (F-SS1) either alone or agglutinated with UEA-I. Negative controls received PBS, while positive controls were vaccinated with either H. pylori lysate (HpL) plus CT or F-SS1 plus CT. Four weeks after the second dose, all mice were challenged with H. pylori SS1, and the bacterial burden was determined a further 4 weeks later by colony-forming assay. H. pylori colonization levels were calculated as CFU per stomach. Box plots present the median level of colonization (horizontal bar), the interquartile range (boxed region), and the 10th and 90th percentile values (error bars). Data shown are from a single experiment and are representative of two separate experiments. *, significantly reduced colonization compared with the negative control (P < 0.001 by ANOVA). #, significantly reduced colonization compared with both the unvaccinated control group and the group receiving formalin-fixed bacteria alone (P < 0.01 by ANOVA).
FIG. 5.
Mucosal and systemic antibody responses induced by oral delivery of UEA-I-agglutinated live C. jejuni. BALB/c mice (n = 16) were orally dosed with live C. jejuni 81-176, either mixed with PBS or agglutinated by UEA-I. One and two weeks later, sera and intestinal scrapings were collected from eight mice and specific anti-C. jejuni antibody end point titers determined by ELISA. Box plots present the median antibody titers (horizontal bar), the interquartile range (boxed region), and the 10th and 90th percentile values (error bars). UEA-I-mediated agglutination significantly elevated the levels of anti-C. jejuni IgG and IgA in sera and intestines compared with the group receiving bacteria alone as determined by ANOVA.
FIG. 6.
Intestinal colonization following oral delivery of lectin-agglutinated C. jejuni. BALB/c mice (n = 16) were orally dosed with live C. jejuni 81-176, either mixed with PBS or agglutinated by UEA-I. One and two weeks later, intestines were removed from eight mice and bacterial colonization levels determined by colony-forming assay. Colonization levels for individual mice are shown, with the median level of colonization for each group (horizontal bar). Delivery of UEA-I-agglutinated C. jejuni significantly reduced colonization levels at both time points compared with nonagglutinated controls as determined by ANOVA.
FIG. 7.
Induction of protective immunity against C. jejuni by oral vaccination with UEA-I-agglutinated bacteria. BALB/c mice (n = 8) were orally vaccinated twice with formalin-fixed C. jejuni 81-176 either alone or agglutinated with UEA-I. Negative controls received PBS. Four weeks after the second vaccination, all mice were challenged with C. jejuni 81-176, and the bacterial burden was determined a further 4 weeks later by colony-forming assay. Colonization levels for individual mice are shown, with the median level of colonization for each group (horizontal bar). Oral vaccination with fixed C. jejuni induced significant protection against subsequent live challenge compared with unvaccinated controls as determined by ANOVA. Protection was significantly increased when vaccinating bacteria were agglutinated with UEA-I (as determined by ANOVA).
References
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