Secretory IgA-mediated protection against V. cholerae and heat-labile enterotoxin-producing enterotoxigenic Escherichia coli by rice-based vaccine

Abstract

Cholera and enterotoxigenic Escherichia coli (ETEC) are among the most common causes of acute infantile gastroenteritis globally. We previously developed a rice-based vaccine that expressed cholera toxin B subunit (MucoRice-CTB) and had the advantages of being cold chain-free and providing protection against cholera toxin (CT)-induced diarrhea. To advance the development of MucoRice-CTB for human clinical application, we investigated whether the CTB-specific secretory IgA (SIgA) induced by MucoRice-CTB gives longstanding protection against diarrhea induced by Vibrio cholerae and heat-labile enterotoxin (LT)-producing ETEC (LT-ETEC) in mice. Oral immunization with MucoRice-CTB stored at room temperature for more than 3 y provided effective SIgA-mediated protection against CT- or LT-induced diarrhea, but the protection was impaired in polymeric Ig receptor-deficient mice lacking SIgA. The vaccine gave longstanding protection against CT- or LT-induced diarrhea (for > or = 6 months after primary immunization), and a single booster immunization extended the duration of protective immunity by at least 4 months. Furthermore, MucoRice-CTB vaccination prevented diarrhea in the event of V. cholerae and LT-ETEC challenges. Thus, MucoRice-CTB is an effective long-term cold chain-free oral vaccine that induces CTB-specific SIgA-mediated longstanding protection against V. cholerae- or LT-ETEC-induced diarrhea.

Fig. 1.

Critical role of antigen-specific SIgA induced by oral MucoRice-CTB vaccine in protection against CT- or LT-induced diarrhea. Cross-protective antigen-specific antibody immune responses were examined and compared among oral MucoRice-CTB (100 mg)–immunized WT mice (gray columns), oral MucoRice-CTB–immunized pIgR-deficient mice (white columns), and WT rice–fed WT mice (black columns). (A) Antibody immune responses against CTB. (B) ELISPOT assay. Frequency of CTB-specific IgA AFCs in intestinal LP was elevated in MucoRice-CTB–immunized WT mice (gray squares), and markedly increased in MucoRice-CTB–immunized pIgR-deficient mice (white triangles), but absent in WT rice–fed mice. (C) Oral CT challenge (20 μg). WT rice–fed WT mice (black column) or MucoRice-CTB–immunized pIgR-deficient mice (white column) had severe fluid accumulation, whereas MucoRice-CTB–immunized WT mice (gray column) had markedly reduced fluid accumulation. (D) Cross-protective specific serum IgG and fecal IgA against LTB were induced in mice by oral MucoRice-CTB immunization. (E) Oral LT challenge: 30 μg of LT was intragastrically administered to mice. WT rice–fed WT mice (black column) or MucoRice-CTB–immunized pIgR-deficient mice (white column) had severe fluid accumulation, whereas MucoRice-CTB–immunized WT mice (gray column) had markedly reduced fluid accumulation. Data represent means ± SD. *P < 0.0001.

Fig. 2.

Induction of long-term SIgA-mediated immunity against toxin by oral MucoRice-CTB vaccine. We examined the longevity of antigen-specific antibody immune responses and protection against CT-induced diarrhea, as well as the boosting effect of oral MucoRice-CTB. Booster (100 mg of MucoRice-CTB containing 150 μg CTB) was administered 24 weeks after the final immunization. One week later, boosted immune responses were measured and monitored for the next 16 weeks. (A) Antibody titers were simultaneously evaluated. (B) ELISPOT. Numbers of CTB-specific IgA AFCs in the intestinal LP of each mouse were evaluated over the same time period. (C) Oral CT challenge. Mice were immunized and challenged over the same time course as described earlier. Data represent means ± SD. *P < 0.0001.

Fig. 3.

Oral MucoRice-CTB–induced antigen-specific SIgA provides cross-protective immunity against V. cholerae– and LT-ETEC–induced diarrhea. Murine intestinal loop assay using V. cholerae (10⁹ cells) and LT-ETEC (10⁹ cells) was executed in WT mice orally immunized with MucoRice-CTB or WT rice. Unlike WT rice, oral MucoRice-CTB vaccination markedly reduced the incidence of V. cholerae– and LT-ETEC–induced diarrhea. When the ratio of fluid to length was greater than 30 μL/cm, the intestinal loop was considered positive for diarrhea. The positive loop ratio is shown in the parentheses as a percentage of the total number of mice examined. (a) P = 0.004 compared with WT rice–fed mice. (b) P = 0.04 compared with WT rice–fed mice.