Combined Oral and Intravenous Immunization Stimulates Strong IgA Responses in Both Systemic and Mucosal Compartments

<div><p>To investigate the influence of immunization routes onIgG, IgA and IgM production in systemic and mucosal compartments, we immunized mice with keyhole limpet hemocyanin (KLH) via oral, intranasal (i.n.) or subcutaneous (s.c.) routes alone or combined with the intravenous (i.v.) route. We found that administering antigen intravenously could affect antibody production and formation of antibody secreting cells (ASCs) depending on the immunization route previously used. Combined oral/i.v. immunization but not s.c./i.v. immunization caused a great increase of IgA ASCs in the spleen and enhanced IgA production in the small intestine and serum. Combined i.n./i.v. immunization could also increase IgA ASCs in the spleen and enhance IgA production in serum but had no effect on IgA production in the small intestine. Oral/i.v. immunization caused increase of IgG ASCs in both the spleen and bone marrow. In comparison, combined i.n./i.v. and s.c./i.v. immunization could increase IgG ASCs in the spleen but not in bone marrow. Intravenous administration of KLH in mice that had been immunized via oral, i.n. or s.c. routes caused some increase of IgM ASCs in the spleen but not in bone marrow. In conclusion, combined oral and i.v. administration of an antigen can induce fast and strong immune responses, especially for IgA, in both systemic and mucosal compartments.</p></div

IHC analysis of IgA ASCs in the spleen.

<p>BALB/c mice were immunized with KLH via oral, i.n. or s.c. routes. Three days before sacrificing, mice were administered with KLH orally, intranasally, subcutaneously or intravenously. Spleens of mice were embedded in paraffin and sectioned into 3-μm slices and stained using anti-IgA antibody (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168037#sec002" target="_blank">Materials and Methods</a>). Top row, mice were immunized with KLH orally, intranasally or subcutaneously. Three days before sacrificing, the mice received PBS via oral, i.n. or i.v. routes. The negative control was from mice that did not receive inoculations. Middle row, mice were immunized with KLH and received a final immunization with KLH before sacrificing either orally, intranasally or subcutaneously. Bottom row, mice were immunized orally, intranasally or subcutaneously and administered a final immunization with KLH intravenously. The isotype control was stained using rat IgG1 anti-human fibrinogen antibody. Data are representative ofeightmice(The s.c./s.c. group in the middle row were five mice). Scale bar = 50 μm.</p

Levels of KLH-specific IgA antibodies in serum and feces after final administration of KLH.

<p>Oral and i.n. immunizations were performed four or five times at one-week intervals. Subcutaneous immunization was performed three times at three-week intervals. Three days before sacrificing, mice were received KLH via different routes. Serum and feces were collected, and the KLH-specific IgA antibodies were measured by ELISA in 96-well microtiter plates coated with KLH. Serum was 1 in 2 serially diluted (A, B, C). Feces were extracted by PBS (10 μl PBS per 1 mg feces) for 10 h and supernatants were collected (D). The anti-KLH IgA antibody was detected by HRP conjugated rat anti-mouse IgA. Group control means the mice did not receive inoculations. Group imm in D means the mice administered with KLH via the previous immunization routes (oral, i.n. or s.c.). Data shown are mean ± SEM for eightmice per group(The s.c./s.c. group in C and D were fivemice).</p

ELISPOT analysis for KLH-specific ASCs in bone marrow.

<p>BALB/c mice were immunized with KLH via oral, i.n. or s.c. routes. Three days before sacrificing, mice were administered with PBS via the previous immunization routesor administered with KLH either via the previous immunization routes (imm) or via the i.v. route. Then, bone marrow cells were analyzed by ELISPOT for KLH-specific cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168037#sec002" target="_blank">Materials and Methods</a>). (A) Representative results of eight experiments for IgG ASCs (The s.c./s.c. group were of five experiments). 10⁶ cells were added each well. (B) ELISPOT counts of KLH-specific IgG ASCs. (C) Representative results of eight experiments for IgA ASCs (The s.c./s.c. group were of five experiments). 5×10⁶ cells were added each well for the top and middle rows, and 10⁶ cells were added each well for the bottom row. (D) ELISPOT counts of KLH-specific IgA ASCs. (E) Representative results of eight experiments for IgM ASCs (The s.c./s.c. group were of five experiments). 10⁶ cells were added each well. (F) ELISPOT counts of KLH-specific IgM ASCs. The results presented in (B), (D), (F) are the mean ± SEM for eight mice per group (The s.c./s.c. group were five mice).<i>P</i>> 0.05 was treated as not significant (ns).</p

Vaccination schedule using different routes.

<p>BALB/c mice were immunized with KLH orally, intranasally or subcutaneously. The oral group was first immunized with 2 mg KLH and 10 μg CTB. Four boost immunizations were performed once a week with 1 mg KLH and 10 μg CTB. The i.n. group was first immunized with 30 μg KLH and 10 μg CTB intranasally. Three boost immunizations were performed once a week with 15 μg KLH and 10 μg CTB. The s.c. group was first injected with 30 μg KLH mixed with 100 μl CFA. Two boost immunizations were performed using 15 μg KLH mixed with 100 μl IFA at three-week intervals. Three days before sacrificing, mice were administered with KLH or PBS mucosally, subcutaneously or intravenously.</p

IHC analysis of IgA ASCs in the small intestines.

<p>BALB/c mice were immunized via oral, i.n. or s.c. routes. Three days before sacrificing, mice were administered with KLH orally, intranasally, subcutaneously or intravenously. The small intestines of mice were embedded in paraffin, sectioned into 3-μm slices and stained using an anti-IgA antibody (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168037#sec002" target="_blank">Materials and Methods</a>). Top row, mice were immunized with KLH orally, intranasally or subcutaneously. Three days before sacrificing, they were received PBS via oral, i.n. or i.v. routes. The negative control was from mice that did not receive inoculations. Middle row, mice were immunized with KLH. Three days before sacrificing, they were received KLH orally, intranasally or subcutaneously. Bottom row, mice were immunized orally, intranasally or subcutaneously and finally administered with KLH intravenously. The isotype control was stained using rat IgG1 anti-human fibrinogen antibody. Data are representative of eightmice(The s.c./s.c. group in the middle row were five mice). Scale bar = 50 μm.</p