Enhanced mucosal and systemic immune responses to Helicobacter pylori antigens through mucosal priming followed by systemic boosting immunizations

Abstract

It is estimated that Helicobacter pylori infects the stomachs of over 50% of the world's population and if not treated may cause chronic gastritis, peptic ulcer disease, gastric adenocarcinoma and gastric B-cell lymphoma. The aim of this study was to enhance the mucosal and systemic immune responses against the H. pylori antigens cytotoxin-associated gene A (CagA) and neutrophil-activating protein (NAP), through combinations of mucosal and systemic immunizations in female BALB/c mice. We found that oral or intranasal (i.n.) followed by i.m. immunizations induced significantly higher serum titres against NAP and CagA compared to i.n. alone, oral alone, i.m. alone, i.m. followed by i.n. or i.m. followed by oral immunizations. However, only oral followed by i.m. immunizations induced anti-NAP antibody-secreting cells in the stomach. Moreover, mucosal immunizations alone or in combination with i.m., but not i.m. immunizations alone, induced mucosal immunoglobulin A (IgA) responses in faeces. Any single route or combination of immunization routes with NAP and CagA preferentially induced antigen-specific splenic interleukin-4-secreting cells and far fewer interferon-gamma-secreting cells in the spleen. Moreover, i.n. immunizations alone or in combination with i.m. immunizations induced predominantly serum IgG1 and far less serum IgG2a. Importantly, we found that while both i.n. and i.m. recall immunizations induced similar levels of serum antibody responses, mucosal IgA responses in faeces were only achieved through i.n. recall immunization. Collectively, our data show that mucosal followed by systemic immunization significantly enhanced local and systemic immune responses and that i.n. recall immunization is required to induce both mucosal and systemic memory type responses.

Figure 1

Enhanced systemic and mucosal humoral responses following oral priming and systemic boosting immunizations. Mice were immunized five times intramuscularly (i.m.) with NAP and CagA purified proteins in MF59 adjuvant or five times orally with a mixture of NAP and CagA purified proteins and LTR72 adjuvant, or three times orally followed by twice i.m., or twice i.m. followed by three times orally. Sera and faecal pellets were collected 6 days after the final immunization. All immunizations were performed at 2-week intervals. Single-cell suspensions were prepared from stomach, spleen and mesenteric lymph nodes (MLN) 7 days after the final immunization for the ELISPOT assay. (a) Serum IgG titres; the results are shown as mean anti-NAP and anti-CagA serum titres from 10 mice as measured by a standard colorimetric ELISA. (b) Anti-NAP and (c) anti-CagA antibody-secreting cells (ASC). The results are shown as the mean number of anti-NAP or anti-CagA ASC from pools of 10 mice + SD of a minimum of four ELISPOT wells per group per tissue. (d) Anti-NAP IgA titres in faecal extracts. The results are shown as mean anti-NAP faecal IgA titres from 10 mice as measured by a chemiluminescence ELISA. The results are representative of three independent experiments with similar results.

Figure 2

Induction of Th2-type cytokine-secreting cells following oral and/or intramuscular immunizations with CagA and NAP. Mice were immunized with five intramuscular (i.m.) immunizations with NAP and CagA purified proteins in MF59 adjuvant or primed with three oral immunizations with a mixture of NAP and CagA purified proteins and LTR72 adjuvant and boosted i.m. with NAP and CagA purified proteins in MF59 adjuvant. All immunizations were performed at 2-week intervals. The mice were killed 7 days after the final immunization in each group. The number of IL4-secreting cells and IFNγ-secreting cells specific for a mixture of CagA and NAP was measured by the ELISPOT assay as described in the Materials and methods section. The results are shown as the mean number of cytokine-secreting cells from pools of 10 mice per group + SD of a minimum of four ELISPOT wells per group. The results are representative of three independent experiments with similar results.

Figure 3

Enhanced humoral responses following intranasal (i.n.) priming and systemic boosting immunizations. Mice were immunized with five intramuscular (i.m.) immunizations with NAP and CagA purified proteins in MF59 adjuvant (NCMF59) or five i.n. immunizations with a mixture of NAP and CagA purified proteins and LTK63 adjuvant (NCLTK63), or primed i.m. with NCMF59 and boosted i.n. with NCLTK63, or primed i.n. with NCLTK63 and boosted i.m. with NCMF59. All immunizations were performed at 2-week intervals. Sera and faecal pellets were collected 6 days after the final immunization. (a) Mean anti-NAP (solid bars) or anti-CagA (shaded bars) serum end-point ELISA titres + SD of 10 mice per group. The results are representative of three independent experiments with 10 mice in each group with similar results. (b) Mean anti-NAP or anti-CagA faecal IgA titres from two subgroups of 10 mice each per group as measured by a chemiluminescence ELISA. The data are representative of two independent experiments with similar results.

Figure 4

Induction of Th2-type cytokine-secreting cells following intranasal (i.n.) and/or intramuscular (i.m.) immunizations with CagA and NAP. Mice were immunized with five i.m. immunizations with NAP and CagA purified proteins in MF59 adjuvant (NCMF59) or five i.n. immunizations with a mixture of NAP and CagA purified proteins and LTK63 adjuvant (NCLTK63), or primed i.n. with NCLTK63 and boosted i.m. with NCMF59. All immunizations were performed at 2-week intervals. The mice were killed 7 days after the final immunization in each group. (a) The number of IL-4-secreting cells and IFN-γ-secreting cells specific for a mixture of CagA and NAP was measured by ELISPOT as described in the Materials and methods section. The results are shown as the mean number of IL-4-secreting cells from pools of 10 mice per group + SD of a minimum of four ELISPOT wells per group. The results are representative of three independent experiments with similar results. (b) Mean anti-NAP IgG1 (solid bars) or IgG2a (shaded bars) serum end-point ELISA titres + SD of 10 mice per group. The results are representative of three independent experiments with 10 mice in each group with similar results

Figure 5

Mucosal and systemic memory-type responses following nasal priming and systemic boosting can be recalled by intranasal re-boost. Mice were primed intranasally (i.n.) with a mixture of NAP and CagA purified proteins and LTK63 adjuvant (NCLTK63) and boosted intramuscularly (i.m.) with a mixture of NAP and CagA purified proteins in MF59 adjuvant (NCMF59) and rested for 4 months before given either an i.n. or i.m. re-boost with NCLTK63 or NCMF59, respectively. All immunizations were performed at 2-week intervals. Serum and faecal pellets were collected 6 days after the final immunization in each group. Cells were prepared from mice killed 7 days after the final immunization for the ELISPOT assay. (a) Serum IgG titres. The results are shown as mean anti-NAP and anti-CagA serum titres from 10 mice as measured by a standard colorimetric ELISA. (b) Anti-NAP and anti-CagA antibody-secreting cells (ASC) in spleen (SP) and mesenteric lymph nodes (MLN) as measured by the ELISPOT assay. The ASC data are means of pools of two subgroups of five mice (total of 10 mice per group) + SD of the two pools of five mice per subgroup. The results are representative of two independent experiments with 10 mice in each group with similar results. (c) Mean anti-NAP IgA end-point titres in pools of faecal extracts from 10 mice + SD of five mice per subgroup as measured by a chemiluminescence ELISA. The results are representative of two independent experiments with similar results.