A lipopolysaccharide from pantoea agglomerans is a promising adjuvant for sublingual vaccines to induce systemic and mucosal immune responses in Mice via TLR4 pathway

A lipopolysaccharide from Pantoea agglomerans (LPSpa) has been applied to various fields for human use as a Toll-like receptor 4 ligand and its safety has been confirmed. Here, we showed for the first time the application of LPSpa as an effective mucosal adjuvant for activating vaccine-induced antigen specific immune responses. Mice sublingually immunized with influenza vaccine (HA split vaccine) with LPSpa induced both HA-specific IgG (systemic) and IgA (mucosal) antibody responses, which led to a significant increase in survival rate against lethal influenza virus challenge compared with subcutaneous vaccination. After sublingual administration of ovalbumin with LPSpa, ovalbumin-specific mucosal IgA responses were induced at both mucosal surfaces close to the immunized site and at remote mucosal surfaces. Sublingual administration of LPSpa evoked local antigen-uptake by dendritic cells in cervical lymph nodes. LPSpa induced cytokine production and the maturation and proliferation of innate immune cells via Toll-like receptor 4 in dendritic cells. Collectively, these results suggest that LPSpa can be used as an effective mucosal adjuvant to stimulate and activate local innate immune cells to improve and enhance mucosal vaccine potency against various pathogens

Enhanced Ag uptake of DCs in cervical lymph nodes in the presence of LPSpa.

<p>BALB/c mice, divided into 3 groups, were s.l. administered with 50 μg Alexa 647 OVA with or without LPSpa (5 μg) (n = 3). After 18 h, cervical lymph nodes were removed and cells were assessed by flow cytometry. Results are representative of three or more independent experiments. <i>*P</i><0.05, by one-way ANOVA with Tukey’s test. N.D., not detectable.</p

Cytokine production, proliferation and maturation of BMDCs after stimulation with LPSpa.

<p>BMDCs were obtained from C57BL/6 mice-derived bone marrow cells cultured with GM-CSF. Cells were stimulated for 24 h with MPL (0.1, 1, 10, 100, 1000 ng/ml) or LPSpa (0.1, 1, 10, 100, 1000 ng/ml) and (A) IL-6 or (B) TNF levels in supernatants were measured by ELISA (n = 3). (C) Total cell numbers were counted (n = 3) and (D) the maturation of BMDCs was assessed by measuring the expression levels of CD40, CD80 and CD86 by flow cytometry (n = 3). (E) Cells were stimulated for 8 h with LPSpa (1000 ng/ml) and then stained with propidium iodide and analyzed by flow cytometry (n = 3). Results are representative of three (C, D), four (E) or five (A, B) independent experiments. <i>*P</i><0.05, <i>**P</i><0.01, <i>***P</i><0.001, by Two-tailed student <i>t</i>-test when comparing between two groups and one-way ANOVA with Dunnett’s test when comparing three or more groups.</p

Seasonal influenza split s.l. vaccination with LPSpa induced HA-specific immune responses in serum and mucosa.

<p>BALB/c mice were divided into 4 groups and immunized twice s.c. or s.l. with H1N1, H3N2 and B strain-derived split vaccine (0.1 μg) with or without LPSpa (1 μg) at weekly intervals (n = 4–7). The titers of HA-specific (A) serum IgG and (B) nasal IgA were assessed by ELISA. In s.c. and s.l. with LPSa groups, at 1, 4, 8 and 12 weeks after the last immunization, HA (H1N1)–specific (C) serum IgG and (D) nasal IgA were assessed by ELISA (n = 4–5). (E) HI titers in 3 groups were measured in serum by HI assay (n = 5). Results were representative of at least three experiments. <i>***P</i><0.001, by one-way ANOVA with Tukey’s test (H1, H3 and B were assessed independently [A, B]). N.S., not significant. N.D., not detectable.</p

Induction of OVA-specific IgG and IgA in serum or various mucosal areas by LPSpa.

<p>BALB/c mice, divided into 3 groups, were s.l. immunized with 10 μg OVA or a mixture of OVA (10 μg) and 1 μg LPSpa twice at weekly intervals (n = 4). Seven days after the last immunization, samples were collected and the titers of (A) serum IgG, (B) nasal IgA, (C) BALF IgA, (D) fecal IgA and (E) vaginal IgA were assessed by ELISA. Results are representative of three independent experiments. <i>*P</i><0.05, <i>***P</i><0.001, by one-way ANOVA with Tukey’s test. N.D., not detectable.</p

S.l. vaccination of PR8 split vaccine with LPSpa protected mice from viral challenge.

<p>BALB/c mice were divided into 3 groups and immunized twice s.c. with 0.1 μg PR8 vaccine or s.l. with PR8 vaccine (0.1 μg) and 5 μg LPSpa at weekly intervals. The titer of PR8-specific (A) serum IgG and (B) nasal IgA were assessed by ELISA (n = 4). Seven days after the last immunization, mice were challenged with a lethal dose (100×LD₅₀) of PR8 virus and (C) survival rates were recorded (n = 8–10). (D) Lung sections at 5 days after virus challenge were stained with hematoxylin-eosin to evaluate cellular infiltration (n = 3). Similar experiments were performed at least three times and one representative experiment is shown. <i>***P</i><0.001, by the log-rank test. N.D., not detectable.</p

Cytokine production from mouse macrophages after stimulation with LPSpa via TLR4.

<p>Thioglycollate-elicited peritoneal macrophages were collected from BALB/c mice. Cells were stimulated for 24 h with MPL (0.1, 1, 10, 100, 1000 ng/ml) or LPSpa (0.1, 1, 10, 100, 1000 ng/ml) and (A) IL-6 or (B) TNF levels in supernatants were measured by ELISA (n = 3). Macrophages were treated with isotype Ab, TLR2 mAb or TLR4 mAb for 2 h before stimulation with LPSpa (1 ng/ml) and 24 h later, (C) IL-6 production of supernatants was measured by ELISA (n = 3). Results are representative of two (C) or three (A, B) independent experiments. <i>*P</i><0.05, <i>**P</i><0.01, <i>***P</i><0.001, by Two-tailed student <i>t</i>-test when comparing between two groups and one-way ANOVA with Tukey’s test when comparing between three groups.</p