Nano selenium-enriched probiotic Lactobacillus enhances alum adjuvanticity and promotes antigen-specific systemic and mucosal immunity

Abstract

Nano selenium-enriched probiotics have been identified to improve immune responses, such as alleviating inflammation, antioxidant function, treatment of tumors, anticancer activity, and regulating intestinal flora. However, so far, there is little information on improving the immune effect of the vaccine. Here, we prepared nano selenium-enriched Levilactobacillus brevis 23017 (SeL) and heat-inactivated nano selenium-enriched L. brevis 23017 (HiSeL) and evaluated their immune enhancing functions on the alum-adjuvanted, inactivated Clostridium perfringens type A vaccine in mouse and rabbit models, respectively. We found that SeL enhanced immune responses of the vaccine by inducing a more rapid antibody production, eliciting higher immunoglobulin G (IgG) antibody titers, improving secretory immunoglobulin A (SIgA) antibody level and cellular immune response, and regulating Th1/Th2 immune response, thus helping to induce better protective efficacy after challenge. Moreover, we confirmed that the immunoenhancement effects are related to regulating oxidative stress, cytokine secretion, and selenoprotein expression. Meanwhile, similar effects were observed in HiSeL. In addition, they show enhanced humoral immune response at 1/2 and 1/4 standard vaccine doses, which confirms their prominent immune enhancement effect. Finally, the effect of improving vaccine immune responses was further confirmed in rabbits, which shows that SeL stimulates the production of IgG antibodies, generates α toxin-neutralizing antibodies rapidly, and reduces the pathological damage to intestine tissue. Our study demonstrates that nano selenium-enriched probiotics improve the immune effect of the alum adjuvants vaccine and highlight its potential usage in remedying the disadvantages of alum adjuvants.

Keywords: SIgA antibody; alum adjuvants; immunoenhancement; nano selenium-enriched L. brevis 23017; selenoprotein.

Figure 1

Synthesis and characterization of nano selenium-enriched L. brevis 23017. L. brevis 23017 cultivated under sodium selenite stress (30 μg/ml) led to reduction of selenite ions (Se^IV) into extracellular elemental Se (Se⁰) nanoparticles observed by transmission electron microscopy (TEM), and visible as red color. The red arrow in the figure refers to SeNPs.

Figure 2

Effect of SeL and HiSeL on the humoral immune response and mucosal immune response induced by the immunization of mice. Sera and fecal samples of mice were gathered on days 7, 10, 14, and 28 after immunization to detect specific IgG and SIgA antibody response. Intestinal mucus samples of mice were gathered on day 28 after immunization to detect total SIgA concentration. (A) The titer of specific IgG antibody in mice sera. (B) The titer of specific SIgA antibodies in mice fecal and total SIgA concentration in intestinal mucus. Each experimental group consisted of five mice per group. Data were presented as means ± SD (*P < 0.05, and **P < 0.01). The statistical analysis was performed using one-way ANOVA.

Figure 3

Effect of SeL and HiSeL on the secretion of cytokines related to SIgA induced by the immunization of mice. Sera samples of mice were gathered on days 1 and 28 after immunization to detect cytokines related to SIgA. (A) Results of cytokine in sera on the first day after immunization. (B) Results of cytokine in sera on the 28th day after immunization. Each experimental group consisted of five mice per group. Data were presented as means ± SD (*P < 0.05, and **P < 0.01). The statistical analysis was performed using one-way ANOVA.

Figure 4

Effect of SeL and HiSeL on the expression of cytokine genes related to SIgA induced by the immunization of mice. Jejunal tissue samples of mice were gathered on days 1 and 28 after immunization to detect cytokine genes related to SIgA. (A) Results of mRNA level of cytokines in jejunal cells on the first day after immunization. (B) Results of mRNA level of cytokines in jejunal cells on the 28th day after immunization. Each experimental group consisted of five mice per group. Data were presented as means ± SD (*P < 0.05, and **P < 0.01). The statistical analysis was performed using one-way ANOVA.

Figure 5

Effect of SeL and HiSeL on the mRNA levels of selenoprotein-related functional genes induced by the immunization of mice. Spleen samples of mice were gathered on days 1 and 28 after immunization to detect selenoprotein-related functional genes. (A) Results of mRNA level of selenoprotein-related functional genes in spleen on the first day after immunization. (B) Results of mRNA level of selenoprotein-related functional genes in spleen on the 28th day after immunization. Each experimental group consisted of five mice per group. Data were presented as means ± SD (*P < 0.05, **P < 0.01, and ***P < 0.001). The statistical analysis was performed using one-way ANOVA.

Figure 6

Effect of SeL or HiSeL on the pathological changes of jejunal tissue induced by the immunization of mice and rabbits after challenge. Jejunal tissue samples of mice and rabbits were gathered on day 28 after immunization to detect pathological changes. The pathological changes were examined by HE staining (magnification of ×40), the black arrows point at the lesion location. (A) Control group of mice. (B) Vaccine group of mice. (C) L + Vac group of mice. (D) SeL + Vac group of mice. (E) HiSeL + Vac group of mice. (F) Control group of rabbits. (G) Vaccine group of rabbits. (H) SeL + Vac group of rabbits.

Figure 7

Effect of SeL or HiSeL on the humoral immune response induced by the immunization of mice and rabbits. Sera samples of mice were gathered on days 0 and14 after immunization with 1/2 and 1/4 standard vaccine doses to detect specific IgG antibody response. Sera samples of rabbits were gathered on days 7, 10, 14, and 28 after immunization to detect specific IgG antibody response. (A) The titer of specific IgG antibody in mice sera immunized with 1/2 and 1/4 standard vaccine doses. (B) The titer of specific IgG antibody in rabbit sera. Each experimental group consisted of five mice or rabbits per group. Data were presented as means ± SD (*P < 0.05, and **P < 0.01). The statistical analysis was performed using one-way ANOVA or two-way ANOVA.

Figure 8

Effect of SeL on the clinical symptoms of immunized rabbits after challenge. The clinical symptoms of rabbits were watched and recorded on days 0–6 after challenge. The results were expressed as DAI score. Each experimental group consisted of five rabbits per group. Data were presented as means ± SD (**P < 0.01). The statistical analysis was performed using two-way ANOVA.