Intramuscular primary immunization by nucleic acid vaccine pcDNA/Gpd-IL-2 and enhanced immunization with mucosal adjuvant CpG-ODN and Gpd-IL-2 recombinant protein effectively induced strong mucosal immune responses and immune protective effects against Treponema pallidum skin infection

Abstract

The present study aimed to evaluate the immune effect of intramuscular primary immunization by the nucleic acid vaccine pcDNA/glycerophosphodiester phosphodiesterase-interleukin-2 (pcDNA/Gpd-IL-2) and enhanced immunization 2 weeks later with the combination of mucosal adjuvant CpG-oligodeoxynucleotides (ODN) and Gpd-IL-2 recombinant protein on skin infection caused by Treponema pallidum (Tp) in New Zealand rabbits. At week 8 following immunization, MTT assay was used to detect spleen cell proliferation, while enzyme-linked immunosorbent assay was performed to detect the cytokine and secretory immunoglobulin A (SIgA) levels. At week 10 after primary immunization, rabbits were inoculated with 10⁵ Tp (Nichols strain). Alterations in the skin redness, swelling and ulceration were recorded for 0-60 days. In addition, positive rate of Tp in skin lesions and ulcer formation rate were examined using dark field and silver staining. The results indicated that intramuscular primary immunization by nucleic acid vaccine pcDNA/Gpd-IL-2 followed by enhanced immunization via nasal feeding with mucosal adjuvant CpG-ODN and Gpd-IL-2 recombinant protein induced the higher levels of Tp Gpd specific antibodies, increased the secretion of IL-2 and interferon-γ, and promoted the proliferation of T cells in the first 8 weeks after immunization. Furthermore, this immunization strategy stimulated the production of mucosa specific SIgA antibody. Thus, this strategy led to the lowest Tp positive and ulcer formation rates at the Tp infection sites, as well as healing of skin lesions on the earliest time point (day 42). In conclusion, immunization by nucleic acid vaccine pcDNA/Gpd-IL-2 followed by enhanced immunization with a combination of mucosal adjuvant CpG-ODN and Gpd-IL-2 recombinant protein is an effective immune strategy to induce strong mucosal immune responses and immune protective effects.

Keywords: CpG-oligodeoxynucleotides; Gpd-interleukin-2; Treponema pallidum; nucleic acid vaccine.

Figure 1.

Alterations in the levels of IgG antibodies in the serum of experimental rabbits following immunization. At 0, 2, 4, 6, and 8 weeks after immunization, blood was collected from the rabbit ear veins and the anti-Tp Gpd antibody levels in the serum were determined by enzyme-linked immunosorbent assay. Absorbance was measured at 450 nm. *P<0.05 vs. A1 group at the same time point; ^#P<0.05 vs. C2 group at the same time point; ^$P<0.05 vs. B1 group at the same time point. A1, 2× intramuscular immunization with pcDNA3.1 (100 µg); A2, 2× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg); B1, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with pcDNA/Gpd-IL-2 (100 µg); B2, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with pcDNA/Gpd-IL-2 (100 µg) and CpG-ODN (10 µg); C1, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with Gpd-IL-2 recombinant protein (100 µg); C2, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with Gpd-IL-2 recombinant protein (100 µg) and CpG-ODN (10 µg); ODN, oligodeoxynucleotides; TP, Treponema pallidum; IL-2, interleukin-2; Gpd, glycerophosphodiester phosphodiesterase.

Figure 2.

Concentrations of cytokines (A) IL-2 and (B) IFN-γ secreted from spleen cells following Tp Gpd stimulation in vitro. One-way analysis of variance was used for statistical analysis, while pairwise comparison was also performed among A1 to C2 groups. IL-2 (A1, A2, B1, B2, C1 and C2): F=17.268, P<0.05; IFN-γ (A1, A2, B1, B2, C1 and C2): F=113.727, P<0.05. A1, 2× intramuscular immunization with pcDNA3.1 (100 µg); A2, 2× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg); B1, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with pcDNA/Gpd-IL-2 (100 µg); B2, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with pcDNA/Gpd-IL-2 (100 µg) and CpG-ODN (10 µg); C1, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with Gpd-IL-2 recombinant protein (100 µg); C2, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with Gpd-IL-2 recombinant protein (100 µg) and CpG-ODN (10 µg); TP, Treponema pallidum; IL-2, interleukin-2; IFN-γ, interferon-γ; Gpd, glycerophosphodiester phosphodiesterase. *P<0.05 vs. A2/B1/B2/C1/C2; **P<0.05 vs. B1; ***P<0.05 vs. C2; ^#P<0.05 vs. B2/C1/C2.

Figure 3.

SI of spleen cells. One-way analysis of variance was used for statistical analysis, while pairwise comparison was performed among A1 to C2 groups. SI (A1, A2, B1, B2, C1 and C2): F=74.675, P<0.05. A1, 2× intramuscular immunization with pcDNA3.1 (100 µg); A2, 2× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg); B1, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with pcDNA/Gpd-IL-2 (100 µg); B2, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with pcDNA/Gpd-IL-2 (100 µg) and CpG-ODN (10 µg); C1, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with Gpd-IL-2 recombinant protein (100 µg); C2, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with Gpd-IL-2 recombinant protein (100 µg) and CpG-ODN (10 µg); SI, stimulation index. *P<0.05 vs. A2/B1/B2/C1/C2; **P<0.05 vs. B1; ***P<0.05 vs. B2/C1/C2; ^#P<0.05 vs. C2.

Figure 4.

Levels of SIgA antibody in (A) nasopharyngeal and (B) vaginal fluid from immunized rabbits. One-way analysis of variance was used for statistical analysis, and pairwise comparison was performed among A1 to C2 groups. Levels of SIgA antibody in nasopharyngeal fluid (A1, A2, B1, B2, C1 and C2): F=46.703, P<0.05. Levels of SIgA antibody in vaginal fluid (A1, A2, B1, B2, C1 and C2): F=113.679, P<0.05. A1, 2× intramuscular immunization with pcDNA3.1 (100 µg); A2, 2× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg); B1, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with pcDNA/Gpd-IL-2 (100 µg); B2, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with pcDNA/Gpd-IL-2 (100 µg) and CpG-ODN (10 µg); C1, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with Gpd-IL-2 recombinant protein (100 µg); C2, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with Gpd-IL-2 recombinant protein (100 µg) and CpG-ODN (10 µg); SIgA, secretory immunoglobulin A. *P<0.05 vs. A2/B1/B2/C1/C2; **P<0.05 vs. B1/B2/C1/C2; ***P<0.05 vs. B2/C2; ^#P<0.05 vs. C2.

Figure 5.

Measurements of erythematous lesion diameters in rabbits infected with Tp on week 10 after immunization at 3-day intervals (between 0–60 days). At week 10, 15 out of 18 rabbits in each group received Tp Gpd DNA vaccine immunization intradermally at eight sites on their shaved backs with 10⁵ Tp (Nichols) spirochetes. Each bar represents the mean ± standard error of diameter of erythematous lesion. A1, 2× intramuscular immunization with pcDNA3.1 (100 µg); A2, 2× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg); B1, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with pcDNA/Gpd-IL-2 (100 µg); B2, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with pcDNA/Gpd-IL-2 (100 µg) and CpG-ODN (10 µg); C1, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with Gpd-IL-2 recombinant protein (100 µg); C2, 1× intramuscular immunization with pcDNA/Gpd-IL-2 (100 µg) + 1× nasal immunization with Gpd-IL-2 recombinant protein (100 µg) and CpG-ODN (10 µg); TP, Treponema pallidum; Gpd, glycerophosphodiester phosphodiesterase.