Enhancement of adaptive immunity to Neisseria gonorrhoeae by local intravaginal administration of microencapsulated interleukin 12

Abstract

Gonorrhea remains one of the most frequent infectious diseases, and Neisseria gonorrhoeae is emerging as resistant to most available antibiotics, yet it does not induce a state of specific protective immunity against reinfection. Our recent studies have demonstrated that N. gonorrhoeae proactively suppresses host T-helper (Th) 1/Th2-mediated adaptive immune responses, which can be manipulated to generate protective immunity. Here we show that intravaginally administered interleukin 12 (IL-12) encapsulated in sustained-release polymer microspheres significantly enhanced both Th1 and humoral immune responses in a mouse model of genital gonococcal infection. Treatment of mice with IL-12 microspheres during gonococcal challenge led to faster clearance of infection and induced resistance to reinfection, with the generation of gonococcus-specific circulating immunoglobulin G and vaginal immunoglobulin A and G antibodies. These results suggest that local administration of microencapsulated IL-12 can serve as a novel therapeutic and prophylactic strategy against gonorrhea, with implications for the development of an effective vaccine.

Keywords: IL-12; Neisseria gonorrhoeae; cytokine therapy; immunity; microencapsulation.

Figure 1.

Effect of intravaginal IL-12 microsphere (ms) treatment on primary gonococcal infection in BALB/c mice. A, interleukin 12 (IL-12) ms dose optimization experiment. Microspheres containing the stated doses of IL-12 were given on days 0, 2, 4, 6, and 8 (8 mice per group). The Neisseria gonorrhoeae burden was monitored daily by vaginal swab culture. Significant differences in infection burdens were found between mice treated with 2.0 µg (P < .01), 1.0 µg (P < .01), or 0.5 µg (P < .05) of microencapsulated IL-12 and controls (analysis of variance). B, Time course of infection in mice treated with IL-12 ms, soluble IL-12, interleukin 17 (IL-17) ms, or control ms or in untreated mice (cytokine dose, 1.0 µg given on days –1, 1, 3, 5, and 7; 8 mice per group). Significant differences in infection burdens were found between mice treated with IL-12 ms (P < .01) or IL-17 ms (P < .02) and controls (analysis of variance). C, Data from the experiment shown in B, plotted as percentage of mice remaining infected with the indicated cytokine treatments. Infection was cleared significantly faster in mice treated with IL-12 ms (P < .001) or IL-17 ms (P < .001) than in controls (Kaplan-Meier analysis). D, Cytokine expression in isolated iliac lymph node cells from sham-infected or infected mice with IL-12 ms, IL-17 ms, or control ms treatment (7 mice per group). Expression of interferon (IFN) γ, interleukin 4 (IL-4), and IL-17 in CD4⁺ T cells isolated at day 5 after infection was analyzed with flow cytometry. E, Reverse-transcription polymerase chain reaction (RT-PCR) analysis of IFN-γ, IL-4, and IL-17 messenger RNA (mRNA) levels in vaginal tissue harvested at day 3 from sham-infected or infected mice with IL-12 ms, IL-17 ms, or control ms treatment (7 mice per group). Cytokine gene expression levels detected with RT-PCR were normalized relative to expression of β-actin and set at 1.0 for the sham-infected group. F, Phenotypic profile of vaginal cells isolated on day 5 from sham-infected or infected mice treated with IL-17 ms or control ms (7 mice per group). G, H, Vaginal (G) and (H) serum anti-gonococcal immunoglobulin A (IgA) and immunoglobulin G (IgG) antibody responses in sham-infected or infected mice with IL-12 ms, IL-17 ms, or control ms treatment (7 mice per group). Vaginal wash and serum samples were collected 15 days after inoculation, and gonococcus-specific and total IgA and IgG were measured with enzyme-linked immunosorbent assay. Results from 1 of 3 independent experiments are shown. Data shown as means ±SEM. ^#P < .05; *P < .01 (unpaired t test in D–H).

Figure 2.

Effect of intravaginal interleukin 12 (IL-12) microsphere (ms) treatment during primary infection on secondary Neisseria gonorrhoeae infection. A, Time course of secondary infection in mice treated with IL-12 ms, soluble IL-12, interleukin 17 (IL-17) ms, or control ms during primary infection or in previously sham-infected mice with or without IL-12 ms treatment (8 mice per group). Significant differences in infection burdens were found between mice previously treated with IL-12 ms (P < .02) and controls (analysis of variance). B, Data from the experiment shown in A plotted as percentage of mice remaining infected after reinfection under the indicated treatments during primary infection. Infection was cleared significantly faster in mice previously treated with IL-12 ms (P < .001) than in controls (Kaplan-Meier analysis). C, Flow cytometric analysis of cytokine expression in iliac lymph node CD4⁺ T cells isolated at day 5 from reinfected mice treated with IL-12 ms, IL-17 ms, or control ms during primary infection, or from mice that were sham infected in both primary and secondary phases (“sham reinfected”) (7 mice per group). D, Reverse-transcription polymerase chain reaction (RT-PCR) analysis of interferon (IFN) γ, interleukin 4 (IL-4), and IL-17 messenger RNA (mRNA) levels in vaginas harvested at day 3 from sham-reinfected or reinfected mice treated with IL-12 ms, IL-17 ms, or blank ms during primary infection (7 mice per group). Cytokine gene expression levels detected by RT-PCR were normalized relative to expression of β-actin and set at 1.0 for sham-reinfected group. E, F, Vaginal (E) and (F) serum anti-gonococcal immunoglobulin A (IgA) and immunoglobulin G (IgG) antibody responses to secondary infection in sham-reinfected or reinfected mice treated with IL-12 ms, IL-17 ms, or blank ms during primary infection (7 mice per group). Vaginal wash and serum samples were collected 15 days after inoculation, and gonococcus-specific and total IgA and IgG were measured with enzyme-linked immunosorbent assay. Results from 1 of 3 independent experiments are shown. Data shown as means ±SEM. ^#P < .05; *P < .01 (unpaired t test in C–F).

Figure 3.

Effect of intravaginal anti–transforming growth factor (TGF) β antibody or anti–interleukin 10 (IL-10) antibody microsphere (ms) treatment during primary infection on primary and secondary genital Neisseria gonorrhoeae infection in BALB/c mice. A, Time course of primary infection in mice treated with anti–TGF-β ms, anti–IL-10 ms, or control ms (8 mice per group). Significant differences in infection burdens were found between mice treated with anti–TGF-β ms (P < .02) or anti–IL-10 ms (P < .05) and controls (analysis of variance). B, Data from experiment shown in A plotted as percentage of mice remaining infected with the indicated treatments. Infection was cleared significantly faster in mice treated with anti–TGF-β ms (P < .01) or anti–IL-10 ms (P < .01) than in controls (Kaplan-Meier analysis). C, Flow cytometric analysis of cytokine expression in isolated iliac lymph node CD4⁺ T cells from sham-infected or infected mice with anti–TGF-β antibody ms, anti–IL-10 antibody ms, or control ms treatment (7 mice per group). D, Time course of secondary infection in mice treated with anti–TGF-β antibody ms, anti–IL-10 antibody ms, or control ms during primary infection (8 mice per group). Significant differences in infection burdens were found between mice previously treated with anti–TGF-β ms (P < .02) or anti–IL-10 ms (P < .02) and controls (analysis of variance). E, Data from experiment shown in D plotted as percentage of mice remaining infected after reinfection under the indicated treatments during primary infection. Infection was cleared significantly faster in mice previously treated with anti–TGF-β ms (P < .01) or anti–IL-10 ms (P < .001) than in controls (Kaplan-Meier analysis). F, Flow cytometric analysis of cytokine expression in iliac lymph node CD4⁺ T cells isolated at day 5 from sham-reinfected or reinfected mice treated with anti–TGF-β antibody ms, anti–IL-10 antibody ms, or control ms during primary infection (7 mice per group). Results from 1 of 3 independent experiments are shown. Data shown as means ±SEM. Abbreviations: IFN, interferon; IL-4, interleukin 4. ^#P < .05; *P < .01 (unpaired t test in C and F).