Membrane-anchored CCL20 augments HIV Env-specific mucosal immune responses

Abstract

Background: Induction of broad immune responses at mucosal site remains a primary goal for most vaccines against mucosal pathogens. Abundance of evidence indicates that the co-delivery of mucosal adjuvants, including cytokines, is necessary to induce effective mucosal immunity. In the present study, we set out to evaluate the role of a chemokine, CCL20, as an effective mucosal adjuvant for HIV vaccine.

Methods: To evaluate the role of CCL20 as a potent adjuvant for HIV vaccine, we examined its effects on antigen-specific antibody responses, level of antibody-secreting cells, cytokine production and intestinal homing of plasma cells in vaccine immunized mice.

Results: CCL20-incorporated VLP administered by mucosal route (intranasal (n = 10, p = 0.0085) or intravaginal (n = 10, p = 0.0091)) showed much higher potency in inducing Env-specific IgA antibody response than those administered by intramuscular route (n = 10). For intranasal administration, the HIV Env-specific IFN-γ(751 pg/ml), IL-4 (566 pg/ml), IL-5 (811 pg/ml) production and IgA-secreting plasma cells (62 IgA-secreting plasma cells/10⁶ cells) in mucosal lamina propria were significantly augmented in CCL20-incorporated VLP immunized mice as compared to those immunized with Env only VLPs (p = 0.0332, 0.0398, 0.033, 0.0302 for IFN-γ, IL-4, IL-5, and IgA-secreting plasma cells, respectively). Further, anti-CCL20 mAb partially suppressed homing of Env-specific IgA ASCs into small intestine in mice immunized with CCL20-incorporated VLP by intranasal (62 decreased to 16 IgA- secreting plasma cells/10⁶ cells, p = 0.0341) or intravaginal (52 decreased to 13 IgA- secreting plasma cells/10⁶ cells, p = 0.0332) routes.

Conclusion: Our data indicated that the VLP-incorporated CCL20 can enhance HIV Env-specific immune responses in mice, especially those occurring in the mucosal sites. We also found that i.m. prime followed by mucosal boost is critical and required for CCL20 to exert its full function as an effective mucosal adjuvant. Therefore, co-incorporation of CCL20 into Env VLPs when combined with mucosal administration could represent a novel and promising HIV vaccine candidate.

Keywords: Antibody-secreting cells (ASCs); CCL20; Chemokine; HIV vaccine; Mucosal immune responses; Virus-like particles (VLPs).

Fig. 1

Characterization of HIV VLPs. Western blotting analysis of the protein component of HIV VLPs. The VLPs samples were loaded on SDS-PAGE followed examination by western blotting. Protein bands were probed with anti-HIV Gag antibody (panel a); anti-gp120 antibody (panel b); anti-CCL20 antibody (panel c); Lane 1, Gag only VLPs; lane 2, standard VLPs; lane 3, chimeric VLPs. M, molecular weight (kD). the Target bands were indicated by arrows

Fig. 2

Systemic and mucosal Ab responses against HIV-gp120. The mice were immunized with Gag VLPs, standard VLPs(Gag/Env), chimeric VLPs (Gag/Env/ccl20) and standard VLPs mixed with soluble CCL20 (Gag/Env-ccl20) by different immunization routes. The sera and vaginal wash samples were collected 2 weeks after each immunization and final samples were tested in the present study. a, Serum IgG titers; b and c, IgG and IgA titers of vaginal wash, respectively. Assays were performed as described in materials and methods. Results are expressed as means ± standard deviations. P values of less than 0.05 (p < 0.05) were considered to be statistically significant. *, p < 0.05

Fig. 3

HIV gp120-specific Th1/Th2-associated cytokine production. Lamina propria cells isolated from immunized mice (1 × 10⁷cells) were stimulated with gp120 (20μg/ml) for 5 days, and cytokine production was quantified afterward. HIV gp120-specific cytokine production in HIV-VLPs treated mice is shown. Panels a, b and c represent the quantified IFN-γ, IL-4, IL-5 concentrations (pg/ml), respectively, in the supernatants from ex vivo HIV-VLPs re-stimulated lamina propria cells. Results are expressed as means ± standard deviations. P values of less than 0.05 (p < 0.05) were considered to be statistically significant. *, p < 0.05

Fig. 4

Effect of CCL20 on the level of mucosal IgA-ASCs. Distribution of Env-specific IgA ASCs at the mucosal level in BALB/c mice immunized with various HIV VLPs by different routes i.m., i.n., IV is shown in panel a. Lymphocytes were prepared from lamina propria of small intestine. Effect of blocking CCL20 on intestinal homing of Env-specific IgA ASCs was shown in panel b. BALB/c mice were immunized with HIV VLPs in different routes and also injected i.p. with PBS or anti-CCL20 mAb. After 6 days, lymphocytes were prepared from lamina propria of small intestine. IgA ASCs were enumerated by ELISPOT. The results of ELISPOT images were from cVLPs immunized groups. Data represent mean ± SD and statistically significant differences are represented