A Strong Humoral Immune Response Induced by a Vaccine Formulation Containing rSm29 Adsorbed to Alum Is Associated With Protection Against Schistosoma mansoni Reinfection in Mice

Abstract

The helminth Schistosoma mansoni is one of main causes of human schistosomiasis, a health and economic concern in some of the world's poorest countries. Current treatment regimens can lead to serious side effects and are not suitable for breastfeeding mothers. As such, efforts have been undertaken to develop a vaccine to prevent infection. Of these, Sm29 is a promising candidate that has been associated with resistance to infection/reinfection in humans and mice. Its ability to induce resistance to reinfection has also been recently demonstrated using a vaccine formulation containing Freund's adjuvant. However, Freund's adjuvant is unsuitable for use in human vaccines. We therefore evaluated the ability of Sm29 to induce protection against S. mansoni reinfection when formulated with either alum or MPLA as an adjuvant, both approved for human use. Our data demonstrate that, in contrast to Sm29 with MPLA, Sm29 with alum reduced parasite burden after reinfection compared to a control. We next investigated whether the immune response was involved in creating the differences between the protective (Sm29Alum) and non-protective (Sm29MPLA) vaccine formulations. We observed that both formulations induced a similar mixed-profile immune response, however, the Sm29 with alum formulation raised the levels of antibodies against Sm29. This suggests that there is an association between a reduction in worm burden and parasite-specific antibodies. In summary, our data show that Sm29 with an alum adjuvant can successfully protect against S. mansoni reinfection in mice, indicating a potentially effective vaccine formulation that could be applied in humans.

Keywords: MPLA-SM; Schistosoma mansoni; Sm29; alum; reinfection; vaccine.

Figure 1

Activation status of dendritic cells (DC) and macrophages. Spleen from 5 to 6 animals was obtained for each group. Dendritic cells and macrophages were labeled to evaluate the expression of activation markers. For dendritic cells analysis, CD11c⁺ and MHC⁺ double positive cells were selected and the mean fluorescence intensity (MFI) of CD86 was determinate (A). For macrophages, F4/80 and CD11b double positive cells were selected and the mean fluorescence intensity of MHCII and CD40 were evaluated (C). Data of CD86 MFI in dendritic cells (B), MHCII MFI (D) and CD40 MFI (E) in macrophages from animals of MPLA (closed triangles), Sm29 MPLA (closed circles), Alum (open triangles), Sm29 Alum (open circles) and infected/treated (gray circles) groups are represented on graphics. Results are representative of one from two independent experiments.

Figure 2

Profile of TCD4 subsets in animals immunized with Sm29 Alum and Sm29 MPL immunization. The percentage of CD4+IL-4+, CD4+IFN-γ+ and CD4+IL-10+ cytokines was evaluated by TCD4 intracellular staining in spleen cells from 5 to 6 animals of each group. Intracellular cytokine staining was performed in the absence of polyclonal or antigen specific stimulated to assesses differentiation induced by the vaccine formulation in vivo. Data analysis was carried out as demonstrated in (A) within singlet cells/lymphocyte region, cells expressing CD4 and CD3 molecules were selected and the percentage of CD4⁺ cells producing IL-4 (B), IL-10 (C) and IFN-γ (D) was evaluated in MPLA (closed triangles), Sm29 MPLA (closed circles), Alum (open triangles), Sm29 Alum (open circles) and infected/treated (gray circles) groups. Results are representative of one from two independent experiments. No significant differences between groups were observed.

Figure 3

Cytokine production by spleen cells culture of immunized mice. Spleen from animals of MPLA (n = 11); Sm29 MPLA (n = 14); Alum (n = 11), Sm29 Alum (n = 15) and Infected and treated (n = 11) groups were obtained. Spleen cells cultured with (black bars) or without (open bars) rSm29 (25 μg/mL) stimulation was assessed to determine cytokine production. The levels of TNF-a (A), IL-6 (B), IL-10 (C), IL-17 (D), and IFN-γ (E) were measured by the CBA Th1/Th2/Th17 Kit. Statistically significant differences are denoted on the graphics. Bars represents the mean + SD values of three independent experiments.

Figure 4

Frequency of B memory cells in immunized mice. Spleen from 5 to 6 animals was obtained for each group to determine the frequency of B memory cells. Data analysis was carried out as demonstrated (A): within singlet cells/lymphocyte population, CD19⁺ and CD3⁻ cells were selected and the percentage of B cells was determined. B memory cells were assessed by determining CD19⁺CD27⁺cell frequency. Data represents percentage and number of B Cells (B,D) and B memory cells (C,E) in animals from MPLA (closed triangles), Sm29 MPLA (closed circles), Alum (open triangles), Sm29 Alum (open circles) and infected/treated (gray circles) groups. Statistically significant differences are denoted on the graphics. Results are representative of one from two independent experiments.

Figure 5

Frequency of T memory cells in immunized mice. Spleen from 5 to 6 animals was obtained for each group to determine the frequency of T memory cells. Data analysis was carried out as demonstrated (A): within singlet cells/lymphocyte population, CD4⁺CD44^high cells were selected and, within that population, the percentage of CD127⁺CD62^low cells representing CD4⁺ T effector memory cells, CD127⁺CD62^high population representing the CD4⁺ T central memory cells were determined. Data represents percentage and number of CD4⁺ T central memory cells (B,D) effector memory cells (C,E) in animals from MPLA (closed triangles), Sm29 MPLA (closed circles), Alum (open triangles), Sm29 Alum (open circles) and infected/treated (gray circles) groups. Results are representative of one from two independent experiments.

Figure 6

Production of Sm29-specific antibodies in immunized mice. Sera from mice were obtained 15 days after each immunization dose and were assessed to determine the levels of IgG (A), IgG1 (B), IgG2a (C), and IgE (D) antibodies against rSm29 in the animals inoculated with alum (open bars), MPLA (bright-gray bars), Sm29Alum (black bars) or Sm29/MPLA (dark-gray bar). Significant differences between adjuvant control and experimental groups are indicated by an asterisk (p < 0.05). Significant difference between Sm29/Alum and Sm29/MPLA groups are denoted by the letter a (p < 0.05). Significant differences compared to the first and are second immunization dose are denoted by letters b and c, respectively. Results are representative of one from two independent experiments.