Investigating Immunization With Nucleotide Enzymes of Schistosoma mansoni: Nucleoside Diphosphate Kinase and Adenylosuccinate Lyase as New Antigenic Targets Against Schistosomiasis

Abstract

Schistosomiasis, caused by Schistosoma mansoni trematode worm, affects more than 1.5 million people in Brazil. The current treatment consists in the administration of Praziquantel, the only medicine used for treatment for more than 40 years. Some of the limitations of this drug consist in its inactivity against schistosomula and parasite eggs, the appearance of resistant strains and non-prevention against reinfection. Thus, the objective of this study was to evaluate the effect of immunization with recombinant functional enzymes of the purine salvage pathway of S. mansoni, Nucleoside Diphosphate Kinase (NDPK) and Adenylosuccinate Lyase (ADSL), to evaluate the host immune response, as well as the parasite load after vaccination. For this, Balb/c mice were divided into 5 groups: control (uninfected and untreated), non-immunized/infected, NDPK infected, ADSL infected, and NDPK + ADSL infected. Immunized groups received three enzyme dosages, with a 15-day interval between each dose, and after 15 days of the last application the animals were infected with 80 cercariae of S. mansoni. On the 47th day after the infection, fecal eggs were counted and, on the 48th day after the infection, the evaluation of leukocyte response, parasite load, antibody production, cytokines quantification, and histopathological analysis were performed. The results showed that immunizations with NDPK, ADSL or NDPK + ADSL promoted a discreet reduction in eosinophil counts in lavage of peritoneal cavity. All immunized animals showed increased production and secretion of IgG1, IgG2a, and IgE antibodies. Increased production of IL-4 was observed in the group immunized with the combination of both enzymes (NDPK + ADSL). In addition, in all immunized groups there were reductions in egg counts in the liver and intestine, such as reductions in liver granulomas. Thus, we suggest that immunizations with these enzymes could contribute to the reduction of schistosomiasis transmission, besides being important in immunopathogenesis control of the disease.

Keywords: Adenylosuccinate Lyase; Nucleoside Diphosphate Kinase; Schistosoma mansoni; immunization; schistosomiasis.

FIGURE 1

Experimental design for immunization of the animals with the recombinant proteins NDPK and ADSL.

FIGURE 2

Number of eggs in feces (A) and adult worms recovered from the hepatic portal system (B) from two independent experiments (n = 6–7 animals/group/experiment). The numbers of eggs/gram of feces data represent the first and third quartiles at the top and bottom of the box plot graph, the middle line represents the median value and the minimum and maximum values are represented by the error bars (whiskers). The numbers of adult worms recovered data represent the mean ± SD. There was no statistical difference between the results of the immunized groups when compared with the INF group. The geometric figures represent the dispersion of the data for each group.

FIGURE 3

Number of eosinophils in the blood (A) and in the PCL (B) 48 days after infection with S. mansoni and 93 days after the first immunization. The upper and lower part of the Box plot graph represents the first and third quartiles, the middle line represents the median value and the minimum and maximum values are represented by the error bars (whiskers). (#) Represents a statistically significant difference from two independent experiments (n = 6–7 animals) using the Kruskal–Wallis non-parametric test followed by Dunn’s post-test between the results of the experimental groups when compared with the CTRL group; ##p < 0.01; ####p < 0.0001. The geometric figures represent the dispersion of the data for each group.

FIGURE 4

Concentrations of IFN-γ (A) and IL-4 (B) cytokines in plasma pool 48 days after infection with S. mansoni and 93 days after the first immunization of two independent experiments (n = 6–7 animals). The IFN-γ cytokine data represent the first and third quartiles at the top and bottom of the box plot graph, the middle line represents the median value and the minimum and maximum values are represented by the error bars (whiskers). The IL-4 cytokine data represent the mean ± SD. (#) represents statistically significant difference using the One-Way ANOVA parametric test followed by the Tukey’s post-test between the results of the experimental groups when compared with the CTRL group; ^#p < 0.05. The geometric figures represent the dispersion of the data for each group.

FIGURE 5

Detection of IgG1 (A–C), IgG2a (D–F), and IgE (G–I) antibodies in a plate sensitized with NDPK, ADSL, and NDPK+ADSL in the plasma pool of mice 48 days after infection with S. mansoni, and 93 days after the first immunization of two independent experiments (n = 6–7 animals). Nonparametric data were analyzed using the Kruskal–Wallis test followed by Dunn’s post-test and are represented in Box plot, with the lower and upper lines being the first and third quartiles, the middle line being the median value and the minimum and maximum values are represented by error bars (whiskers). Parametric data were analyzed by the One-Way ANOVA test followed by the Tukey’s post-test and are represented in the bar graph through the mean ± SD. (#) represents a statistically significant difference between the results of the experimental groups when compared with the CTRL group; ^#p < 0.05; ^##p < 0.01. (*) It represents a statistically significant difference between the results of the experimental groups when compared with the INF group; *p < 0.05; **p < 0.01; ***p < 0.001. The geometric figures represent the dispersion of the data for each group.

FIGURE 6

Histopathological sections of the liver from the groups CTRL (A,B), INF (C,D), NDPK (E,F), ADSL (G,H), and NDPK + ADSL (I,J). The images on the left were stained with hematoxylin-eosin (HE) and those on the right with Masson trichrome (TM). Increase: 300X. The arrows indicate the presence of the granuloma around the egg.