Evaluation of the Immunomodulatory Effect of the Recombinant 14-3-3 and Major Antigen Proteins of Strongyloides stercoralis against an Infection by S. venezuelensis

Abstract

Strongyloidiasis, caused by Strongyloides stercoralis, is a neglected parasitic disease that represents a serious public health problem. In immunocompromised patients, this parasitosis can result in hyperinfection or disseminated disease with high levels of mortality. In previous studies, the mRNAs encoding for the 14-3-3 and major antigen proteins were found to be expressed at high levels in S. stercoralis L3 larvae, suggesting potential key roles in parasite-host interactions. We have produced them as recombinant proteins (rSs14-3-3 and rSsMA) in a bacterial protein expression system. The serum levels of anti-rSs14-3-3 and anti-rSsMA IgGs are increased upon infection with S. venezuelensis, validating the use of the mouse model since the native 14-3-3 and MA proteins induce an immune response. Each recombinant protein was formulated in the adjuvant adaptation (ADAD) vaccination system and injected twice, subcutaneously, in CD1 mice that were experimentally infected with 3000 S. venezuelensis L3 to evaluate their protective and immunomodulatory activity. Our results, including the number of parthenogenetic females, number of eggs in stool samples and the analysis of the splenic and intestinal indexes, show that the vaccines did not protect against infection. The immunization with rSs14-3-3 induced changes in the cytokine profile in mice, producing higher expression of IL-10, TGF-β, IL-13 and TNF-α in the spleen, suggesting a Th2/Treg-type response with an increase in TNF-α levels, confirming its role as an immunomodulator.

Keywords: 14-3-3; ADAD; Strongyloides stercoralis; cytokines; major antigen; vaccination.

Figure 1

Synthesis and purification of rSs14-3-3 and rSsMA in E. coli. Purified recombinant Ss14-3-3 and SsMA proteins (10 µg/lane) were separated on SDS-PAGE and visualized using Coomassie blue stain. Molecular weight markers in kDa are shown (Precision Plus Protein^TM Dual Color Standards (BIO-RAD, Hercules, CA, USA)).

Figure 2

Number of eggs per gram (EPG) in mice feces after immunization with rSs14-3-3 and rSsMA formulated in ADAD vaccination system and challenged with 3000 S. venezuelensis L3 determined at 5, 6, and 7 days post-infection (PI). Bars represent means ± SEM. Differences between groups were determined by two-way ANOVA F_(3,84) = 4.18; p = 0.0083. Post-hoc Tukey’s honestly significant difference (HSD) test p-values are shown in the graph.

Figure 3

Number of S. venezuelensis parthenogenetic females after immunization with rSs14-3-3 and rSsMA formulated in ADAD vaccination system and challenged with 3000 L3. Bars represent means ± SEM. The number of recovered females under each experimental condition was evaluated by one-way ANOVA analysis with respect to the infection control group (F_(3,27) = 1; p < 0.0001). Post-hoc Tukey’s honestly significant difference (HSD) test p-values are shown in the graph.

Figure 4

Effect of the immunization in the Splenic (A) and Intestinal (B) indexes after infection of mice with S. venezuelensis. Bars represent means ± SEM. Data were evaluated by one-way ANOVA analysis against infection and ADAD controls, but there were not significant differences (F_(3,28) = 0; p = 0.5086 for the splenic index and F_(3,28) = 0.5649; p = 0.6427 for the intestinal index).

Figure 4

Effect of the immunization in the Splenic (A) and Intestinal (B) indexes after infection of mice with S. venezuelensis. Bars represent means ± SEM. Data were evaluated by one-way ANOVA analysis against infection and ADAD controls, but there were not significant differences (F_(3,28) = 0; p = 0.5086 for the splenic index and F_(3,28) = 0.5649; p = 0.6427 for the intestinal index).

Figure 5

Serum levels of anti-14-3-3 IgG (A) and anti-MA IgG (B) in mice before and after the treatment (immunization and infection). IgG levels were measured by indirect ELISA by coating the plates with 2 µg/mL of 14-3-3 protein or 2 µg/mL MA protein per well, respectively. (A) IgG levels against rSs14-3-3 protein; (B) IgG levels against rSsMA protein. Bars represent means ± SEM. Differences between groups were determined by two-way ANOVA. The IgG levels are significantly increased after the assay ((A): F_(1,42) = 70.24; p < 0.0001; (B): F_(1,42) = 37.08; p < 0.0001). Post-hoc Tukey’s honestly significant difference (HSD) test p-values are shown in the graph.

Figure 6

Expression levels of IL-10, TGF-β, IL-13 and TNF-α in spleen of mice immunized with rSs14-3-3 and rSsMA formulated in ADAD and challenged with 3000 S. venezuelensis infective L3. (A) IL-10 mRNA levels; (B) TGFβ mRNA levels; (C) IL-13 mRNA levels; (D) TNFα mRNA levels. Bars represent means ± SEM. Data were evaluated by one-way ANOVA analysis for each cytokine IL-10 (F_(4,9) = 13; p = 0.0007); TGF-β (F_(4,19) = 1; p = 0.0001); IL-13 (F_(4,10) = 5; p = 0.0152; TNF-α (F_(4,6) = 12; p = 0.0042). Post-hoc Tukey’s honestly significant difference (HSD) test p-values are shown in the graphs.