Schistosoma mansoni Venom Allergen Like Proteins Present Differential Allergic Responses in a Murine Model of Airway Inflammation

The Schistosoma mansoni Venom Allergen Like proteins (SmVALs) have been identified in the Transcriptome and Post-Genomic studies as targets for immune interventions. Two secreted members of the family were obtained as recombinant proteins in the native conformation. Antibodies produced against them showed that SmVAL4 was present mostly in cercarial secretions and SmVAL26 in egg secretions and that only the native SmVAL4 contained carbohydrate moieties. Due to concerns with potential allergic characteristics of this class of molecules, we have explored the mouse model of airway inflammation in order to investigate these properties in a more confined system. Sensitization and challenge with rSmVAL4, but not rSmVAL26, induced extensive migration of cells to the lungs, mostly eosinophils and macrophages; moreover, immunological parameters were also characteristic of an allergic inflammatory response. Our results showed that the allergic potential of this class of proteins can be variable and that the vaccine candidates should be characterized; the mouse model of airway inflammation can be useful to evaluate these properties

Molecular characterization of nucleotide pyrophosphatases/ phosphodiesterases of Schistosoma mansoni and their investigation as vaccine antigens.

Neste trabalho caracterizou-se a família das nucleotídeo pirofosfatases/ fosfodiesterases do parasita S. mansoni visando avaliá-los como antígenos vacinais. O parasita possui quatro proteínas desta família (SmNPP-5a, SmNPP-5b, SmNPP-5c e SmNPP-6), sendo que duas delas possuem maior expressão gênica nos estágios que infectam o homem. Dos anticorpos produzidos, apenas o anti-SmNPP-5a apresentou especificidade para a proteína nativa e utilizando-os demonstrou-se que a SmNPP-5a é uma glicoproteína associada às membranas do tegumento dos vermes adultos. Também se verificou que esses anticorpos inibiram parcialmente a atividade da enzima em parasitas vivos. Assim avaliamos a SmNPP-5a recombinante como antígeno vacinal juntamente com uma apirase (SmATPDase) e com a fosfatase alcalina (SmAP), outras duas nucleotidases presentes no tegumento de parasitas adultos. A SmNPP-5 e a SmNTDPase apresentaram menor imunogenicidade que a SmAP. Porém só verificamos a redução da carga parasitária em animais imunizados com a SmAP e tratados com doses subcurativas de praziquantel.In this work the family of nucleotide pyrophosphatases/phosphodiesterases (NPP) from S. mansoni was characterized in order to evaluate them as vaccine antigens. The parasite has four proteins of this family (SmNPP-5a, SmNPP-5b, SmNPP-5c and SmNPP-6), whereas two of them present higher gene expression in stages that infect humans. Only anti-SmNPP-5a anitbodies showed specificity for the native protein. We use them to characterize SmNPP-5a as a glycoprotein associated with tegument membranes from adult worms. It was also found that these antibodies were able to partially inhibit the activity of the enzyme in live parasites. Thus we evaluated SmNPP-5a as recombinant vaccine antigen together with an apyrase (SmATPDase) and alkaline phosphatase (SmAP), two other nucleotidases involved in nucleotide metabolism and present in the tegument of adult parasites. SmNTDPase and SmNPP-5 were less immunogenic than SmAP. However, we only verified the reduction of parasite burden in mice immunized with SmAP, when the animals also received a subcurative treatment with praziquantel

Characterization of phosphodiesterase-5 as a surface protein in the tegument of Schistosoma mansoni.

Schistosoma mansoni is a major causative agent of schistosomiasis, an important parasitic disease that constitutes a severe health problem in developing countries. Even though an effective treatment exists, it does not prevent re-infection and the development of an effective vaccine still remains the most desirable means of control for this disease. In thisworkwe describe the cloning and characterization of a S. mansoni nucleotidepyrophosphatase/phosphosdiesterase type 5(SmNPP-5), previously identifiedinthe tegument by proteomic studies. In silico analysis predicts an N-terminal signal peptide, three N-glycosylation sites and a C-terminal transmembrane domain similar to that described for mammalian isoforms. Real-time quantitative RT-PCR andWestern blot analyses determined that SmNPP-5 is significantly upregulated in the transition from free-living cercaria to schistosomulum and adult worm parasitic stages; additionally, the native protein was demonstrated to be N-glycosylated. Immunolocalization experiments and tegument surface membrane preparations confirm the protein as a tegument surface protein. Furthermore, the ectolocalization of this enzyme was corroborated through the hydrolysis of the phosphodiesterase specific substrate (_-Nph-5_-TMP) by living adult and 21-day-old worms. Interestingly, pre-incubation of adult and 21-day-old worms with anti-rSmNPP-5 antibody was able to reduce by 50–60% the enzyme activity. These results suggest that SmNPP-5 is closely associated with the new tegument surface generation after cercarial penetration, and being located at the host–parasite interface, is a potential target for immune intervention

Immunoblotting of released proteins using anti-rSmVALs polyclonal antibodies.

<p>(A) Released proteins by newly transformed schistosomulum (RP) cultured 0–6 h or correspondent parasite extract (PE) hybridized with anti-rSmVAL4 (RP - total released proteins by 1000 parasites was loaded in each lane, PE - 10 µg of protein extract was loaded in each lane). (B) EGG, eggs; MIR, miracidia; Hf, hatched fluid containing released proteins by hatching eggs, hybridized with anti-rSmVAL26; (C) ESP, secreted proteins by 72 h cultured viable mature eggs, hybridized with anti-rSmVAL26, (20 µg of protein was loaded in each lane); P, positive control rSmVALs (50 ng).</p

Molecular weight and isoeletric point of SmVALs investigated in this study.

a<p>Exp Mw = Expected Molecular weight.</p>b<p>Obs Mw = Approximate Observed Molecular weight.</p

Evaluation of rSmVALs induction of airway inflammation and lung histopathology.

<p>(A) BAL total and differential cell counts for animals immunized with rSmVAL4 or rSmVAL4-Pro (rSmVAL4 protein after treatment with Pronase) and (B) rSmVAL26. Control group received only the intranasal challenge with rSmVAL and naïve mice received only PBS. Results are expressed as means ± SEM for groups of four mice and are representative of two experiments. *Significant differences (p<0.05) when compared to Control (mice that were only challenged with the respective proteins). (C) Lung sections of mice that were only challenged. (D) Representative lung sections of rSmVAL4-induced allergic airway inflammation in BALB/c mice, revealing the marked infiltration of inflammatory cells in the peribronchiolar space. (E) Lung sections of mice that received rSmVAL4-Pro, showing patterns similar to the control group; pictures at 10× of magnification.</p

rSmVALs specific antibody production.

<p>BALB/c were sensitized s.c. with rSmVALs/Alum, rSmVAL4-Pro/Alum or PBS/Alum (Control) and then challenged i.n. with the proteins. The experiments were performed 24 h after the last challenge. (A) rSmVALs-specific IgG1 and (B) rSmVALs-specific IgE were determined in the sera by sandwich ELISA. Results are expressed as means ± SEM for groups of four mice and are representative of two experiments. a, b or #, significant differences (p<0.05) when compared to Control group (mice that were challenged with protein only).</p

Immunoblotting of protein extracts from <i>S. mansoni</i> stages and tegument fraction using anti-rSmVALs polyclonal antibodies.

<p>(A) Anti-rSmVAL4, and (B) anti-rSmVAL26. CER, cercariae; EGG, eggs; MIR, miracidia; AD, adult worms (female and male); SCH, 7-day old schistosomula; TEG, tegument; (C) Immunoblotting of Cercariae extracts before (−) and after (+) treatment with PNGase F using anti-rSmVAL4; and (D) Immunoblot of Egg extracts before (−) and after (+) treatment with PNGase F using anti-rSmVAL26 (20 µg of protein was loaded in each lane); P, positive control rSmVALs (50 ng). Positions of molecular mass standards (kDa) are indicated inside or on the side the autoradiogram film.</p