Immunoprotection of Mice against Schistosomiasis Mansoni Using Solubilized Membrane Antigens

<div><p>Background</p><p>Schistosomiasis continues to be one of the most prevalent parasitic diseases in the world. Despite the existence of a highly effective antischistosome drug, the disease is spreading into new areas, and national control programs do not arrive to complete their tasks particularly in low endemic areas. The availability of a vaccine could represent an additional component to chemotherapy. Experimental vaccination studies are however necessary to identify parasite molecules that would serve as vaccine candidates. In the present work, C57BL/6 female mice were subcutaneously immunized with an <i>n</i>-butanol extract of the adult worm particulate membranous fraction (AWBE) and its protective effect against a <i>S. mansoni</i> challenge infection was evaluated.</p><p>Methodology and Findings</p><p>Water-saturated <i>n</i>-butanol release into the aqueous phase a set of membrane-associated (glyco)proteins that are variably recognized by antibodies in schistosome-infected patients; among the previously identified AWBE antigens there is Alkaline Phosphatase (SmAP) which has been associated with resistance to the infection in mice. As compared to control, a significantly lower number of perfuse parasites was obtained in the immunized/challenged mouse group (P<0.05, <i>t</i> test); and consequently, a lower number of eggs and granulomas (with reduced sizes), overall decreasing pathology. Immunized mice produced high levels of sera anti-AWBE IgG recognizing antigens of ∼190-, 130-, 98-, 47-, 28-23, 14-, and 9-kDa. The ∼130-kDa band (the AP dimer) exhibited <i>in situ</i> SmAP activity after addition of AP substrate and the activity was not apparently inhibited by host antibodies. A preliminary proteomic analysis of the 25-, 27-, and 28-kDa bands in the immunodominant 28–23 kDa region suggested that they are composed of actin.</p><p>Conclusions</p><p>Immunization with AWBE induced the production of specific antibodies to various adult worm membrane molecules (including AP) and a partial (43%) protection against a challenging <i>S. mansoni</i> infection by mechanism(s) that still has to be elucidated.</p></div

Synthetic peptides sequences derived from known <i>S. mansoni</i> soluble protein antigens tested by MABA against sera of the different treated mouse groups.

<p>1–3: Pre-immunization sera; 4–6: sera of AWBE-immunized mice; 7–9 sera of mice immunized with AWBE and then infected with <i>S. mansoni</i>; 10–12: sera of mice inoculated with ADJ; 13–15: sera of mice inoculated with ADJ and then infected with <i>S. mansoni</i>.</p

Microscopic appearance of liver in the different mouse groups.

<p>Microscopic appearance of histological sections of livers of C57BL/6 mice dissected eight weeks after infection with 150 live <i>S. mansoni</i> (JL strain) cercariae. (A) Healthy (250×) (B) Animals immunized with AWBE/ADJ and infected (200×); (C) Animals injected with ADJ and infected (200×). Arrows indicate parasite eggs.</p

Hepatomegaly measurements of the livers of the different C57BL/6 experimental groups (<i>n</i> = 10 per group) after a challenge infection with 150 <i>S. mansoni</i> (JL strain) cercariae.

<p>Average increase in weight (A) and volume (B) of livers from the differently treated mouse groups after <i>S. mansoni</i> challenge infection, in relation to control livers from non-inoculated/non-infected healthy mice, matched for age and sex with mice of the experimental groups. B = buffer; ADJ = adjuvant; AWBE = Adult Worm <i>n</i>-Butanol Extract. (C) Hepatomegaly expressed as differential over increase percent (%) of weight and volume in livers of <i>S. mansoni</i>-infected mouse groups, compared to absence of hepatomegaly (livers of non-inoculated/non-infected mice).</p

SDS-PAGE/WB analyses of AWBE against sera of treated mouse groups.

<p>(<b>A</b>) Non-reducing SDS-PAGE (15%, 30 µg/lane)/Western Blot (IgG) profile of <i>S. mansoni</i> (JL strain) AWBE against sera from the different mouse groups; on the right, corresponding Coomassie Blue stained 1D gel. (<b>B</b>) Non-reducing SDS-PAGE (7.5%, 30 µg/lane)/Western Blot (IgG) profile of <i>S. mansoni</i> (JL strain) AWBE against sera from the different mouse groups; on the right: 1D gel stained incubated with alkaline phosphatase substrate (nitrobluetetrazolium/5-bromo-4-chloro-3′-indolylphosphate <i>p</i>-toluidine salt) in 100 mM diethylethanolamine pH 9.5). (<b>C</b>) Determination of anti-<i>S. mansoni</i> (IgG) alkaline phosphatase by APIA. Anti-<i>S. mansoni</i> (JL strain) alkaline phosphatase IgG antibodies in sera of the different C57BL/6 mouse groups (6 mice/group). ADJ: adjuvant; AWBE: Adult Worm <i>n</i>-Butanol Extract. Mouse groups were exposed to 150 live <i>S. mansoni</i> cercariae. Significant differences are denoted by one asterisk * (<i>p</i><0.05, <i>t</i> test). Points: individual mice.</p

Protective effect of AWBE antigens against a challenge infection with 150 live <i>S. mansoni</i> cercariae in C57BL/6 mice.

1<p>Average n° of adult worms (males and females) recovered in the perfusion fluid of liver and small mesenteric veins from each experimental group (Mean ± SEM, <i>n</i> = 10).</p><p>The reduction of the parasitic load was calculated according to formula: % Protection = (1−B/A)×100 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002254#pntd.0002254-Shuxian1" target="_blank">[31]</a> where A is the average n° of worms in the AWBE-immunized group and, B the average n° of worms in the adjuvant (ADJ) inoculated group (Control). AW: Adult Worms; AWBE: Adult Worm <i>n</i>-Butanol Extract; ADJ: adjuvant inoculated mice; B: mice injected with buffer. Significance by <i>t</i> student test (compared to group of ADJ-inoculated animals):</p>*<p>significant (<i>p</i>≤0.05).</p

Mortality in C57BL/6 mice immunized with AWBE/ADJ and infected 5 days later with a high parasitic load (∼450 live <i>S. mansoni</i> cercariae, JL strain).

<p>Mortality in C57BL/6 mice immunized with AWBE/ADJ and infected 5 days later with a high parasitic load (∼450 live <i>S. mansoni</i> cercariae, JL strain).</p

Post-treatment ECG/Holter findings with statistical significance according to age of children with oral Chagas disease, Chacao, Caracas, Venezuela, 2008–2017 (Abnormalities without statistical significance were not shown).

Post-treatment ECG/Holter findings with statistical significance according to age of children with oral Chagas disease, Chacao, Caracas, Venezuela, 2008–2017 (Abnormalities without statistical significance were not shown).</p

Absolute number of evaluated patients with oral Chagas disease according to age group before and after the first treatment, Chacao, Caracas, Venezuela 2007–2017.

Absolute number of evaluated patients with oral Chagas disease according to age group before and after the first treatment, Chacao, Caracas, Venezuela 2007–2017.</p

S4 Data -

Incidence of clinical (A) and ECG/Holter (B) findings after treatment in 106 patients with Chagas disease orally acquired, Chacao, Caracas, Venezuela, 2008–2017. (TIF)</p