Mice immunized with Mf in alum have reduced numbers of Mf.

<p>Mice were immunized three times s.c. with 100,000 Mf in alum. Control mice received alum alone. <i>L. sigmodontis</i> infection was performed one week after the last immunization. Microfilaraemia was monitored twice a week throughout patency. (A) Kinetics of Mf load of sham-treated (dashed line) and immunized (black line) mice in the peripheral blood. One representative of three independent experiments with ten mice per group is shown (2-way ANOVA, mean ± SEM), including both Mf⁻ and Mf⁺ mice. For additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s003" target="_blank">figure S3A</a>, B. (B) Percentage of Mf⁺ mice of three independent experiments was analyzed using Student's t-test. Each mouse with peripheral Mf at any given time point was defined as Mf⁺. (C, D) Mf burden in the pleural space days 70 (C) and 90 (D) p.i.. Graphs show one representative of three (C) and two (D) independent experiments (at least seven mice each group, see also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s003" target="_blank">Figure S3C</a>–E) and were analyzed with Welch-corrected t-test. Numbers below the symbols indicate the number of Mf⁺ mice (median, * <i>P</i><0.05, ** <i>P</i><0.005).</p

Immunization inhibits embryogenesis in female worms.

<p>Mice were immunized three times s.c. with 100,000 Mf in alum. Control mice received alum alone. <i>L. sigmodontis</i> challenge infection was performed one week after the last immunization. Seventy days after infection female worms were analyzed for their embryonic stages. Representative pictures of oocyte (A; micron bar 10 µm), divided egg (B; 10 µm), pretzel stage (C; 15 µm) and stretched Mf (D; 30 µm) are shown. (E) Embryogram illustrating the composition of embryonic stages in female worms. If present, three female worms of each mouse were investigated (27 females in the control group, 28 females from the immunized group, additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s004" target="_blank">Figure S4</a>). Statistical analysis was performed with Mann-Whitney U-test (mean ± SEM, ** <i>P</i><0.01, *** <i>P</i><0.001).</p

Immunization induces Mf-specific IgG1 and IgG2.

<p>Mice were immunized three times s.c. with 100,000 Mf in alum (Al-Mf/naïve, Al-Mf/Inf). Control mice received alum alone (Al/naïve, Al/Inf). <i>L. sigmodontis</i> challenge infection was performed one week after the last immunization (Al/Inf, Al-Mf/Inf) or left uninfected (Al/naïve, Al-Mf/naïve). Plasma levels of Mf-specific IgG1 (A) and IgG2a/b (B) were measured. Two-way ANOVA was used for statistical analysis, day 0 indicates day of challenge infection. Asterisks indicate significant differences between the immunized and infected, and the corresponding control group (*** <i>P</i><0.001) and pound signs between the immunized but uninfected, and the corresponding control group (^#<i>P</i><0.05, ^##<i>P</i><0.01, ^###<i>P</i><0.001). (C–F) Pleural space lavage was analyzed for specific IgG1 and IgG2a/b on days 22 (C, D) and 70 p.i. (E, F). Data analyzed with Welch-corrected t-test (mean, *** <i>P</i><0.001). Graphs show representatives of three independent experiments with eight to ten mice each group (additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s006" target="_blank">Figure S6A, B, E–J</a>).</p

Immunization reduces adult worm burden, but does not affect their development.

<p>Mice were immunized three times s.c. with 100,000 Mf in alum. Control mice received alum alone. <i>L. sigmodontis</i> challenge infection was performed one week after the last immunization. Numbers of worms on days 15 (A), 56 (B), 70 (C) and 90 (D) p.i. (additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s005" target="_blank">Figure S5A</a>–C), gender balance (E) (individual experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s005" target="_blank">Figure S5D</a>, E), as well as length of males (F) and females (G) at day 90 p.i. (10/90 percentile, outliers are indicated, individual experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s005" target="_blank">Figure S5F</a>–I) were analyzed with Student's t-test (** <i>P</i><0.01, *** <i>P</i><0.001).</p

Immunization strategies that failed to protect mice from peripheral microfilaraemia.

<p>Mice were immunized with 100,000 Mf either three times i.v. (A, B) or first s.c. followed by an i.p. and i.v. immunization (C, D). All control mice received PBS. <i>L. sigmodontis</i> challenge infection was performed one week after the last immunization. (B) After immunization mice were treated i.v. with IVM. (D) Mice were immunized with irradiated (400 Gy) Mf. Microfilaraemia was monitored throughout patency. Data obtained from single experiments with at least six mice per group are shown. Two-way ANOVA (mean ± SEM) was used for statistical analysis including both Mf⁻ and Mf⁺ mice.</p

Immunization enhances IFN-γ responses.

<p>(A) At day 22 p.i. the pleural lavage was analyzed for IL-5 and IFN-γ. Combined data of three independent experiments with five mice each group are shown. (B, C) At day 22 p.i. cells from the site of infection were restimulated for 72 h with 5 µg/ml Concanavalin A (ConA), 100 µg/ml complete adult (Ls) or microfilarial (Mf) crude extract of <i>L. sigmodontis</i> and IFN-γ (B) and IL-5 (C) secretion were measured (mean ± SEM). Representative data of two independent experiments with five mice each group. Analysis was done using the 2-way ANOVA, for significances see text.</p