Schistosome survival <i>in vivo</i> in mice following treatment with SGTP siRNA.

<p>Schistosomula were treated with the indicated siRNA and used to infect mice. Worm numbers recovered from individual mice in each group are shown as dots. The lines indicate the mean for each group and P values are given at top.</p

Relative viability of schistosomes treated with SGTP siRNA.

<p>Schistosomula (mean ± SD) were treated with the indicated siRNA and their viability was established following culture for 14 days in RPMI medium either containing high glucose (10 mM, grey bars) or low glucose (0.05 mM, white bars).</p

Expression of SGTP1 and SGTP4 (mean ± SE) in schistosomula at different times after treatment with the indicated siRNA.

<p>Parasites were either maintained in culture for 7 days after treatment (white bars) or for 4 week after treatment (grey bars), or were recovered from infected mice 4 weeks after treatment (black bars). A, SGTP1; B, SGTP4.</p

<i>Schistosoma mansoni</i> Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins

<div><p>Schistosomiasis is a major disease of the developing world for which no vaccine has been successfully commercialized. While numerous <i>Schistosoma mansoni</i> worm antigens have been identified that elicit antibody responses during natural infections, little is known as to the identities of the schistosome antigens that are most prominently recognized by antibodies generated through natural infection. Non-reducing western blots probed with serum from schistosome-infected mice, rats and humans on total extracts of larval or adult schistosomes revealed that a small number of antigen bands predominate in all cases. Recognition of each of these major bands was lost when the blots were run under reducing condition. We expressed a rationally selected group of schistosome tegumental membrane antigens in insect host cells, and used the membrane extracts of these cells to unambiguously identify the major antigens recognized by <i>S</i>. <i>mansoni</i> infected mouse, rat and human serum. These results revealed that a limited number of dominant, reduction-sensitive conformational epitopes on five major tegumental surface membrane proteins: SmTsp2, Sm23, Sm29, SmLy6B and SmLy6F, are primary targets of mouse, rat and human <i>S</i>. <i>mansoni</i> infection sera antibodies. We conclude that, <i>Schistosoma mansoni</i> infection of both permissive (mouse) and non-permissive (rat) rodent models, as well as humans, elicit a dominant antibody response recognizing a limited number of conformational epitopes on the same five tegumental membrane proteins. Thus it appears that neither infecting schistosomula nor mature adult schistosomes are substantively impacted by the robust circulating anti-tegumental antibody response they elicit to these antigens. Importantly, our data suggest a need to re-evaluate host immune responses to many schistosome antigens and has important implications regarding schistosome immune evasion mechanisms and schistosomiasis vaccine development.</p></div

<i>S</i>. <i>mansoni</i> infected mouse, rat and human sera recognition of selected <i>S</i>. <i>mansoni</i> antigens expressed as recombinant proteins by insect cells.

<p>Insect cells engineered to express selected <i>S</i>. <i>mansoni</i> membrane antigens were extracted in a mild detergent buffer and resolved by SDS-PAGE under non-reducing conditions. <b>A.</b> Replicate filters containing SmLy6A, SmLy6B, SmLy6C, SmLy6F and Sm29 were probed by mouse, rat or human infection sera (1:2000). <b>B</b>. Replicate filters containing SmTsp2 and Sm23 were probed by mouse, rat or human infection sera. Detection employed the appropriate HRP/anti-mouse, -rat or -human IgG reagent. Numbers indicate the positions of migration of molecular mass markers (kDa).</p

Insect extracts containing recombinant tetraspanins, SmTsp2 and Sm23 block recognition of a major 20 kDa reduction-sensitive <i>S</i>. <i>mansoni</i> antigen on western blots by mouse, rat and human infection sera.

<p>Total extracts from juvenile or adult mammalian-stage <i>Schistosoma mansoni</i> worms were resolved by SDS-PAGE under non-reducing conditions and transferred to filters. Pooled mouse, rat or human infection sera (1:2000) were pre-incubated for 1 hr with control lysate buffer or lysates of insect cells expressing SmTsp2 or Sm23 (as indicated) prior to overnight incubation with the filter strips. Detection employed the appropriate HRP/anti-mouse, -rat or -human IgG reagent. The arrow indicates the position of migration of SmTsp2 and Sm23. Numbers indicate the positions of migration of molecular mass markers (kDa).</p

Insect extracts containing recombinant SmLy6F block recognition of a major 15 kDa reduction-sensitive <i>S</i>. <i>mansoni</i> antigen on western blots by mouse, rat and human infection sera.

<p>Total extracts from juvenile or adult mammalian-stage <i>Schistosoma mansoni</i> worms were resolved by SDS-PAGE under non-reducing conditions and transferred to filters. Pooled mouse, rat or human infection sera (1:2000) were pre-incubated for 1 hr with control lysate buffer (-) or lysates of insect cells expressing SmLy6F (+) prior to overnight incubation with the filter strips. Detection employed the appropriate HRP/anti-mouse, -rat or -human IgG reagent. The arrow indicates the position of migration of SmLy6F. Numbers indicate the positions of migration of molecular mass markers (kDa).</p

Anti-<i>S</i>. <i>mansoni</i> tegument scFv, Teg5, recognizes reduction-sensitive epitopes on SmLy6C.

<p>Insect cells engineered to express recombinant SmLy6A, B, C or F were extracted in a mild detergent buffer and resolved by SDS-PAGE under non-reducing or reducing conditions and transferred to filters. Filters were probed by Teg5 scFv and recognition detected with HRP/anti-E-tag. Numbers indicate the positions of migration of molecular mass markers (kDa).</p

Anti-tegumental rat scFvs recognize distinct juvenile and adult <i>S</i>. <i>mansoni</i> antigens when resolved on non-reducing gels.

<p>Total extracts from juvenile or adult mammalian-stage <i>Schistosoma mansoni</i> worms were resolved by SDS-PAGE under non-reducing conditions and transferred to filters. Filters were probed by one of four different anti-tegumental scFvs (Teg1, S3. Teg4 and Teg5, all at 1 μg/ml) previously obtained from <i>S</i>. <i>mansoni</i> infected rats. Detection was by HRP/anti-E-tag recognition of a peptide E-tag expressed with each scFv. Numbers indicate the positions of migration of molecular mass markers (kDa).</p

Serum from <i>S</i>. <i>mansoni</i> infected mice, rats and humans recognize a limited number of reduction-sensitive epitopes on western blots of total worm extracts.

<p>Total extracts from juvenile (5 day cultured schistosomula) or adult mammalian-stage <i>Schistosoma mansoni</i> worms were resolved by SDS-PAGE under reducing (0.1% βME) or non-reducing conditions and transferred to filters. Filters were probed with serum from mice, rats or humans (1:2000) that were infected by <i>S</i>. <i>mansoni</i>. Detection employed the appropriate HRP/anti-mouse, -rat or -human IgG reagent. A. Filters from a non-reducing gel probed by pooled sera from ten chronically infected mice; pooled sera from five, twice-infected Fischer rats, or; pooled serum from 8 human patients identified as having schistosomiasis. B. Filters from a reducing gel probed with the same sera employed in A. C. Filters from a non-reducing gel probed with five different pools of human sera (NR, SR, I, RR, NI), each derived from eight patients displaying common apparent susceptibility or resistance to schistosome re-infection (for details on these sera, see Pinheiro et al, 2014 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005306#pntd.0005306.ref013" target="_blank">13</a>]). Numbers represent molecular mass markers (kDa).</p