Evidence That Rhesus Macaques Self-Cure from a <i>Schistosoma japonicum</i> Infection by Disrupting Worm Esophageal Function: A New Route to an Effective Vaccine?

<div><p>Background</p><p>Rhesus macaques are unusual among schistosome hosts, self-curing from an established infection and thereafter manifesting solid immunity against a challenge, an ideal model for vaccine development. Previously, the immunological basis of self-cure was confirmed; surviving worms had ceased feeding but how immunological pressure achieved this was unclear. The schistosome esophagus is not simply a conduit for blood but plays a central role in its processing. Secretions from the anterior and posterior esophageal glands mix with incoming blood causing erythrocyte lysis and tethering and killing of leucocytes.</p><p>Methodology/Principal Findings</p><p>We have analysed the self-cure process in rhesus macaques infected with <i>Schistosoma japonicum</i>. Faecal egg output and circulating antigen levels were used to chart the establishment of a mature worm population and its subsequent demise. The physiological stress of surviving females at perfusion was especially evident from their pale, shrunken appearance, while changes in the structure and function of the esophagus were observed in both sexes. In the anterior region electron microscopy revealed that the vesicle secretory process was disrupted, the tips of lining corrugations being swollen by greatly enlarged vesicles and the putative sites of vesicle release obscured by intense deposits of IgG. The lumen of the posterior esophagus in starving worms was occluded by cellular debris and the lining cytoplasmic plates were closely adherent, also potentially preventing secretion. Seven proteins secreted by the posterior gland were identified and IgG responses were detected to some or all of them. Intrinsic rhesus IgG colocalized with secreted SjMEGs 4.1, 8.2, 9, 11 and VAL-7 on cryosections, suggesting they are potential targets for disruption of function.</p><p>Conclusions/Significance</p><p>Our data suggest that rhesus macaques self-cure by blocking esophagus function with antibody; the protein products of the glands provide a new class of potential vaccine targets.</p></div

<i>S</i>. <i>japonicum</i> MEG and VAL proteins are localized to the posterior esophageal gland.

<p>Localization of target proteins on permeabilized whole worms (green, A, B and E to F), and cryosections (red, C and D) by immunocytochemistry, showing that all seven proteins, (A) MEG-4.1, (B) MEG-4.2, (C) MEG-8.2, (D) VAL-7, (E) MEG-11, (F) MEG-14 and (G) MEG-9 were solely expressed in the esophageal gland (on C & D, nuclei are blue and muscle is orange). Only the cytoplasm of the gland cell bodies (EG) was strongly positive, whilst the individual nuclei (N in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003925#pntd.0003925.g007" target="_blank">Fig 7F</a>) appeared as dark unstained spheres. The association of at least three proteins (MEG-4.2, MEG-8.2 and MEG-9) with host leucocytes in the lumen (starred, B and G) and the oral cavity (starred, G), or by a coating on both anterior and posterior esophageal linings (arrowed, C), confirmed that they were secreted. Scale bars: 50 μm (A, C, D), 20 μm (B, E, F, G).</p

Antibody targets the worm esophagus and shows distinct response patterns to worm and egg antigens.

<p>Permeabilized whole worms recovered from rhesus macaques, (A) female and (B) male, reacted with FITC-labeled anti-rhesus IgG, showed intrinsic antibodies (green) bound to the tegument, particularly in the head region, but more strikingly along the whole length of the esophageal lumen (arrowed). (C) Time course of antibody response against soluble worm antigen (SWAP), and egg antigen (SEA) preparations. Binding of antibody to both preparations was barely detected during the first 4 weeks. Thereafter the anti-SEA response rose dramatically to reach a high plateau at week 8, but the decline from week 10 onwards was not significant. In contrast, anti-SWAP reactivity rose only slowly from week 4, with its peak appearing as late as week 18. Scale bars: 50 μm (A), 20 μm (B).</p

The monkey IgG response to esophageal gland proteins is heterogeneous.

<p>Data plotted by (A) animal and (B) proteins, respectively. (A) shows that all monkeys responded to at least three esophageal proteins but variation was apparent between individuals. Monkeys 4 and 5 recognized all tested proteins, some with a strong response while the repertoire of Monkeys 1, 2 and 6 was much less impressive. (B) shows that three esophageal proteins, MEG-4.2, MEG-11and VAL-7 were recognized by all monkeys, although with different intensities. MEG-9 was recognized by IgG from five monkeys, MEG-14 by four and MEG-8.2 by three.</p

Microexon gene transcriptional profiles and evolution provide insights into blood processing by the <i>Schistosoma japonicum</i> esophagus

<div><p>Background</p><p>Adult schistosomes have a well-developed alimentary tract comprising an oral sucker around the mouth, a short esophagus and a blind ending gut. The esophagus is not simply a muscular tube for conducting blood from the mouth to gut but is divided into compartments, surrounded by anterior and posterior glands, where processing of ingested blood is initiated. Self-cure of rhesus macaques from a <i>Schistosoma japonicum</i> infection appears to operate by blocking the secretory functions of these glands so that the worms cease feeding and slowly starve to death. Here we use subtractive RNASeq to characterise the genes encoding the principal secretory products of <i>S</i>. <i>japonicum</i> esophageal glands, preparatory to evaluating their relevance as targets of the self-cure process.</p><p>Methodology/Principal findings</p><p>The heads and a small portion of the rear end of male and female <i>S</i>. <i>japonicum</i> worms were separately enriched by microdissection, for mRNA isolation and library construction. The sequence reads were then assembled <i>de novo</i> using Trinity and those genes enriched more than eightfold in the head preparation were subjected to detailed bioinformatics analysis. Of the 62 genes selected from the male heads, more than one third comprised MEGs encoding secreted or membrane-anchored proteins. Database searching using conserved motifs revealed that the MEG-4 and MEG-8/9 families had counterparts in the bird schistosome <i>Trichobilharzia regenti</i>, indicating an ancient association with blood processing. A second group of MEGs, including a MEG-26 family, encoded short peptides with amphipathic properties that most likely interact with ingested host cell membranes to destabilise them. A number of lysosomal hydrolases, two protease inhibitors, a secreted VAL and a putative natterin complete the line-up. There was surprisingly little difference between expression patterns in males and females despite the latter processing much more blood.</p><p>Significance/Conclusions</p><p>The mixture of approximately 40 proteins specifically secreted by the esophageal glands is responsible for initiating blood processing in the adult worm esophagus. They comprise the potential targets for the self-cure process in the rhesus macaque, and thus represent a completely new cohort of secreted proteins that can be investigated as vaccine candidates.</p></div

Reproductive capacities of female worms from rhesus macaques were diminished.

<p>(A) Egg number in the uterus of females from two populations. Those from rhesus macaques (blue) had a wide range from zero to 141, the majority having from 20 to 40 eggs. Those from permissive rabbit and mouse hosts also had a wide range of egg numbers, but with the majority between 140 and 160. (B) and (C) illustrate that ovary size and body length correlate positively with egg number in the uterus, with almost no overlap. Females from rhesus macaques (blue), had smaller ovaries (B), shorter bodies (C) and fewer eggs, compared to control females (red).</p

The profile of the self-cure process.

<p>(A) Fecundity of the worm population, revealed by fecal egg output reached its peak at week 8, dropped sharply thereafter and then more slowly reached zero, at the end of the study. The level of soluble circulating anodic antigen (CAA, expressed as the ratio of test/control) released in the bloodstream of rhesus macaques, an indicator of worm blood feeding and gut function, was already high at week 4 but started declining after week 12. Values are mean + or—SE, n = 6 animals. (B) The body weight of monkeys increased during the first 4 weeks after infection, declined thereafter reaching the minimum value at week 8 but then gradually recovered towards the weak 4 peak weight by the end of the study. Values are mean + or–SD, n = 6 animals.</p

Females are more susceptible to immunological pressure than males in rhesus macaques.

<p>The body lengths of females (A) and males (B) from rhesus macaques, were shorter than their equivalents from rabbits, but the two male populations overlapped considerably (B) whilst the females did not (A). The detailed difference in females from the two hosts, a mature female from a rabbit (C) and three from rhesus macaques (D to F) all to scale (bar = 1mm), are compared. (C) The rabbit worm has black hemozoin pigment present along the whole length of the body. Females from monkeys were smaller and showed different levels of pigment in the gut: (D), Black group were full of pigment from ovary to tail, plus traces in the anterior; (E), Intermediate group had incomplete pigment only in the posterior region; (F), White group had virtually no pigment anywhere.</p

Host intrinsic antibody and esophageal gland proteins are co-localised.

<p>Immunocytochemistry on permeabilized whole worms from rhesus macaques revealed the localization of host intrinsic antibody (green), six esophageal proteins of <i>S</i>. <i>japonicum</i> (red) and worm muscle (orange). (A) MEG-4.1, (B) MEG-4.2, (C) MEG-9, (D) MEG-11, (E) MEG8.2 and (F) VAL-7. The triplet images display the distribution of host IgG in the first column, the esophageal proteins in the second, and the two overlaid in the third where the lemon yellow color indicates areas of co-localization. The pattern differed for individual esophageal proteins. Colocalization was only partial in MEG-4.2 (B), as free proteins (red) were visible in the anterior esophageal lumen. MEG-4.1 (A), MEG-9 (C) and MEG-11 (D) showed almost complete colocalization with the IgG and protein superimposed, and no free protein visible in the lumen. MEG-8.2 (E) colocalized with IgG in the posterior lining whilst VAL-7 (F) was present only in the posterior esophageal lumen. Scale bars: 50 μm (A, B, E), 20 μm (C, D, F).</p

Host antibody binds to the posterior esophagus which shows an altered cellular morphology.

<p>(A) TEM image of the posterior esophagus, showing the luminal plates (P) tightly packed and closely adherent. The lumen appears congested, occluded by debris and host cells (HC) in the center; electron dense structures (arrowed) are also visible around the edges of the debris. (B) SEM image of the plates in the posterior esophagus, showing that intervening spaces are largely absent. (C) TEM image at high magnification reveals the parallel tramline appearance of posterior esophageal plates. Where two adjacent plates are particularly closely adherent, membranous material is visible (MM, arrowed) bound to the luminal surface. The two lighter lines in the center of each plate denote the basal invagination (BI). (D) High magnification image of electron dense structures revealing they are aggregates of membranous material. Confocal images of male worm heads: (E) a longitudinal section, stained with AF488 labeled anti-monkey IgG, and (F) a transverse section, stained with Cy3 labelled anti-monkey IgG. (E) is counterstained with DAPI (blue) and phalloidin (orange) to highlight the nuclei and musculature and (F) with DAPI only. Both images reveal that intrinsic antibody (green in E, red in F) has bound to the post esophageal lining (PEL). The luminal edges of lining plates are strongly positive whilst the surface of the plates is more weakly stained. Scale bars: 20 μm (A, E, F), 2 μm (B), 200 nm (C, D).</p