Prime-Boost Vaccine Regimen for SjTPI and SjC23 Schistosome Vaccines, Increases Efficacy in Water Buffalo in a Field Trial in China

Schistosomiasis remains a serious zoonotic disease in China and the Philippines. Water buffalo and cattle account for the majority of transmission. Vaccination of water buffalo is considered a key strategy to reduce disease prevalence. Previously, we showed that vaccination of water buffalo with SjC23 or SjCTPI plasmid DNA vaccines, induced 50% efficacy to challenge infection. Here, we evaluated several parameters to determine if we can develop a two dose vaccine that maintains the efficacy of the three dose vaccine. We performed four trials evaluating: (1) lab produced vs. GLP grade vaccines, (2) varying the time between prime and boost, (3) the influence of an IL-12 adjuvant, and (4) a two dose heterologous (DNA-protein) prime-boost. We found the source of the DNA vaccines did not matter, nor did increasing the interval between prime and boost. Elimination of the IL-12 plasmid lowered homologous DNA-DNA vaccine efficacy. A major finding was that the heterologous prime boost improved vaccine efficacy, with the prime-boost regimen incorporating both antigens providing a 55% reduction in adult worms and 53% reduction in liver eggs. Vaccinated buffalo produced vaccine-specific antibody responses. These trials suggest that highly effective vaccination against schistosomes can be achieved using a two dose regimen. No adjuvants were used with the protein boost, and the potential that addition of adjuvant to the protein boost to further increase efficacy should be evaluated. These results suggest that use of these two schistosome vaccines can be part of an integrated control strategy to reduce transmission of schistosomiasis in Asia

On the three-finger protein domain fold and CD59-like proteins in Schistosoma mansoni

Background: It is believed that schistosomes evade complement-mediated killing by expressing regulatory proteins on their surface. Recently, six homologues of human CD59, an important inhibitor of the complement system membrane attack complex, were identified in the schistosome genome. Therefore, it is important to investigate whether these molecules could act as CD59-like complement inhibitors in schistosomes as part of an immune evasion strategy. Methodology/Principal Findings: Herein, we describe the molecular characterization of seven putative SmCD59-like genes and attempt to address the putative biological function of two isoforms. Superimposition analysis of the 3D structure of hCD59 and schistosome sequences revealed that they contain the three-fingered protein domain (TFPD). However, the conserved amino acid residues involved in complement recognition in mammals could not be identified. Real-time RT-PCR and Western blot analysis determined that most of these genes are up-regulated in the transition from free-living cercaria to adult worm stage. Immunolocalization experiments and tegument preparations confirm that at least some of the SmCD59-like proteins are surface-localized; however, significant expression was also detected in internal tissues of adult worms. Finally, the involvement of two SmCD59 proteins in complement inhibition was evaluated by three different approaches: (i) a hemolytic assay using recombinant soluble forms expressed in Pichia pastoris and E. coli; (ii) complement-resistance of CHO cells expressing the respective membrane-anchored proteins; and (iii) the complement killing of schistosomula after gene suppression by RNAi. Our data indicated that these proteins are not involved in the regulation of complement activation. Conclusions: Our results suggest that this group of proteins belongs to the TFPD superfamily. Their expression is associated to intra-host stages, present in the tegument surface, and also in intra-parasite tissues. Three distinct approaches using SmCD59 proteins to inhibit complement strongly suggested that these proteins are not complement inhibitors and their function in schistosomes remains to be determined.Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, Grant Number:04/12872-3)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)National Institute of Health, National Institute of Allergy and Infectious Diseases (NIH-NIAID), Grant AI-095893NIH-NIAID Grant AI-056273FAPESP 00/11624-

Schistosomes Impede ATP-Induced T Cell Apoptosis In Vitro: The Role of Ectoenzyme SmNPP5

Schistosomes (blood flukes) can survive in the bloodstream of their hosts for many years. We hypothesize that proteins on their host-interactive surface impinge on host biochemistry to help ensure their long-term survival. Here, we focus on a surface ectoenzyme of Schistosoma mansoni, designated SmNPP5. This ~53 kDa glycoprotein is a nucleotide pyrophosphatase/phosphodiesterase that has been previously shown to: (1) cleave adenosine diphosphate (ADP) and block platelet aggregation; and (2) cleave nicotinamide adenine dinucleotide (NAD) and block NAD-induced T cell apoptosis in vitro. T cell apoptosis can additionally be driven by extracellular adenosine triphosphate (ATP). In this work, we show that adult S. mansoni parasites can inhibit this process. Further, we demonstrate that recombinant SmNPP5 alone can both cleave ATP and impede ATP-induced T cell killing. As immunomodulatory regulatory T cells (Tregs) are especially prone to the induction of these apoptotic pathways, we hypothesize that the schistosome cleavage of both NAD and ATP promotes Treg survival and this helps to create a less immunologically hostile environment for the worms in vivo

Schistosome migration in the definitive host.

Schistosomes are parasitic blood flukes that infect >200 million people around the world. Free-swimming larval stages penetrate the skin, invade a blood vessel, and migrate through the heart and lungs to the vasculature of the liver, where maturation and mating occurs. From here, the parasite couples migrate to their preferred egg laying sites. Here, we compare and contrast what is known about the migration patterns within the definitive host of the three major species of human schistosome: Schistosoma mansoni, S. japonicum, and S. haematobium. We conclude that intravascular schistosomes are inexorable colonizers whose migration and egg laying strategy is profligate; all three species (and their eggs) can be found throughout the mesenteric venules, the rectal venous plexus, and, to a greater or lesser extent, the urogenital venous plexuses. In addition, it is common for parasite eggs to be deposited in locations that lack easy access to the exterior, further demonstrating the relentless exploratory nature of these intravascular worms

Plasminogen (PLMG) interaction with rSmEno and with schistosome lysates (A) by ELISA or (B) by western blotting.

<p>(<b>A)</b> ELISA plates were coated with r<i>Sm</i>Eno (0.5 μg/well) or the indicated parasite extracts (1.0 μg/well each) and wells were incubated with increasing concentrations of PLMG (0–1 μg) in triplicate. As a negative control, some wells were coated with BSA (0.5 μg/well). Anti-PLMG antibody was used to detect PLMG following standard ELISA conditions. The lines represent the mean absorbance values at OD 450 nm (± SD). (<b>B</b>) Detection by western blot of schistosome PLMG-binding proteins (left panel). Lanes contain extracts from males (M), females (F) and schistosomula (S), as well as pure r<i>Sm</i>Eno (E), BSA (“-”, negative control) and commercially-obtained PLMG (“+”, a control for anti-PLMG antibody binding). Multiple bands in the schistosome extracts bind PLMG. The arrow indicates the position of migration of <i>Sm</i>Eno, here revealed to be a PLMG-binder. No binding to the negative control protein (BSA) is seen. The membrane was stripped and re-probed with anti-ENO1 antibody (right panel). A single, prominent ~47-kDa SmEno band is detected (arrow) in the extracts of males (M), females (F) and schistosomula (S) and in the case of purified rSmEno (E). Images are representative of three replicate experiments.</p

<i>SmEno</i> gene suppression using RNA interference.

<p>(<b>A</b>) Mean level of <i>SmEno</i> gene expression (+/-SD, n = 3) in cultured adult schistosome males (left), females (center) or schistosomula (right) at 72 hours after treatment with control, irrelevant siRNA (“Cont” black bars, set at 100%) or siRNA targeting <i>Sm</i>Eno (“Eno”, white bars), as determined by qRT-PCR. (<b>B</b>) Detection by western blot of <i>Sm</i>Eno protein (top row), in extracts prepared from parasites 72 h after treatment with <i>Sm</i>Eno (Eno) or control (Cont) siRNAs. Diminished levels of <i>Sm</i>Eno protein is seen in the first lane of each group of samples. Western blot analysis detecting a control schistosome protein (lower row) shows roughly equivalent protein amounts per lane. (<b>C</b>) Mean (+/-SD, n = 3) surface <i>Sm</i>Eno enzyme activity in live adult male (left) or female (center) parasites or schistosomula (right) after treatment with control siRNA (“Cont”, black bars) or siRNA targeting <i>Sm</i>Eno (“Eno”, white bars). Significant differences between suppressed compared to control parasites are denoted by ***, p <0.001. (<b>D</b>) Plasmin activity (mean OD₄₀₅ value +/- SD, n = 3) detected in live adult male (left) or female (center) parasites or schistosomula (right) after treatment with control siRNA (“Cont”, black bars) or siRNA targeting <i>Sm</i>Eno (“Eno”, white bars). All conditions contain plasminogen (PLMG), and tissue plasminogen activator (tPA).</p

Plasminogen (PLMG) activation by schistosomes in the presence of tissue plasminogen activator (tPA).

<p>(<b>A</b>) Plasmin activity (mean OD₄₀₅ value +/- SD; 60 min) detected in the presence (+) or absence (-) of live schistosomula (1000 parasites per well, n≥5). (<b>B</b>) Plasmin activity (mean OD₄₀₅ value +/- SD; 60 min) detected in the absence (white bar) or presence of male (black bar) or female (gray bar) adult schistosomes. In each case, ≥5 adult worms were evaluated individually. (<b>C</b>) Plasmin activity (mean OD₄₀₅ value +/- SD; 30 min) detected in the presence of different numbers of male parasites as indicated, n≥5. (<b>D</b>) Plasmin activity (mean OD₄₀₅ value +/- SD; 60 min) detected in the absence (white bar) or presence of individual male <i>S</i>. <i>japonicum</i> (Sj), <i>S</i>. <i>haematobium</i> (Sh) or <i>S</i>. <i>mansoni</i> (Sm), n ≥10. Significant differences from control conditions (reagents themselves without parasites) are denoted by *, p<0.05, and ***, p <0.001.</p

Heterologous expression, purification and kinetics of r<i>Sm</i>Eno.

<p>(<b>A</b>) Heterologous expression of r<i>Sm</i>Eno in <i>E</i>. <i>coli</i> BL21 Star (DE3). Coomassie-stained gel showing SDS-PAGE resolution of lysates of <i>E</i>. <i>coli</i> bacteria harboring pTrcHisB::<i>Sm</i>Eno before (Gel, left lane) or 4 hours after (Gel, right lane) protein expression induction. The arrow indicates r<i>Sm</i>Eno in the induced lane. In western blot analysis of these lysates probed with monoclonal anti-His tag antibody, a prominent ~50 kDa protein (<i>Sm</i>Eno) is detected (Western Blot, anti-His, arrow). Recombinant <i>Sm</i>Eno protein was purified from bacterial lysate by immobilized metal affinity chromatography. A single ~50 kDa pure protein (<i>Sm</i>Eno) is resolved following Coomassie brilliant blue staining of an SDS-PAGE gel and this protein binds anti-ENO antibody, as determined by western blot analysis (right lane, arrow). Positions of migration of molecular mass markers are indicated on the left (kDa). Michaelis-Menten kinetic curve generated using PGA as substrate (<b>B</b>, catalyzing the forward reaction) or using PEP as substrate (<b>C</b>, catalyzing the reverse reaction). The apparent Km and Vmax values shown represent the mean +/- SD of three independent experiments. (<b>D</b>) Recombinant <i>Sm</i>Eno activity in a buffer system covering the pH range 5.5–9.5. Enzymatic activity is maximal at pH 7.5. (<b>E</b>) Impact of divalent ion (Mg²⁺ or Ca²⁺, as indicated) concentration on mean r<i>Sm</i>Eno activity (± SD). The highest activity value (at 100 mM Mg²⁺) was set at 100% and relative activities were calculated and are presented. Significant differences relative to equivalent measurements containing Ca²⁺ are denoted by *** for p <0.001. EDTA is Ethylenediaminetetraacetic acid. (<b>F</b>) r<i>Sm</i>Eno activity in the presence of increasing concentrations of NaF (white bars) compared to its activity in the absence of inhibitors (set at 100%, black bar). Significant differences relative to the untreated control are denoted by *** for p <0.001. (<b>G</b>) Influence of increasing concentrations of mefloquine (MFQ) on r<i>Sm</i>Eno activity (left bars) or on schistosomula lysate enolase activity (right bars). Activity measured in the absence of MFQ was set at 100% and relative activities were calculated. Significant differences relative to the untreated control are denoted by * for p<0.05 and ** for p<0.01. In E-F, bars represent mean relative activity ± SD, n = 3.</p