Can antibody conjugated nanomicelles alter the prospect of antibody targeted therapy against schistosomiasis mansoni?

BACKGROUND: CLA (conjugated linoleic acid)-mediated activation of the schistosome tegument-associated sphingomyelinase and consequent disruption of the outer membrane might allow host antibodies to access the apical membrane antigens. Here, we investigated a novel approach to enhance specific antibody delivery to concealed surface membrane antigens of Schistosoma mansoni utilising antibody-conjugated-CLA nanomicelle technology. METHODOLOGY/PRINCIPAL FINDINGS: We invented and characterised an amphiphilic CLA-loaded whey protein co-polymer (CLA-W) as an IV injectable protein nanocarrier. Rabbit anti-Schistosoma mansoni infection (anti-SmI) and anti-Schistosoma mansoni alkaline phosphatase specific IgG antibodies were purified from rabbit sera and conjugated to the surface of CLA-W co-polymer to form antibody-conjugated-CLA-W nanomicelles (Ab-CLA-W). We investigated the schistosomicidal effects of CLA-W and Ab-CLA-W in a mouse model of Schistosoma mansoni against early and late stages of infection. Results showed that conjugation of nanomicelles with antibodies, namely anti-SmI, significantly enhanced the micelles' schistosomicidal and anti-pathology activities at both the schistosomula and adult worm stages of the infection resulting in 64.6%-89.9% reductions in worm number; 72.5-94% and 66.4-85.2% reductions in hepatic eggs and granulomas, respectively. Treatment induced overall improvement in liver histopathology, reducing granuloma size and fibrosis and significantly affecting egg viability. Indirect immunofluorescence confirmed CLA-W-mediated antigen exposure on the worm surface. Electron microscopy revealed extensive ultrastructural damage in worm tegument induced by anti-SmI-CLA-W. CONCLUSION/SIGNIFICANCE: The novel antibody-targeted nano-sized CLA delivery system offers great promise for treatment of Schistosoma mansoni infection and control of its transmission. Our in vivo observations confirm an immune-mediated enhanced effect of the schistosomicidal action of CLA and hints at the prospect of nanotechnology-based immunotherapy, not only for schistosomiasis, but also for other parasitic infections in which chemotherapy has been shown to be immune-dependent. The results propose that the immunodominant reactivity of the anti-SmI serum, Schistosoma mansoni fructose biphosphate aldolase, SmFBPA, merits serious attention as a therapeutic and vaccine candidate.

Author Correction: Antigenic cross-reactivity between Schistosoma mansoni and allergenic invertebrates putatively due to shared glycanic epitopes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Antigenic cross-reactivity between Schistosoma mansoni and allergenic invertebrates putatively due to shared glycanic epitopes.

Previous studies have shown that rabbit IgG antibodies against Schistosoma mansoni egg antigens (SmSEA) cross-react with allergens in natural rubber latex, peanuts and grass and tree pollens. Here we describe antigenic molecules that cross-react with rabbit anti-S. mansoni IgG antibodies in extracts of the house dust mite (HDM) Dermatophagoides farinae, the Australian cockroach (ACR) Periplaneta australasiae and in the venom of the honey bee Apis mellifera (HBV). Tandem mass spectrometry identified the cross-reactive allergens as Der f 15 in HDM, two homologues of the Periplaneta americana cockroach allergen Cr-PI/Per a 3 in ACR and two isoforms of the allergen Api m 1 (phospholipase A2: PLA2) in HBV. Cross-reactive rabbit anti-SmSEA IgG antibodies eluted from the three invertebrate allergens reacted with S. mansoni egg antigens and variably with schistosome cercarial and worm antigens. Treatment of the electroblotted allergens with sodium metaperiodate abrogated most of the cross-reactivity of the rabbit anti-SmSEA antibodies, suggesting it was due to cross-reactive carbohydrate determinants (CCDs). Furthermore, analyses of the allergens' amino acid sequences indicated that they had potential for both N- and O-linked glycosylation. A potential role for the CCDs shared by the schistosome and invertebrates in inducing an allergy-protective effect, as proposed by the hygiene hypothesis, is discussed.

Biological and proteomic studies of Schistosoma mansoni with decreased sensitivity to praziquantel.

The impact of the laboratory induced Schistosoma mansoni with decreased PZQ sensitivity on the biological performance of its different developmental stages and the concomitant structural changes of adult worms' total proteins were investigated. PZQ exposed snails showed stoppage of cercarial shedding for eight weeks followed by progressive significant reduction of cercarial production along four successive weeks. In the vertebrate host, in comparison to Schistosoma mansoni susceptible isolate, inoculated cercariae with decreased PZQ sensitivity led to an evident decrease in male to female ratio associated with significant reduction in tissue egg counts and significant increase in dead egg percentage. Significant reduction in the fecundity was also determined. Interestingly, eggs from adult worms with decreased PZQ sensitivity showed two unique features as they found to be smaller and more spherical in addition to the observation of hourglass shaped miracidium in about 10% of the detected mature eggs. Proteomic analysis of adult worms with decreased sensitivity to PZQ using mass spectrometry revealed up-regulation of Ca2+ ATPase 2 and Hsp70. This study can point to the increase incidence of the neuroschistosomiasis due to the small size eggs of Schistosoma mansoni with reduced PZQ sensitivity. These worms can also impact the epidemiology in the field. The study can also provide help to elucidate underlying potential molecular mechanisms of resistance that could lead to possible strategies to reverse drug resistance.

Antigenic cross-reactivity between Schistosoma mansoni and pollen allergens from the birch tree (Betula verrucosa) and Timothy grass (Phleum pratense): involvement of shared glycan epitopes and implications for the hygiene hypothesis.

Previous studies have shown that schistosome infection can protect against allergic symptoms, but the underlying mechanisms are still not fully understood. Here we have shown that rabbit IgG antibodies raised against Schistosoma mansoni soluble egg antigens (SmSEA) are cross-reactive with a wide array of molecules in Timothy grass pollen (TGP) and birch tree pollen (BTP). Five of the cross-reactive pollen molecules (two from TGP and three from BTP) were selected randomly and identified by tandem mass spectrometric (TMS) analysis to be, respectively, the TGP allergens Phl p 1 and Phl p 5b, and BTP glutathione S-transferase (GST), and the BTP allergens Bet v 1 and Bet v 6.0102. Rabbit anti-SmSEA IgG antibodies that cross-reacted with each of the five allergens were found to be reactive with three major S. mansoni egg antigens, IPSE/alpha-1, omega-1 and kappa-5. Pairwise alignment of the amino acid sequences of each of the five TMS-identified pollen allergens with each of the three egg antigens revealed a low level of amino acid sequence identity. Further experiments indicated that the schistosome antigen/allergen cross-reactivity was mostly due to similar glycans present in helminths and plants, but not in mammals: so called cross-reactive carbohydrate determinants (CCDs). Previously, CCDs have been implicated in the cross-reactivity between many plants and invertebrates. Furthermore, pollen-induced anti-CCD IgGs have been found in sera of patients undergoing allergen-specific immunotherapy (SIT) and implicated in the treatment of the allergy. Thus, our finding provides not only possible explanations for the allergy-protective effect of helminth/schistosome infections as explained by the hygiene hypothesis, but also a potential starting point for improved SIT.

Treatment of Schistosoma mansoni with miltefosine in vitro enhances serological recognition of defined worm surface antigens.

BACKGROUND: Miltefosine, an anti-cancer drug that has been successfully repositioned for treatment of Leishmania infections, has recently also shown promising effects against Schistosoma spp targeting all life cycle stages of the parasite. The current study examined the effect of treating Schistosoma mansoni adult worms with miltefosine on exposure of worm surface antigens in vitro. METHODOLOGY/PRINCIPAL FINDINGS: In an indirect immunofluorescence assay, rabbit anti-S.mansoni adult worm homogenate and anti-S. mansoni infection antisera gave strong immunofluorescence of the S. mansoni adult worm surface after treatment with miltefosine, the latter antiserum having previously been shown to synergistically enhance the schistosomicidal activity of praziquantel. Rabbit antibodies that recognised surface antigens exposed on miltefosine-treated worms were recovered by elution off the worm surface in low pH buffer and were used in a western immunoblotting assay to identify antigenic targets in a homogenate extract of adult worms (SmWH). Four proteins reacting with the antibodies in immunoblots were purified and proteomic analysis (MS/MS) combined with specific immunoblotting indicated they were the S. mansoni proteins: fructose-1,6 bisphosphate aldolase (SmFBPA), Sm22.6, alkaline phosphatase and malate dehydrogenase. These antibodies were also found to bind to the surface of 3-hour schistosomula and induce immune agglutination of the parasites, suggesting they may have a role in immune protection. CONCLUSION/SIGNIFICANCE: This study reveals a novel mode of action of miltefosine as an anti-schistosome agent. The immune-dependent hypothesis we investigated has previously been lent credence with praziquantel (PZQ), whereby treatment unmasks parasite surface antigens not normally exposed to the host during infection. Antigens involved in this molecular mechanism could have potential as intervention targets and antibodies against these antigens may act to increase the drug's anti-parasite efficacy and be involved in the development of resistance to re-infection.

Schistosoma mansoni cercarial elastase (SmCE): differences in immunogenic properties of native and recombinant forms.

The Schistosoma mansoni cercarial elastase (SmCE) has previously been shown to be poorly immunogenic in mice. However, a minority of mice were able to produce antibodies against SmCE after multiple immunizations with crude preparations containing the enzyme. These mice were partially protected against challenge infections of S. mansoni. In the present study, we show that in contrast to the poor immunogenicity of the enzymatically active native form of SmCE derived from a crude preparation (cercarial transformation fluid), immunization of CBA/Ca mice with two enzymatically inactive forms, namely purified native SmCE or a recombinant SmCE fused to recombinant Schistosoma japonicum glutathione S-transferase (rSmCE-SjGST), after adsorption onto aluminum hydroxide adjuvant, induced specific anti-SmCE immunoglobulin G (IgG) in all mice within 2 weeks of the second immunization. The IgG antibody response to rSmCE-SjGST was mainly of the IgG1 subclass. These results suggest that inactive forms of the antigen could be used to obtain the optimum immunogenic effects as a vaccine candidate against schistosomiasis. Mice immunized with the rSmCE-SjGST on alum had smaller mean worm burdens and lower tissue egg counts when compared with adjuvant alone- and recombinant SjGST-injected controls. The native SmCE was antigenically cross-reactive with homologous enzymes of Schistosoma haematobium and Schistosoma margrebowiei.

Antigenic cross-reactivity between Schistosoma mansoni and peanut: a role for cross-reactive carbohydrate determinants (CCDs) and implications for the hygiene hypothesis.

The antigenic reactivity of constituents of Schistosoma mansoni and peanut (Arachis hypogaea) was investigated to determine whether identical antigenic epitopes possessed by both organisms provided a possible explanation for the negative correlation between chronic schistosome infection and atopy to allergens. Aqueous extracts of peanuts were probed in Western immunoblots with rabbit IgG antibodies raised against the egg, cercarial and adult worm stages of S. mansoni. Several molecules in the peanut extract were antigenically reactive with antibodies from the various rabbit anti-schistosome sera. A pair of cross-reactive peanut molecules at ~30 000-33 000 molecular weight was purified and both proteins were identified by mass spectrometric analysis as the peanut allergen Ara h 1. Anti-S. mansoni soluble egg antigen antibodies that were eluted off the peanut molecules reacted with two S. mansoni egg antigens identified by mass spectrometry as IPSE/α-1 and κ-5. Alignments of the amino acid sequences of Ara h 1 and either IPSE/α-1 or κ-5 revealed a low level of peptide sequence identity. Incubation of nitrocellulose paper carrying electrophoresed peanut molecules, six constituents of other allergic plants and S. mansoni egg antigens in a mild solution of sodium metaperiodate before probing with antibodies, inhibited most of the cross-reactivities. The results are consistent with the antigenic cross-reactive epitopes of S. mansoni egg antigens, peanut and other allergic plants being cross-reactive carbohydrate determinants (CCDs). These findings are novel and an explanation based on 'blocking antibodies' could provide an insight for the inverse relationship observed between schistosome infection and allergies.

Cross-Reactivity between Schistosoma mansoni Antigens and the Latex Allergen Hev b 7: Putative Implication of Cross-Reactive Carbohydrate Determinants (CCDs).

IgG antibodies produced by rabbits immunized against S. mansoni antigens cross-reacted with aqueous soluble constituents of a variety of allergens. The antibody cross-reactivity was largely sensitive to degradation by treatment of the target antigens with sodium meta-periodate, suggesting the cross-reactivity was due to carbohydrate determinants that were common to both the schistosome and the allergens (CCDs). The reaction between the rabbit antibodies and a 43 kDa molecule in a rubber latex extract was analysed further: tandem mass spectrometry identified the latex molecule as allergen Hev b 7. Rabbit anti-schistosome IgG antibodies purified by acid-elution from solid-phase latex Hev b 7 reacted with the S. mansoni egg antigens IPSE/alpha-1 and kappa-5 and cercarial antigens SPO-1 and a fatty acid-binding protein. Moreover, purified anti-S. mansoni egg, latex cross-reactive antibodies reacted with antigenic constituents of some fruits, a result of potential relevance to the latex-fruit syndrome of allergic reactions. We propose that IgG anti-schistosome antibodies that cross-react with allergens may be able to block IgE-induced allergic reactions and thus provide a possible explanation for the hygiene hypothesis.

Purification of a chymotrypsin-like enzyme present on adult Schistosoma mansoni worms from infected mice and its characterization as a host carboxylesterase.

A serine protease-like enzyme found in detergent extracts of Schistosoma mansoni adult worms perfused from infected mice has been purified from mouse blood and further characterized. The enzyme is approximately 85 kDa and hydrolyses N-acetyl-DL-phenylalanine ß-naphthyl-ester, a chromogenic substrate for chymotrypsin-like enzymes. The enzyme from S. mansoni worms appears to be antigenically and enzymatically similar to a molecule that is present in normal mouse blood and so is seemingly host-derived. The enzyme was partially purified by depleting normal mouse serum of albumin using sodium chloride and cold ethanol, followed by repeated rounds of purification by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified material was subjected to tandem mass spectrometry and its derived peptides found to belong to mouse carboxylesterase 1C. Its ability to hydrolyse α- or ß-naphthyl acetates, which are general esterase substrates, has been confirmed. A similar carboxylesterase was purified and characterized from rat blood. Additional evidence to support identification of the enzyme as a carboxylesterase has been provided. Possible roles of the enzyme in the mouse host-parasite relationship could be to ease the passage of worms through the host's blood vessels and/or in immune evasion.

Efficacy of Synriam™, a new antimalarial combination of OZ277 and piperaquine, against different developmental stages of Schistosoma mansoni.

Control of schistosomiasis relies on a single drug, praziquantel (PZQ). Given the rising concerns about the potential emergence of PZQ-resistant strains, it has now become necessary to search for novel therapeutics. However, the current pace for anti-schistosomal drug discovery is slow; hence, repositioning of existing approved drugs can offer a safe, rapid and cost-effective solution. The anti-malarial synthetic artemisinin-derivatives trioxolanes demonstrated anti-schistosomal efficacies against the three major species infecting humans and, unlike PZQ, showed activities against both juvenile and adult worm stages. The 1,2,4-trioxolane/OZ277 (arterolane maleate) in combination with a partner drug: piperaquine phosphate was recently developed as an anti-malarial drug and manufactured by Ranbaxy (India) as Synriam™ (SYN). Herein, the in vivo activities of SYN were investigated in a mouse model of Schistosoma mansoni (S. mansoni), compared to PZQ. We show that a single fixed dose of 240mg/kg SYN (40mg/kg arterolane and 200mg/kg piperaqine) induced significant protective effects in mice, in terms of reduction in worm and tissue egg burdens, which were evident against all schistosome developmental stages. Extensive alterations in the tegument and subtegumental tissues of SYN-exposed worms were revealed by both scanning and transmission electron microscopes. Progressive decrease in worm activity and occurrence of death were noticed in vitro upon exposure to the drug - more pronounced in the presence of haemin. This report provides the first evidence of the efficacy of a combination of 1,2,4-trioxolane and piperaquine against S. mansoni in mice. Being effective against young stages, SYN could be used to prevent early Schistosoma infection.

Polymeric human Fc-fusion proteins with modified effector functions.

The success of Fc-fusion bio-therapeutics has spurred the development of other Fc-fusion products for treating and/or vaccinating against a range of diseases. We describe a method to modulate their function by converting them into well-defined stable polymers. This strategy resulted in cylindrical hexameric structures revealed by tapping mode atomic force microscopy (AFM). Polymeric Fc-fusions were significantly less immunogenic than their dimeric or monomeric counterparts, a result partly owing to their reduced ability to interact with critical Fc-receptors. However, in the absence of the fusion partner, polymeric IgG1-Fc molecules were capable of binding selectively to FcγRs, with significantly increased affinity owing to their increased valency, suggesting that these reagents may prove of immediate utility in the development of well-defined replacements for intravenous immunoglobulin (IVIG) therapy. Overall, these findings establish an effective IgG Fc-fusion based polymeric platform with which the therapeutic and vaccination applications of Fc-fusion immune-complexes can now be explored.