Research Article: Human IgG1 Responses to Surface Localised Schistosoma mansoni Ly6 Family Members Drop following Praziquantel Treatment

Date Published: July 6, 2015

Publisher: Public Library of Science

Author(s): Iain W. Chalmers, Colin M. Fitzsimmons, Martha Brown, Christine Pierrot, Frances M. Jones, Jakub M. Wawrzyniak, Narcis Fernandez-Fuentes, Edridah M. Tukahebwa, David W. Dunne, Jamal Khalife, Karl F. Hoffmann, Jeffrey Michael Bethony.

Abstract: BackgroundThe heptalaminate-covered, syncytial tegument is an important anatomical adaptation that enables schistosome parasites to maintain long-term, intravascular residence in definitive hosts. Investigation of the proteins present in this surface layer and the immune responses elicited by them during infection is crucial to our understanding of host/parasite interactions. Recent studies have revealed a number of novel tegumental surface proteins including three (SmCD59a, SmCD59b and Sm29) containing uPAR/Ly6 domains (renamed SmLy6A SmLy6B and SmLy6D in this study). While vaccination with SmLy6A (SmCD59a) and SmLy6D (Sm29) induces protective immunity in experimental models, human immunoglobulin responses to representative SmLy6 family members have yet to be thoroughly explored.Methodology/Principal FindingsUsing a PSI-BLAST-based search, we present a comprehensive reanalysis of the Schistosoma mansoni Ly6 family (SmLy6A-K). Our examination extends the number of members to eleven (including three novel proteins) and provides strong evidence that the previously identified vaccine candidate Sm29 (renamed SmLy6D) is a unique double uPAR/Ly6 domain-containing representative. Presence of canonical cysteine residues, signal peptides and GPI-anchor sites strongly suggest that all SmLy6 proteins are cell surface-bound. To provide evidence that SmLy6 members are immunogenic in human populations, we report IgG1 (as well as IgG4 and IgE) responses against two surface-bound representatives (SmLy6A and SmLy6B) within a cohort of S. mansoni-infected Ugandan males before and after praziquantel treatment. While pre-treatment IgG1 prevalence for SmLy6A and SmLy6B differs amongst the studied population (7.4% and 25.3% of the cohort, respectively), these values are both higher than IgG1 prevalence (2.7%) for a sub-surface tegumental antigen, SmTAL1. Further, post-treatment IgG1 levels against surface-associated SmLy6A and SmLy6B significantly drop (p = 0.020 and p < 0.001, respectively) when compared to rising IgG1 levels against sub-surface SmTAL1.Conclusions/SignificanceCollectively, these results expand the number of SmLy6 proteins found within S. mansoni and specifically demonstrate that surface-associated SmLy6A and SmLy6B elicit immunological responses during infection in endemic communities.

Partial Text: Human schistosomiasis is estimated to affect more than 200 million people living in developing countries, with 120 million people symptomatic and 20 million suffering severe illness [1]. With a further 600 million individuals at risk of infection from Schistosoma mansoni, Schistosoma haematobium and Schistosoma japonicum (the three main human-infective species) and up to 70 million disability-adjusted life years (DALYs) lost annually, this neglected tropical disease (NTD) is one of the most important on the planet [2]. Schistosomiasis control is predominantly facilitated by mass drug administration (MDA) of praziquantel, a safe and potent chemotherapy developed in the late 1960’s [3]. However, mono-chemotherapy control of schistosomiasis raises the spectre of drug resistance [4] and highlights the need for developing an immunoprophylactic anti-schistosomal vaccine. Throughout the past several decades, progress in schistosome vaccine development has proved disappointing, with six promising candidates failing to induce sufficient protection in independent experimental models [5]. However, with the increased use of genomic, transcriptomic and proteomic methodologies to characterize schistosome biology, the number of novel and, perhaps more potent, vaccine candidates has vastly expanded [6–12].

Unless otherwise stated, all chemicals and reagents were purchased from Sigma-Aldrich, United Kingdom.

As Ly6 proteins share little sequence identity [16,18], we performed a systematic search of the S. mansoni genome using position-specific iterated BLAST search (PSI-BLAST; [30]). First iteration searches were performed using five representative Ly6 proteins (HsCD59, HsuPAR, DmBoudin, DmRtv and CeODR-2) as query sequences. Sequences for use in subsequent iterations were selected by identifying regions between 55–115aa in length containing 10 cysteines, where Cys1 and Cys2 were separated by any two residues (C1-X-X-C2) and Cys10 was followed by an Asn residue (C10N) (see Materials and Methods for detailed description of PSI-BLAST parameters). This comprehensive search of the S. mansoni genome, using canonical characteristics of the Ly6 family, resulted in the identification of eleven S. mansoni Ly6 members named SmLy6A-K (see Table 1). All eleven SmLy6 transcript sequences were verified by PCR (PCR primers listed in S1 Table) from parasite cDNA, with complete open reading frames (ORF) verified for nine transcripts (SmLy6A-H and J) and partial ORFs confirmed for two (SmLy6I, K). Of these eleven Ly6 members, eight have been previously reported—SmLy6A and SmLy6B in a tegumental proteomic study ([11]; SmCD59a and b respectively), SmLy6D, which is the tegumental vaccine candidate Sm29 [44,45] and seven SmLy6 members (SmLy6A-F, I and K) in a recent publication also characterizing this family [27]. SmLy6G, Ly6H and Ly6J represent novel S. mansoni family members.

Investigating the human immune responses directed against schistosome surface proteins is a logical step in the progression of next-generation vaccine candidates to combat schistosomiasis [49]. Here we have focused our investigation on the Ly6 protein family as previously studies have indicated that three members are found on the schistosome surface tethered by a GPI-anchor (SmLy6A, SmLy6B and SmLy6D; [11]), two are capable of inducing protective immunity in the mouse model (SmLy6B [50] and SmLy6D [44]) and one is recognised during human schistosomiasis (SmLy6D; [45]).



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