Schistosome membrane proteins as vaccines

International Journal for Parasitology 37 (2007) 257–263 www.elsevier.com/locate/ijpara Invited review Schistosome membrane proteins as vaccines Alex Loukas *, Mai Tran, Mark S. Pearson Helminth Biology Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research Brisbane, Qld 4006, Australia1 Received 19 October 2006; received in revised form 28 November 2006; accepted 3 December 2006 Abstract Schistosomes are parasitic blood flukes that infect approximately 200 million people and are arguably the most important human helminth in terms of mortality. The outermost surface of intra-mammalian stages of the parasite, the tegument, is the key to the parasite’s success, but it is also generally viewed as the most susceptible target for vaccines and drugs. Over the past 2 years the proteome of the Schistosoma mansoni tegument has been investigated and these studies revealed surprisingly few proteins that are predicted to be accessible to the host immune response, namely proteins with at least one membrane-spanning domain. However, of this handful of proteins, some are showing great promise as recombinant vaccines against schistosomiasis at a pre-clinical level. In particular, the tetraspanin family of integral membrane proteins appears to be abundantly represented in the tegument, and convergent data using the mouse vaccine model and correlates of protective immunity in naturally exposed people suggests that this family of membrane proteins offer great promise for schistosomiasis vaccines. With the recent advances in schistosome genomics and proteomics, a new suite of potential vaccine antigens are presented and these warrant detailed investigation and appropriate funding over the next few years. Ó 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Schistosoma; Vaccine; Praziquantel; Tetraspanin; Transmembrane; Tegument; Proteome 1. Introduction Schistosomes are parasitic blood flukes that infect approximately 200 million people (World health Organization, 2002). Female worms deposit eggs in the blood vessels surrounding the gut and liver (Schistosoma mansoni and Schistosoma japonicum) or bladder (Schistosoma haematobium), and the ensuing granulomatous response to them causes the symptoms associated with schistosomiasis. Schistosomes are the most important human helminth in terms of morbidity and mortality, and a recent meta-analysis by King assigned 2–15% disability weight to this pandemic (King et al., 2005). The Bill and Melinda Gates Foundation fund the Schistosomiasis Control Initiative (Garba et al., 2006; Kabatereine et al., 2006), an organisation which advocates mass chemotherapy * Corresponding author. Tel.: +61 7 3845 3702; fax: +61 7 3845 3507. E-mail address: [email protected] (A. Loukas). 1 Australian Society for Parasitology 2006 Bancroft-Mackerras Medal Oration. throughout Africa to control schistosomiasis. Although there is evidence that wide-spread chemotherapy with praziquantel (PZQ) is effective in reducing infection intensity and prevalence (Kabatereine et al., in press), to sustain their effects, these drugs must be applied periodically and for an indefinite period of time. Moreover, high rates of re-infection after mass treatment limit strategies based on chemotherapy alone. As such, a prophylactic vaccine is the ideal method for sustainable control of schistosomiasis, alone or in combination with anthelmintic drugs (Bergquist et al., 2005). Given the enormous burden of disease related to this organism, relying solely on existing disease control methods – for schistosomiasis, this refers to mass and repeated treatment of exposed populations with the anthelmintic PZQ – may not be feasible. For example, Bergquist and colleagues recently stated that ‘‘the simplistic approach of exclusive drug treatment might not be sufficient and, in the worst-case scenario, might even exacerbate pathology. To meet this challenge, the repositioning of vaccines within the totality of disease control through 0020-7519/$30.00 Ó 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2006.12.001 258 A. Loukas et al. / International Journal for Parasitology 37 (2007) 257–263 the combined use of chemotherapy and vaccination is recommended as the basis for a novel, more-versatile approach to control’’ (Bergquist et al., 2005). 2. Schistosome vaccines, past Assuming that a schistosomiasis vaccine is necessary to effectively combat the disease, can we look to the past for lessons on what to do, and equally importantly, what not to do? Vaccination with infective larvae (cercariae) of S. mansoni that have been attenuated with ionizing radiation induces high levels of protection in animals (Minard et al., 1978; Stek et al., 1981). While this model raised hopes for the development of molecular vaccines, no single antigen has consistently induced these same levels of protection, particularly in recombinant form (reviewed in Bergquist and Colley, 1998). While widespread human use of an attenuated Plasmodium vaccine is being considered for malaria (Luke and Hoffman, 2003), such an option is unrealistic for a schistosomiasis vaccine. The logistics and cost of producing and distributing a live parasite vaccine exclusively for developing countries is prohibitive. Nonetheless, attenuated cercariae are protective in a range of animal models, including non-human primates (Eberl et al., 2001; Kariuki et al., 2004). Unfortunately, the progress made with attenuated larval vaccines did not equate to advances in the development of recombinant vaccines. An independent test of six candidate antigens funded by the World Health Organization (WHO) in the mid-1990s showed that the levels of protection obtained with these antigens never exceeded the 40% benchmark set by the WHO for progression of an antigen into clinical trials (Bergquist and Colley, 1998). Furthermore, of these six antigens, only one (Sm23) has since been shown to be truly exposed on the surface of labelled worms, using proteomic techniques (Braschi and Wilson, 2006). The past few years have witnessed major advances in schistosome molecular biology – the transcriptome (Verjovski-Almeida et al., 2003), genome (El-Sayed et al., 2004) and much of the tegument proteome of S. mansoni (Knudsen et al., 2005; van Balkom et al., 2005; Braschi et al., 2006; Braschi and Wilson, 2006) have now been mostly characterized. This upsurge in molecular information, in particular the marriage of nucleotide and protein sequence data to rapidly link proteins to mRNAs, is now bearing fruit in terms of a whole new suite of promising vaccine antigens. These proteomic and transcriptomic analyses have also reminded us that the most important target of the schistosome is the tegument. Moreover, this multi-nucleated syncitium contains proteins that are: (i) intracellular, and (ii) membrane bound. It is this latter group, the transmembrane proteins expressed on the tegument surface, and their known/potential efficacy as vaccines, that will be the focus of the remainder of this review. 3. The schistosome tegument – the key to its success but also its achilles heel The adult schistosome body wall differentiates de novo from cells within the parenchyma to replace the embryonic body wall. This mature structure, called the tegument, is a single syncytium that covers the entire body and may be in cytoplasmic continuity with other syncytial linings (Jones, 1998). The tegument is a dynamic host-interactive layer involved in nutrition, immune evasion and modulation, excretion, osmoregulation, sensory reception and signal transduction, and importantly from a vaccine perspective, it constitutes the host–parasite interface (Jones et al., 2004; Van Hellemond et al., 2006). Pioneering studies shed light on the unusual architecture of the tegument (McLaren and Hockley, 1977) and the presence and trafficking of some of the proteins through this unique syncytium (Skelly and Shoemaker, 1996, 2001). More recently, parasitologists (like most other biologists) have embraced recent advances in proteomics, and this has resulted in a comprehensive catalogue of proteins expressed in the tegument. It has also reminded us that ‘‘accessibility’’ of a protein in the tegument is of paramount importance when selecting target molecules for vaccines and drugs. It is critical to bear in mind that not all proteins inside the tegument are exposed to host tissues. Indeed, one might predict that only a few of the tegument proteins are truly ‘‘exposed’’, and these are likely to be the membrane proteins which contain motifs (signal peptides and/or transmembrane domains) that direct their trafficking through the secretory pathway to be lodged in the plasma membrane of the tegument. Early gene-based approaches to vaccine antigen discovery involved immunoscreening of cDNA expression libraries with antisera from humans and animals. More recently, reporter-based techniques to selectively clone schistosome cDNAs encoding secreted and membrane proteins were used (Smyth et al., 2003; Pearson et al., 2005) and these studies identified cDNAs that are now known to encode excellent vaccine candidates (Tran et al., 2006). Given the advanced state of the S. mansoni transcriptome and genome sequencing efforts, it is reasonable to assume that we are now armed with the genetic information to design an effective recombinant vaccine against schistosomiasis. The major difficulties facing us now are how to meaningfully mine this data for suitable vaccine antigens. Taking a protein-first approach, three seminal articles were recently published which identified the major proteins in the tegument of S. mansoni and matched most of these proteins to expressed sequence tags (ESTs). First, tegument-specific proteins were identified by removing the tegument from adult S. mansoni and comparing the protein profiles of tegument with underlying sub-tegumental tissue (van Balkom et al., 2005). A set of 43 proteins was identified which was expressed exclusively in the tegument and not in underlying tissues (van Balkom et al., 2005). Many well studied proteins, some of which had been assessed as vaccines, were shown to be in the tegument, as well as a A. Loukas et al. / International Journal for Parasitology 37 (2007) 257–263 diverse range of intracellular and membrane-bound/associated proteins that had not been previously reported. This study, however, did not address the sub-cellular locations of each protein within the tegument – i.e., which ones associate with the membranes and which are cytoplasmic. In a second pivotal study, tegument proteins were separated by their solubility into cytosolic, cytoskeletal and membraneassociated proteins (Braschi et al., 2006). In a complementary approach, the same researchers labelled the surface of live adult S. mansoni with both long-form and short-form biotin (differing in the depth through which they penetrated the tegument) (Braschi and Wilson, 2006) then purified the labelled proteins by streptavidin-affinity chromatography. Surprisingly, only nine proteins of parasite origin were labelled with long-form biotin, reflecting the relative paucity of proteins in the outer tegument. Not surprisingly, these nine proteins detected on the tegument surface all contained at least one transmembrane domain (Braschi and Wilson, 2006) (Table 1). There is currently no method to reliably separate the tegument plasma membrane from the overlying trilaminate membranocalyx (Hockley and McLaren, 1973; Wilson and Barnes, 1974), so proteins labelled with long-form biotin could be located in either the plasma membrane of the tegument or in the membranocalyx (Braschi and Wilson, 2006), although the former is likely to be the location for most of these proteins given their multiple membrane-spanning domains (Table 1). This has implications for recognition of membrane proteins by antibodies which are thought to be too large to enter through ‘‘pores’’ in the membranocalyx (Braschi and Wilson, 2006), and researchers are still uncertain about the true ‘‘accessibility’’ of protein epitopes to antibodies on the surface of an intra-vascular schistosome with an intact membranocalyx. Using a high throughput proteomics approach, more than 3000 proteins were identified from the Asian schistosome, S. japonicum (Liu et al., 2006), of which 156 were found in the tegument of mixed sex adult worms and 159 from the tegument of hepatic schistosomula. However, the sub-cellular location of these S. japonicum proteins was not determined and it is likely that many of these are intracellular proteins. 4. Tetraspanins – abundant in the tegument and promising vaccine antigens One family of proteins that are prominent in the outer tegument of S. mansoni are the tetraspanins, four transmembrane domain proteins found on the surface of eukaryotic cells including B and T cells. They have two extracellular loops – a short loop 1 of 17–22 residues (EC-1) with little tertiary structure, and a larger 70–90 residue loop 2 (EC-2) which has four or six cysteines that form two or three disulfide bonds (Fig. 1). In general, the extracellular loops mediate specific protein–protein interactions with laterally associated proteins, or in some cases, known ligands (reviewed in Levy and Shoham, 2005). The four 259 transmembrane domains provide stability during biosynthesis and are crucial for assembly and maintenance of the tetraspanin web, a scaffold by which many membrane proteins are laterally organised (Andre et al., 2006). Although their functions are unknown, it is now apparent from proteomic studies that a family of tetraspanins is expressed in the schistosome tegument (van Balkom et al., 2005; Braschi et al., 2006; Braschi and Wilson, 2006), and at least three of these show promise as vaccines (Table 1). Sm23 is a tetraspanin that is expressed in the tegument of S. mansoni (Harn et al., 1985) and one of the independently tested WHO vaccine candidates (Bergquist and Colley, 1998). Sm23 is most efficacious when delivered as a DNA vaccine (Da’Dara et al., 2003) and does not confer protection as a recombinant protein when formulated with alum. More recently, signal sequence trapping in mammalian cells was used to capture two new S. mansoni tetraspanins (Sm-TSP-1 and TSP-2) (Smyth et al., 2003) which were then shown to be expressed in the tegument of S. mansoni (Tran et al., 2006), and immunolocalization of TSP-2 is shown in Fig. 2. TSP-2 in particular provided high levels of protection as a recombinant vaccine in the mouse model of schistosomiasis (Tran et al., 2006). Because TSP-1 and TSP-2 were expressed in the tegument and showed efficacy in a murine vaccine model, they were used to screen for specific antibodies in the sera of individuals who were exposed to S. mansoni and who were either: (i) putatively resistant (PR) (Correa-Oliveira et al., 1989; Correa-Oliveira et al., 2000) or (ii) chronically infected (CI) with the parasite. PR individuals are resistant to infection despite years of exposure to S. mansoni (Correa-Oliveira et al., 1989, 2000; Viana et al., 1994). Levels of IgG1 and IgG3 against TSP-2 were significantly higher in PR sera than in sera from CI individuals (Tran et al., 2006). In fact, CI individuals and unexposed blood donors failed to mount any detectable antibody isotype response to TSP-2. Interestingly, the antibody response mounted by the PR individuals against TSP-2 consisted exclusively of the cytophilic antibodies IgG1 and IgG3 (but not other IgG subclasses nor IgE) – IgG1 and IgG3 are not the antibody isotypes commonly associated with chronic helminth infections (IgG4 and IgE) (Hoffmann et al., 2002). Studies in Brazil (Ribeiro de Jesus et al., 2000) and Egypt (Al-Sherbiny et al., 2003) assessed the immune responses of resistant and susceptible individuals to a panel of S. mansoni vaccine antigens (mostly those tested by the WHO (Bergquist and Colley, 1998)), and no single antigen was uniquely recognized by antibodies from resistant but not chronically infected individuals, further supporting the development of TSP-2 as a vaccine for S. mansoni. In addition to TSP-1, TSP-2 and Sm23, two additional tetraspanins were identified from the outer tegument of S. mansoni adults (Braschi and Wilson, 2006) (Table 1). Sm04463 (homologous to TE736 from S. japonicum (Fan and Brindley, 1998)) and Sm07392 both encoded tetraspanin open reading frames, although Sm07392 was incorrectly defined as having no known homologues (Braschi and 260 A. Loukas et al. / International Journal for Parasitology 37 (2007) 257–263 Table 1 Selected proteins that associate with, or are inserted into, the plasma membrane of the tegument of adult Schistosoma mansoni and their potential as recombinant vaccines Protein or cDNA Location in tegumenta Identity/sequence features Protective vaccine in miceb Protective role in humans Representative publication Sm-TSP-2 (tetraspanin D) Sm-TSP-1 Outer membrane Outer membrane Outer membrane Intracellular but associates with inner membrane Tetraspanin integral membrane protein Tetraspanin integral membrane protein Unknown but has C-terminal transmembrane domain Neutral cysteine protease Yes Tran et al. (2006) No Tran et al. (2006) Yes Cardoso et al. (2006) ND Siddiqui et al. (2003) Outer membrane Outer membrane Unknown but has transmembrane domain and Ig-like receptor domain Tetraspanin integral membrane protein similar to S. japonicum TE736 ++ (recombinant protein) ++ (recombinant protein) ++ (recombinant protein)c + (plasmid DNA)++ (plasmid DNA including cytokines) ND ND Racoosin et al. (1999) ND ND Sm23 Outer membrane Tetraspanin integral membrane protein + (plasmid DNA) annexin Outer tegument ND Sm11921 Outer tegument Potential molecular glue that binds membranocalyx to cell membrane; contains one potential transmembrane domaind Contains integrin-like domain and at least one transmembrane domain No, but is recognized by infected subjects ND Fan and Brindley (1998); Verjovski-Almeida et al. (2003); Braschi and Wilson (2006) Da’dara et al. (2001); Ribeiro de Jesus et al. (2000) ND ND Sm07392 Outer tegument Tetraspanin integral membrane proteine ND ND Sm29 calpain Sm200 Tetraspanin B (Sm04463) Verjovski-Almeida et al. (2003); Braschi and Wilson (2006) Verjovski-Almeida et al. (2003); Braschi and Wilson (2006) Verjovski-Almeida et al. (2003); Braschi and Wilson (2006) a Location data taken mostly from references (Braschi et al., 2006; Braschi and Wilson, 2006). +, 30–50% reduction in worm burdens; ++,>50% reduction in worm burdens; ND, not determined. c Sergio Costa Oliveira, personal communication. d Contains one predicted transmembrane domain (A. Loukas, unpublished data – determined using http://www.ch.embnet.org/software/ TMPRED_form.html) despite publications stating that there are no transmembrane domains Braschi and Wilson (2006). e Sm07392 is listed by Braschi and Wilson (2006) as having no known homologues, however the cDNA entry for this clone in schistoDB (http:// dev.genedb.org/genedb/smansoni/) encodes an open reading frame with identity to tetraspanins (A. Loukas, unpublished data). b Wilson, 2006). Despite their great promise as recombinant vaccines for schistosomiasis, we still do not know the functions of tetraspanins in the parasite. From the literature on mammalian tetraspansins, one might predict that the schistosome proteins might be pivotal in formation of a structural web for maintaining the integrity of the tegument membrane (Levy and Shoham, 2005). Schistosomes coat themselves in host proteins, so alternatively, tetraspanins might function as receptors for host molecules (Tran et al., 2006). The recent successful application of RNA interference to schistosomes and observation of a subsequent phenotype (Correnti et al., 2005) bodes well for the elucidation of the functions of tetraspanins in the schistosome tegument. 5. Other membrane proteins with potential as vaccines The next few years will hopefully see the assessment of some of these new membrane proteins identified in the teg- ument. Table 1 presents a select list of proteins associated with, or anchored in, the outer membrane of the tegument of S. mansoni (Braschi and Wilson, 2006). Only a few of these proteins have been tested as vaccines in the mouse model and fewer again have been assessed for their recognition by naturally exposed people (CI and PR cohorts) in endemic areas. In our opinion, the most obvious vaccine candidates from this list are those which are anchored to the outer membrane but present large extracellular regions, whether they be loops (as in the tetraspanins) or the entire protein other than a terminal transmembrane domain (such as Sm29). Sm29 is of particular interest as a potential vaccine. It has been reported as ‘‘secreted’’ rather than membranebound (Braschi and Wilson, 2006), but transmembrane prediction programs indicate that Sm29 has a C-terminal membrane-spanning domain (Cardoso et al., 2006). Like TSP-2 (Tran et al., 2006), Sm29 is preferentially recogni- A. Loukas et al. / International Journal for Parasitology 37 (2007) 257–263 P C C EC-2 W/F/Y C G C C G C EC-1 E/Q extracellular G/A G/A E/Q 2 1 G/A membrane G/A 3 F/Y 4 E/Q K intracellular Fig. 1. Schematic depiction of the architecture of a tetraspanin in the outer tegument membrane of Schistosoma mansoni. Transmembrane domains are numbered in white circles, conserved residues are in green circles, conserved Cys residues involved in disulfide bond formation are in cyan circles and the two extra Cys residues that form an optional third disulfide bond are in blue circles. EC,extracellular loop. 261 6. Schistosoma mansoni membrane protein vaccines – mechanisms of protection? We are still in the dark when it comes to the desired protective mechanisms required to engineer an efficacious recombinant vaccine for human schistosomiasis. Contrasting and conflicting data is presented from the mouse model and from human field studies. For example, activation of predominantly T helper type 1 (Th1) cells to schistosomula antigens correlates with naturally acquired protection of individuals who are exposed to the parasite but are not infected and have never been treated with PZQ (CorreaOliveira et al., 2000). On the other hand, partial resistance can be induced in some adult individuals with repeated PZQ treatment and this correlates with a predominantly Th2 response (Walter et al., 2006). In mice, recombinant vaccines conferring various levels of protection claim to induce different immune response phenotypes. This is influenced at least in part by the properties of the adjuvants used or the intrinsic immunogenicity of the respective proteins, however a general consensus is lacking. Studies using the irradiated cercariae model in mice suggest that protection can be induced with either a mixed Th1/Th2 response, a polarised Th1 response or even a polarised Th2 response (reviewed in Hewitson et al., 2005). Given that antibodies alone can confer protection in this model (Jankovic et al., 1999), perhaps the phenotype of the response, and even the isotype/subclass of antibody produced, is not the key issue here. Most commercially available vaccines rely specifically on the induction of neutralizing antibodies that block the function of their target protein/s. This also appears to be the case for other helminth vaccines that are showing promise in pre-clinical studies, where neutralising antibodies block proteins that are essential in tissue migration or digestion of the blood meal (Loukas et al., 2006). 7. More money for NTDs but not yet for schistosomiasis vaccines Fig. 2. Detection of Sm-TSP-2 tetraspanin (red fluorescence) in the outer tegument of Schistosoma mansoni adult worms by immunofluorescence microscopy using a mouse antibody raised to recombinant TSP-2. The slide was also stained with 4 0 ,6-diamidino-2-phenylindole to highlight the nuclei (blue). teg, tegument; tub, tubercles of the tegument. sed by antibodies from PR compared with CI individuals (Cardoso et al., 2006), although the extent of selectivity is not as great as that reported for TSP-2. Moreover, preliminary trials in mice suggest that this protein is an efficacious recombinant vaccine (S. Costa Oliveira, personal communication), lending further support to its development as a recombinant vaccine for S. mansoni infection. There is now renewed interest, even referred to as a ‘‘renaissance’’ (Rabinovich, 2006), in the Neglected Tropical Diseases, or NTDs (Hotez et al., 2006a,c). Increased funding is being provided by philanthropic organisations such as the Bill and Melinda Gates Foundation to develop cures for the NTDs and schistosomiasis was not altogether ignored. However, it is noteworthy that the large philanthropies have not yet (recently) invested in the development of a schistosomiasis vaccine, despite investing more than $US30 million into mass PZQ treatment throughout Africa (Garba et al., 2006). Much has changed since the last WHO assessment of vaccines was conducted, not the least of which is the discovery of a new panel of antigens whose subcellular locations in the tegument are known (Table 1), an almost complete genome and proteome, new vaccine delivery technologies, and a better understanding of human and animal immune responses. Philanthropic organisations 262 A. Loukas et al. / International Journal for Parasitology 37 (2007) 257–263 should therefore consider funding a new independent assessment of recombinant vaccines for schistosomiasis. Moreover, there are now numerous public-private partnerships focusing on accelerating the transition of cures for NTDs from bench to bedside (Hotez and Ferris, 2006). A vaccine is essential for schistosomiasis, and progress in vaccine development for related blood-feeding helminths such as hookworm is being facilitated by significant philanthropic investments (Hotez et al., 2006b; Loukas et al., 2006). It is imprudent to think that chemotherapy alone will suffice in the long-term global control of schistosomiasis, and vaccine discovery and development efforts should be encouraged and appropriately funded. Acknowledgements We are grateful to our colleagues, Drs Jeffrey Bethony, Malcolm Jones, Danielle Smyth and Sergio Costa Oliveira for helpful discussions and providing us with unpublished data. Our schistosomiasis research is funded by the National Health and Medical Research Council of Australia (NHMRC). AL is supported by an R.D. Wright fellowship from the NHMRC. References Al-Sherbiny, M., Osman, A., Barakat, R., El Morshedy, H., Bergquist, R., Olds, R., 2003. 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