We believe that we report here the first identification of recombinant antibodies that bind to epitopes on the host-exposed surface of living schistosomes. The antibodies, expressed as scFv domains, derive from rats that are in the active process of rejecting a schistosome infection. Rats are semi-permissive hosts for S. mansoni
and reject most of the parasites approximately 4 weeks following a bolus infection (Knopf et al., 1977
; Phillips et al., 1977
; Cioli et al., 1978
). Following this infection, the rats rapidly reject subsequent challenge employing a protective response that has been associated with antibody effectors (Horta and Ramalho-Pinto, 1984
; Mangold and Dean, 1986
; Cetre et al., 1999
; Khalife et al., 2000
). Clearly, antibodies recognizing host-exposed epitopes must be considered as candidates for immune effectors and antigens containing host-exposed epitopes are strong candidates for vaccine targets. This study reports five scFvs that we believe are excellent reagents in the search for promising vaccine targets, for dissecting the role of antibodies in schistosome immunity and for improved understanding of the host/parasite relationship. These five scFvs likely represent only a fraction of the worm surface binding antibodies present following schistosome infection of rats and we thus expect the scFv library to remain a useful resource for further studies of the host-exposed schistosome tegument.
In this study, we employed the Fischer rat model of schistosomiasis which has previously been shown to be semi-permissive to S. mansoni. We reproduced earlier studies demonstrating that Fischer rats reject large infections of S. mansoni shortly after the fourth week of infection. After 4 weeks, the worms that were recovered from the rat by liver perfusion were much smaller and less robust than worms recovered from mice at the same time p.i.. We also found that rats develop a higher titer antibody response against tegumental antigens by 5–6 weeks p.i. than do mice. This more robust surface antibody response may participate in the worm rejection or it may simply be a consequence of increased worm damage and death caused by some other effector mechanism. Following a second infection, the titer of tegumental antibodies was further boosted and this serum was potent in its ability to immunologically stain living lung stage schistosomula and formaldehyde-fixed adults. The spleens from the twice infected rats were thus a good source of B cells for preparing a scFv-display library to select antibodies recognizing worm surface epitopes.
Five different, unique scFvs were selected from the immune rat scFv-display library for their ability to bind to both apical membrane extracts and intact, formaldehyde-fixed adult worms. The antigen recognized by one of the scFvs, Teg1, was unambiguously identified as SmTSP-2. This antigen was previously shown to be localized to the apical t egument by proteomics (van Balkom et al., 2005
; Braschi et al., 2006
) and IF (Tran et al., 2006
), although these studies did not investigate exposure of SmTSP-2 epitopes on living schistosomes. Our studies demonstrate that SmTSP-2 is clearly exposed on living larval and lung-stage schistosomes. Exposure of SmTSP-2 on adult worms was ambiguous as IFA staining with Teg1 was observed only in discrete bright patches that appear to be regions of damaged tegument.
Another selected scFv, Teg4, produced the brightest staining of both larval and adult schistosomes. This scFv recognizes an antigen migrating at approximately 35 kD on Western blots following SDS-PAGE of apical extracts. It is intriguing that two different mAbs were previously identified from S. mansoni
-infected rats that each recognized a schistosome surface antigen estimated at ~38 kD. One of these rat mAbs was found to protect against cercarial challenge and the other had blocking activity (Dissous et al., 1982
; Grzych et al., 1984
). This antigen was never identified and it is quite possibly the same antigen recognized by Teg4 scFv. In our studies, proteomic MS/MS analysis of the excised 35 kD antigen recognized by Teg4 identified Sm29 as the major membrane protein species in the fraction. The identity of Sm29 is consistent with other results indicating this protein to be a schistosome apical membrane antigen (van Balkom et al., 2005
; Braschi et al., 2006
; Cardoso et al., 2008
). Western blots of anti-Sm29 antisera recognize a band having similar mobility as the Teg4 antigen on SDS-PAGE (Cardoso et al., 2008
). The Teg4 scFv, however, did not recognize recombinant Sm29, expressed in E. coli
, on Western blots (not shown). While these results may indicate that Teg4 recognizes a different apical protein than Sm29, it is also possible that Teg4 recognizes a conformational or non-protein epitope on Sm29 that is not reproduced within bacterial expressed recombinant Sm29. Sm29 protein sequence is strongly suggestive of a glycosylphosphatidylinositol (GPI)-anchored protein. When isolated from schistosomes, Sm29 migrates much slower on SDS-PAGE than would be expected based on its protein MW of 29 kD, further suggesting that it becomes post-translationally modified (Cardoso et al., 2008
). Interestingly, we have recently identified several scFvs from the 2× infected rat library that do recognize recombinant Sm29, confirming that Sm29 is a immunogen following schistosome infection in rat, but these scFvs do not appear to recognize host-exposed schistosome epitopes.
Two recent findings are strongly consistent with our finding that SmTSP-2 and Sm29 are present on the exposed surface of schistosomes. Both of these antigens are among a small group of antigens found to be exposed to surface biotinylation on living schistosomes (Braschi and Wilson, 2006
). Secondly, these are the only two antigens found to date that are selectively recognized by sera from humans identified as putatively resistant to schistosomiasis compared with chronically infected (Cardoso et al., 2006
; Tran et al., 2006
) and both produce a strong protective immune response upon immunization with recombinant protein (Tran et al., 2006
; Cardoso et al., 2008
). In unpublished results, we have shown that SmTSP-2 and Sm29 are recognized by antibodies in schistosome-infected rat sera at significantly higher titer than equivalent infected mouse sera. Furthermore, Sm29 is released from living adult worms by treatment with phosphatidylinositol phospholipase C (Dr. Alan R. Wilson, personal communication). Our results add further evidence in support of SmTSP-2 and Sm29 as promising, host-exposed targets of a schistosomiasis vaccine.
Three of the scFvs that we found to recognize exposed epitopes on living schistosomes, Teg5, 20 and 37, clearly recognize apical-enriched antigens by ELISA but did not recognize discreet apical antigen species on Western blots. It may be that the epitopes recognized by these scFvs are derived from antigens present in the tegumental extracts at a level below detection, or are conformationally sensitive epitopes that are denatured by SDS-PAGE. Alternatively, the antigens recognized by these scFvs may be non-proteinaceous in nature and not migrate as a discrete species on these gels. It is clear that adult schistosomes have non-protein components exposed at the host/parasite interface (Skelly and Wilson, 2006
). Cercariae are known to be coated with a glycocalyx largely composed of carbohydrate (Samuelson and Caulfield, 1985
; Caulfield et al., 1987
; Khoo et al., 1995
) and the exposed surface of living adults is also known to contain substantial carbohydrates (Klabunde et al., 2000
). A valuable feature of the schistosome surface binding scFvs is that these binding proteins are reagents that will permit the purification and characterization of these antigens that comprise the critical host/parasite interface on adult schistosomes.
The role of antibodies in protective immunity to schistosomiasis in the rat model has been clearly demonstrated although the mechanism by which effector function is exerted remains uncertain (reviewed by Capron and Capron, 1986
; Khalife et al., 2000
). Recognition of worm surface antigens seems likely to participate in antibody effector action, and evidence has accumulated in support of this view (Dissous et al., 1982
; Barker et al., 1985
). To date, the specific antigen targets of protective antibodies targeting the exposed surface on living worms have not been identified. Recent studies have found a strong correlation of antibody recognition of two schistosome surface antigens, SmTSP-2 and Sm29, with putative resistance in schistosome-infected people in Brazil (Cardoso et al., 2006
; Tran et al., 2006
). It is probably not a coincidence that these are the same two antigens that we identified in this study as the host-exposed targets of antibodies from schistosome-resistant rats. The availability of scFvs recognizing these antigens will permit identification of the exposed epitopes and the design of peptide immunogens.
The staining of mammalian stage schistosomes by immune sera, or by the rat scFvs selected for worm surface binding, has a patched appearance that is often arrayed in a belt-like pattern in the younger worms. The source or cause of the patchy appearance is unknown at present, although it closely resembles the pattern of newly deposited material formed by the eruption of cytons during the initial formation of the tegument during cercarial transformation (Skelly and Shoemaker, 2001
). It seems possible that the patches of staining observed in this study somehow result from the deposition of the outer tegumental membrane that “erupts” from the underlying cytons. It has been postulated that the outer tegument of schistosomes is formed from multilamellar bodies which initially, upon deposition, appear as membrane stacks (Hockley and McLaren, 1973
). Perhaps this recently deposited material is more available to antibody binding than older tegument or, alternatively, the deposited materials may be enriched in antigens recognized by serum antibodies from infected animals.
A provocative result of this study was the observation that the surface of living schistosomula and lung schistosomules stained well with S. mansoni
-infected rat sera and some of the rat scFvs, while the surface of adult worms at 3 weeks or older did not stain with these agents unless the worms were fixed in formaldehyde. We do not know which tegument characteristics change during worm maturation that allow living adult worms to become refractory to antibody staining. The loss of staining is not due to the loss of antigens at the surface because the worms are readily stained by the antibodies following formaldehyde fixation. Possible explanations for the loss of antibody staining include the secretion of proteases, increased turnover of the tegument and some form of antigen masking that is obviated by formalin treatment. Whatever the mechanism, it seems likely that it plays a role in schistosome immune evasion. In this context, it is interesting to note that the point in schistosome maturation at which they become refractory to antibody staining (~7 days) is the same time at which schistosomes become refractory to killing by immune rat serum in passive transfer experiments (Mangold, 1981
). The worm surface binding scFvs identified in this study should be useful reagents to identify and characterize the tegumental properties that develop after the lung stage and that lead to the inability of antibodies to remain associated with the surface of live adults.