Characterization of a novel microfilarial antigen for diagnosis of Wuchereria bancrofti infections.

BACKGROUND: Lymphatic filariasis (LF) is a neglected tropical disease caused by the filarial nematodes Wuchereria bancrofti, Brugia malayi and Brugia timori. The Global Program to Eliminate LF uses mass drug administration (MDA) of anti-filarial drugs that clear microfilariae (Mf) from blood to interrupt transmission by mosquitos. New diagnostic tools are needed to assess the impact of MDA on bancroftian filariasis, because available serologic tests can remain positive after successful treatment. METHODOLOGY/PRINCIPAL FINDINGS: We identified Wb-bhp-1, which encodes a W. bancrofti homologue of BmR1, the B. malayi protein used in the Brugia Rapid antibody test for brugian filariasis. Wb-bhp-1 has a single exon that encodes a 16.3 kD protein (Wb-Bhp-1) with 45% amino acid identity to BmR1. Immunohistology shows that anti-Wb-Bhp-1 antibodies primarily bind to Mf. Plasma from 124 of 224 (55%) microfilaremic individuals had IgG4 antibodies to Wb-Bhp-1 by ELISA. Serologic reactivity to Wb-Bhp-1 varied widely with samples from different regions (sensitivity range 32-92%), with 77% sensitivity for 116 samples collected from microfilaremic individuals outside of sub-Saharan Africa. This variable sensitivity highlights the importance of validating new diagnostic tests for parasitic diseases with samples from different geographical regions. Individuals with higher Mf counts were more likely to have anti-Wb-Bhp-1 antibodies. Cross-reactivity was observed with a minority of plasma samples from people with onchocerciasis (17%) or loiasis (10%). We also identified, cloned and characterized BmR1 homologues from O. volvulus and L. loa that have 41% and 38% identity to BmR1, respectively. However, antibody assays with these antigens were not sensitive for onchocerciasis or loiasis. CONCLUSIONS: Wb-Bhp-1 is a novel antigen that is useful for serologic diagnosis of bancroftian filariasis. Additional studies are needed to assess the value of this antigen for monitoring the success of filariasis elimination programs.

Broad specificity of immune helminth scFv library to identify monoclonal antibodies targeting Strongyloides.

Antibodies have different chemical properties capable of targeting a diverse nature of antigens. Traditionally, immune antibody libraries are perceived to be disease-specific with a skewed repertoire. The complexity during the generation of a combinatorial antibody library allows for a skewed but diverse repertoire to be generated. Strongyloides stercoralis is a parasite that causes strongyloidiasis, a potentially life-threatening disease with a complex diagnosis that impedes effective control and treatment of the disease. This study describes the isolation of monoclonal antibodies against S. stercoralis NIE recombinant protein using an immune antibody phage display library derived from lymphatic filaria-infected individuals. The isolated antibody clones showed both lambda and kappa light chains gene usage, with diverse amino acid distributions. Structural analysis showed that electropositivity and the interface area could determine the binding affinity of the clones with NIE. The successful identification of S. stercoralis antibodies from the filarial immune library highlights the breadth of antibody gene diversification in an immune antibody library that can be applied for closely related infections.

Self-reactive IgG4 antibodies are associated with blocking of pathology in human lymphatic filariasis.

Lymphatic filariasis, a chronic disfiguring disease exhibits complex pathology. Based on different clinical manifestations, infected individuals are categorized into asymptomatic-carriers and chronic-patients. The mechanism behind differential clinical outcomes remains unclear. Roles of filaria-specific B cell responses in filariasis have been documented, whereas the contribution of B1 cell response and poly-specific IgG and IgA in the context of clinical filariasis is not deciphered. In this study, we measured the poly-specific IgG and IgA levels in different clinical categories of filariasis. Asymptomatic-carriers exhibited increased IgG4 antibodies against both filarial-antigens as well as auto-antigens compared to other clinical categories, although IgG against these auto-antigens remained lower. IgA levels against both filarial and auto-antigens were decreased in asymptomatic-carriers. A positive correlation between anti-filarial IgG4 and IgG4 against auto-antigens were observed, suggesting the synergistic role of poly-specific natural IgG4 with anti-filarial IgG4 in blocking the pathogenesis in asymptomatic microfilarial cases.

Natural Selection on Antihelminth Antibodies in a Wild Mammal Population.

An effective immune response is expected to confer fitness benefits through improved resistance to parasites but also incur energetic costs that negatively impact fitness-related traits, such as reproduction. The fitness costs and benefits of an immune response are likely to depend on host age, sex, and levels of parasite exposure. Few studies have examined the full extent to which patterns of natural selection on immune phenotypes vary across demographic groups and environments in the wild. Here, we assessed natural selection on plasma levels of three functionally distinct isotypes (IgA, IgE, and IgG) of antibodies against a prevalent nematode parasite measured in a wild Soay sheep population over 26 years. We found little support for environment-dependent selection or reproductive costs. However, antibody levels were negatively associated with parasite egg counts and positively associated with subsequent survival, albeit in a highly age- and isotype-dependent manner. Raised levels of antiparasite IgA best predicted reduced egg counts, but this did not predict survival in lambs. In adults increased antiparasite IgG predicted reduced egg counts, and in adult females IgG levels also positively predicted overwinter survival. Our results highlight the potential importance of age- and sex-dependent selection on immune phenotypes in nature and show that patterns of selection can vary even among functionally related immune markers.

Vaccination with DNA encoding ES 43-kDa /45-kDa antigens significantly reduces Trichinella spiralis infection in mice.

Trichinella spiralis is an intestinal nematode parasite that can cause trichinellosis in humans and animals worldwide. The most important known hosts of T. spiralis are pigs, horses, dogs and cats. Pork and its products are the main sources of infection in human trichinellosis. Vaccines against these infections are urgently needed. In this study, the genes encoding the 43-kDa or 45-kDa glycoprotein present in the excretory-secretory (ES) products from T. spiralis muscle larvae (ML) were cloned into the eukaryotic expression vector pVAX1, resulting plasmids pVAX1-Ts43 and pVAX1-Ts45, respectively. Then BALB/c mice were intramuscularly immunized with the DNA vaccine pVAX1-Ts43, pVAX1-Ts45, or both to evaluate their immunogenicity and host protective potential. After the third immunization, mice of each group were challenged with 300 T. spiralis ML. The results showed that the mice immunized with the DNA vaccine pVAX1-Ts43 or pVAX1-Ts45 developed significant numbers of FAS+PNA+ B220+ B cells indicating the formation of the germinal centers (GCs), IFN-γ-secreting (mesenteric lymph nodes, MLN) cells, and IL-4-, and IL-10-secreting splenocytes. Mice immunized with the pVAX1-Ts43 or pVAX1-Ts45 vaccine elicited partial protective immunity against challenge infections with T. spiralis as shown by significant reduction in ML. Most notably, the combined immunity of pVAX1-Ts43 and pVAX1-Ts45 induced better immune responses than either of the DNA vaccines given alone and provided as high as 75.9% reductions in muscle larval burden. These results suggest that the plasmid DNA encoding the 43-kDa or 45-kDa glycoprotein could be considered as a potential vaccine candidate against T. Spiraling infection.

Antibody-Based Protective Immunity against Helminth Infections: Antibody Phage Display Derived Antibodies against BmR1 Antigen.

Helminth parasite infections are significantly impacting global health, with more than two billion infections worldwide with a high morbidity rate. The complex life cycle of the nematodes has made host immune response studies against these parasites extremely difficult. In this study, we utilized two phage antibody libraries; the immune and naïve library were used to identify single chain fragment variable (scFv) clones against a specific filarial antigen (BmR1). The V-gene analysis of isolated scFv clones will help shed light on preferential VDJ gene segment usage against the filarial BmR1 antigen in healthy and infected states. The immune library showed the usage of both lambda and kappa light chains. However, the naïve library showed preferential use of the lambda family with different amino acid distributions. The binding characteristics of the scFv clones identified from this work were analyzed by immunoassay and immunoaffinity pull down of BmR1. The work highlights the antibody gene usage pattern of a naïve and immune antibody library against the same antigen as well as the robust nature of the enriched antibodies for downstream applications.

Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants.

Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of some helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct the helminth glycoproteins omega-1 and kappa-5, two major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein's Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens.

A Recombinant Positive Control for Serology Diagnostic Tests Supporting Elimination of Onchocerca volvulus.

BACKGROUND: Serological assays for human IgG4 to the Onchocerca volvulus antigen Ov16 have been used to confirm elimination of onchocerciasis in much of the Americas and parts of Africa. A standardized source of positive control antibody (human anti-Ov16 IgG4) will ensure the quality of surveillance data using these tests. METHODOLOGY/PRINCIPAL FINDINGS: A recombinant human IgG4 antibody to Ov16 was identified by screening against a synthetic human Fab phage display library and converted into human IgG4. This antibody was developed into different positive control formulations for enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT) platforms. Variation in ELISA results and utility as a positive control of the antibody were assessed from multiple laboratories. Temperature and humidity conditions were collected across seven surveillance activities from 2011-2014 to inform stability requirements for RDTs and positive controls. The feasibility of the dried positive control for RDT was evaluated during onchocerciasis surveillance activity in Togo, in 2014. When the anti-Ov16 IgG4 antibody was used as a standard dilution in horseradish peroxidase (HRP) and alkaline phosphatase (AP) ELISAs, the detection limits were approximately 1ng/mL by HRP ELISA and 10ng/mL by AP ELISA. Positive control dilutions and spiked dried blood spots (DBS) produced similar ELISA results. Used as a simple plate normalization control, the positive control antibody may improve ELISA data comparison in the context of inter-laboratory variation. The aggregate temperature and humidity monitor data informed temperature parameters under which the dried positive control was tested and are applicable inputs for testing of diagnostics tools intended for sub-Saharan Africa. As a packaged positive control for Ov16 RDTs, stability of the antibody was demonstrated for over six months at relevant temperatures in the laboratory and for over 15 weeks under field conditions. CONCLUSIONS: The recombinant human anti-Ov16 IgG4 antibody-based positive control will benefit inter-laboratory validation of ELISA assays and serve as quality control (QC) reagents for Ov16 RDTs at different points of the supply chain from manufacturer to field use.

Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development.

The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv) phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11-12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES) proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST). As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP) relative to an irrelevant protein control (ovalbumin). Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test.

Taenia solium: Development of an Experimental Model of Porcine Neurocysticercosis.

Human neurocysticercosis (NC) is caused by the establishment of Taenia solium larvae in the central nervous system. NC is a severe disease still affecting the population in developing countries of Latin America, Asia, and Africa. While great improvements have been made on NC diagnosis, treatment, and prevention, the management of patients affected by extraparenchymal parasites remains a challenge. The development of a T. solium NC experimental model in pigs that will allow the evaluation of new therapeutic alternatives is herein presented. Activated oncospheres (either 500 or 1000) were surgically implanted in the cerebral subarachnoid space of piglets. The clinical status and the level of serum antibodies in the animals were evaluated for a 4-month period after implantation. The animals were sacrificed, cysticerci were counted during necropsy, and both the macroscopic and microscopic characteristics of cysts were described. Based on the number of established cysticerci, infection efficiency ranged from 3.6% (1000 oncospheres) to 5.4% (500 oncospheres). Most parasites were caseous or calcified (38/63, 60.3%) and were surrounded by an exacerbated inflammatory response with lymphocyte infiltration and increased inflammatory markers. The infection elicited specific antibodies but no neurological signs. This novel experimental model of NC provides a useful tool to evaluate new cysticidal and anti-inflammatory approaches and it should improve the management of severe NC patients, refractory to the current treatments.

Delineation of BmSXP antibody V-gene usage from a lymphatic filariasis based immune scFv antibody library.

Phage display technology is an important tool for antibody generation or selection. This study describes the development of a scFv library and the subsequent analysis of identified monoclonal antibodies against BmSXP, a recombinant antigen for lymphatic filariasis. The immune library was generated from blood of lymphatic filariasis infected individuals. A TA based intermediary cloning approach was used to increase cloning efficiency for the library construction process. A diverse immune scFv library of 10(8) was generated. Six unique monoclonal antibodies were identified from the 50 isolated clones against BmSXP. Analysis of the clones showed a bias for the IgHV3 and Vκ1 (45.5%) and IgHV2 and Vκ3 (27.3%) gene family. The most favored J segment for light chain is IgKJ1 (45.5%). The most favored D and J segment for heavy chain are IgHD6-13 (75%) and IgHJ3 (47.7%). The information may suggest a predisposition of certain V genes in antibody responses against lymphatic filariasis.

Concerted activity of IgG1 antibodies and IL-4/IL-25-dependent effector cells trap helminth larvae in the tissues following vaccination with defined secreted antigens, providing sterile immunity to challenge infection.

Over 25% of the world's population are infected with helminth parasites, the majority of which colonise the gastrointestinal tract. However, no vaccine is yet available for human use, and mechanisms of protective immunity remain unclear. In the mouse model of Heligmosomoides polygyrus infection, vaccination with excretory-secretory (HES) antigens from adult parasites elicits sterilising immunity. Notably, three purified HES antigens (VAL-1, -2 and -3) are sufficient for effective vaccination. Protection is fully dependent upon specific IgG1 antibodies, but passive transfer confers only partial immunity to infection, indicating that cellular components are also required. Moreover, immune mice show greater cellular infiltration associated with trapping of larvae in the gut wall prior to their maturation. Intra-vital imaging of infected intestinal tissue revealed a four-fold increase in extravasation by LysM+GFP+ myeloid cells in vaccinated mice, and the massing of these cells around immature larvae. Mice deficient in FcRγ chain or C3 complement component remain fully immune, suggesting that in the presence of antibodies that directly neutralise parasite molecules, the myeloid compartment may attack larvae more quickly and effectively. Immunity to challenge infection was compromised in IL-4Rα- and IL-25-deficient mice, despite levels of specific antibody comparable to immune wild-type controls, while deficiencies in basophils, eosinophils or mast cells or CCR2-dependent inflammatory monocytes did not diminish immunity. Finally, we identify a suite of previously uncharacterised heat-labile vaccine antigens with homologs in human and veterinary parasites that together promote full immunity. Taken together, these data indicate that vaccine-induced immunity to intestinal helminths involves IgG1 antibodies directed against secreted proteins acting in concert with IL-25-dependent Type 2 myeloid effector populations.

Immune antibodies and helminth products drive CXCR2-dependent macrophage-myofibroblast crosstalk to promote intestinal repair.

Helminth parasites can cause considerable damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination particularly during enteric infection. However, how protective type 2 responses targeted against these tissue-disruptive multicellular parasites might contribute to homeostatic wound healing in the intestine has remained unclear. Here, we observed that mice lacking antibodies (Aid-/-) or activating Fc receptors (Fcrg-/-) displayed impaired intestinal repair following infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb), whilst transfer of immune serum could partially restore chemokine production and rescue wound healing in Aid-/- mice. Impaired healing was associated with a reduced expression of CXCR2 ligands (CXCL2/3) by macrophages (MΦ) and myofibroblasts (MF) within intestinal lesions. Whilst antibodies and helminths together triggered CXCL2 production by MΦ in vitro via surface FcR engagement, chemokine secretion by intestinal MF was elicited by helminths directly via Fcrg-chain/dectin2 signaling. Blockade of CXCR2 during Hpb challenge infection reproduced the delayed wound repair observed in helminth infected Aid-/- and Fcrg-/- mice. Finally, conditioned media from human MΦ stimulated with infective larvae of the helminth Ascaris suum together with immune serum, promoted CXCR2-dependent scratch wound closure by human MF in vitro. Collectively our findings suggest that helminths and antibodies instruct a chemokine driven MΦ-MF crosstalk to promote intestinal repair, a capacity that may be harnessed in clinical settings of impaired wound healing.

Antibodies trap tissue migrating helminth larvae and prevent tissue damage by driving IL-4Rα-independent alternative differentiation of macrophages.

Approximately one-third of the world's population suffers from chronic helminth infections with no effective vaccines currently available. Antibodies and alternatively activated macrophages (AAM) form crucial components of protective immunity against challenge infections with intestinal helminths. However, the mechanisms by which antibodies target these large multi-cellular parasites remain obscure. Alternative activation of macrophages during helminth infection has been linked to signaling through the IL-4 receptor alpha chain (IL-4Rα), but the potential effects of antibodies on macrophage differentiation have not been explored. We demonstrate that helminth-specific antibodies induce the rapid trapping of tissue migrating helminth larvae and prevent tissue necrosis following challenge infection with the natural murine parasite Heligmosomoides polygyrus bakeri (Hp). Mice lacking antibodies (JH (-/-)) or activating Fc receptors (FcRγ(-/-)) harbored highly motile larvae, developed extensive tissue damage and accumulated less Arginase-1 expressing macrophages around the larvae. Moreover, Hp-specific antibodies induced FcRγ- and complement-dependent adherence of macrophages to larvae in vitro, resulting in complete larval immobilization. Antibodies together with helminth larvae reprogrammed macrophages to express wound-healing associated genes, including Arginase-1, and the Arginase-1 product L-ornithine directly impaired larval motility. Antibody-induced expression of Arginase-1 in vitro and in vivo occurred independently of IL-4Rα signaling. In summary, we present a novel IL-4Rα-independent mechanism of alternative macrophage activation that is antibody-dependent and which both mediates anti-helminth immunity and prevents tissue disruption caused by migrating larvae.

Development of specific scFv antibodies to detect neurocysticercosis antigens and potential applications in immunodiagnosis.

We have shown previously that detection of circulating antibodies against mimotopes selected by phage display were useful in neurocysticercosis diagnosis. However, circulating antigens may also be useful in patients' clinical follow-up. Therefore, we aimed to select novel combinatorial antibodies, single-chain variable fragment (scFv), which can be used for specific antigens with pre-defined affinity and specificity without prior immunization. A phage scFv antibody library was selected against Taenia solium mimotopes displayed on phages coupled in beads and total saline extract of T. solium metacestodes (S) immobilized on microtiter plate wells. After two rounds of selection, 96 phage clones were evolved and validated against each target by enzyme linked immunosorbent assay (ELISA), and dot-blot, and three specific antibodies (B6, G10 and A4) were further characterized by sequencing and indirect immunofluorescence (IFI) assays. IFI revealed tegument staining for the B6, while the others showed a non-uniform staining in the whole parasite. The selected scFvs were used to capture their antigen targets that were elucidated through mass spectrometry, and used for antibody detection in NC patients' sera by ELISA, which achieved sensitivities greater than 97% and specificities above 95%. We have successfully developed scFv antibodies against important mimotopes used in NC diagnosis, and can be further explored to detect circulating antigens for clinical follow-up of patients with NC. Our strategy also highlighted the possibility of using this combinatorial approach to select, capture and characterize specific antigens to better understand this intriguing parasite infection and disease evolution.

A prime-boost vaccination of mice with attenuated Salmonella expressing a 30-mer peptide from the Trichinella spiralis gp43 antigen.

Protection against Trichinella infections has been achieved using various parasite antigens and adjuvants. Recently, we reported that immunization of mice with an attenuated Salmonella strain displaying a 30-mer peptide (residues 210-239) from the Trichinella spiralis gp43 antigen using the ShdA autotransporter induced partial protection against T. spiralis infection. To improve the efficacy of vaccination, we used the MisL autotransporter system to display the Ts30mer peptide on the surface of Salmonella enterica ser. Typhimurium in combination with a prime-boost vaccination strategy. This vector and immunization regimen induced superior protection against T. spiralis when compared to our previously reported approach. Data presented herein showed a significant reduction in adult worm and muscle larvae burdens, high IgG titers, and increased production of intestinal mucus with entrapped adult worms. This prime-boost vaccination scheme is a suitable strategy to elicit enhanced protective immunity against T. spiralis.

IL-10R blockade during chronic schistosomiasis mansoni results in the loss of B cells from the liver and the development of severe pulmonary disease.

In schistosomiasis patients, parasite eggs trapped in hepatic sinusoids become foci for CD4+ T cell-orchestrated granulomatous cellular infiltrates. Since the immune response is unable to clear the infection, the liver is subjected to ongoing cycles of focal inflammation and healing that lead to vascular obstruction and tissue fibrosis. This is mitigated by regulatory mechanisms that develop over time and which minimize the inflammatory response to newly deposited eggs. Exploring changes in the hepatic inflammatory infiltrate over time in infected mice, we found an accumulation of schistosome egg antigen-specific IgG1-secreting plasma cells during chronic infection. This population was significantly diminished by blockade of the receptor for IL-10, a cytokine implicated in plasma cell development. Strikingly, IL-10R blockade precipitated the development of portal hypertension and the accumulation of parasite eggs in the lungs and heart. This did not reflect more aggressive Th2 cell responsiveness, increased hepatic fibrosis, or the emergence of Th1 or Th17 responses. Rather, a role for antibody in the prevention of severe disease was suggested by the finding that pulmonary involvement was also apparent in mice unable to secrete class switched antibody. A major effect of anti-IL-10R treatment was the loss of a myeloid population that stained positively for surface IgG1, and which exhibited characteristics of regulatory/anti-inflammatory macrophages. This finding suggests that antibody may promote protective effects within the liver through local interactions with macrophages. In summary, our data describe a role for IL-10-dependent B cell responses in the regulation of tissue damage during a chronic helminth infection.

Isolation of neurocysticercosis-related antigens from a genomic phage display library of Taenia solium.

In this study, the authors have generated a tapeworm Taenia solium genomic DNA expression library where foreign peptides/proteins were fused to N-termini of M13 cpVIII and expressed at a high copy number on the phage surface, and they showed that this library may be used in bioselection against antipathogen immune sera, allowing the identification of disease-related antigens recognizing antibodies present in clinical samples. They isolated 2 phage clones expressing T. solium-derived antigens specifically reacting with antibodies present in plasma and cerebrospinal fluid samples of neuroimaging-confirmed neurocysticercosis patients. The described antigen discovery strategy may be used for the direct identification of antigens useful for host-pathogen interaction studies as well as for the development of molecular vaccines and diagnostics.

Schistosoma mansoni host-exposed surface antigens characterized by sera and recombinant antibodies from schistosomiasis-resistant rats.

Antibodies from Schistosoma mansoni-infected rats, unlike mice, show a higher titer for schistosome apical tegumental antigens compared with non-apical membrane antigens. These antibodies bind to the surface of living lung-stage worms and to formaldehyde-fixed adult worms. We produced a single-chain antibody Fv domain (scFv) phage library displaying the antibody repertoire of rats highly immune to schistosome infection and we selected for scFvs that recognize the host-exposed surface of worms. Five unique rat scFvs (Teg1, Teg4, Teg5, Teg20 and Teg37) were obtained which recognize schistosome surface epitopes. Each of the scFvs recognizes the surface of living schistosomula and lung-stage schistosomules and/or the surface of formaldehyde-fixed adult worms. None of these scFvs reproducibly stained living adult worms. This suggests that a change occurs during the transition from lung schistosomules to 4-week adults such that at least some surface antigens, although remaining on the surface in living adult worms, can no longer be immunologically stained. Teg1 and Teg4 scFvs both recognize specific bands on Western blots. No bands were observed for the other three scFvs, suggesting that these scFvs may recognize non-protein or conformationally-dependent epitopes. Teg1 was unambiguously identified as recognizing the S. mansoni tetraspanin antigen, SmTSP-2, within the large extracellular domain. Teg4 recognizes a 35kDa band tentatively identified as Sm29 by proteomic analysis. These scFvs can now be used to characterize schistosome epitopes at the host-parasite interface, to target worms in vivo, and to study the mechanisms by which these worms naturally evade immune damage to the tegument within permissive hosts.

Intraspecific epitopic variation in a carbohydrate antigen exposed on the surface of Trichostrongylus colubriformis infective L3 larvae.

The carbohydrate larval antigen, CarLA, is present on the exposed surface of all strongylid nematode infective L3 larvae tested, and antibodies against CarLA can promote rapid immune rejection of incoming Trichostrongylus colubriformis larvae in sheep. A library of ovine recombinant single chain Fv (scFv) antibody fragments, displayed on phage, was prepared from B cell mRNA of field-immune sheep. Phage displaying scFvs that bind to the surface of living exsheathed T. colubriformis L3 larvae were identified, and the majority of worm-binding scFvs recognized CarLA. Characterization of greater than 500 worm surface binding phage resulted in the identification of nine different anti-CarLA scFvs that recognized three distinct T. colubriformis CarLA epitopes based on blocking and additive ELISA. All anti-CarLA scFvs were specific to the T. colubriformis species of nematode. Each of the three scFv epitope classes displayed identical Western blot recognition patterns and recognized the exposed surface of living T. colubriformis exsheathed L3 larvae. Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms. Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations. These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes.