Filarial nematode vaccines, polypeptides, and nucleic acids
09994624 ยท 2018-06-12
Assignee
Inventors
- Ben Makepeace (Liverpool Merseyside, GB)
- David Taylor (Edinburgh, GB)
- Simon Babayon (Glasgow, GB)
- Stuart Armstrong (Liverpool, GB)
- Mark Blaxter (Edinburgh, GB)
Cpc classification
A61K45/06
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
A61K39/00
HUMAN NECESSITIES
Abstract
The present invention relates to vaccines comprising a ShK domain of a filarial nematode protein. These vaccines may be used for the prevention and/or treatment of filarial nematode infections. The invention also relates to novel proteins comprising a ShK domain of a filarial nematode protein and pharmaceutical compositions. The invention may be used for the prevention and/or treatment of filarial nematode infections in canine subjects, and also in human subjects.
Claims
1. A polypeptide, comprising at least one polypeptide including: at least one ShK domain of L. sigmoidontis protein nLs_04059 according to SEQ ID NO: 1; and/or at least one polypeptide sharing at least 70 percent identity with said at least one ShK domain, wherein the at least one polypeptide retains 6 cysteine residues with characteristic spacing of a ShK domain; wherein the polypeptide is a chimeric polypeptide comprising an additional antigen from a source other than the filarial nematode from which the Shk domain was obtained.
2. The polypeptide according to claim 1, comprising a plurality of the same ShK domain.
3. The polypeptide according to claim 1, comprising a plurality of different ShK domains.
4. The polypeptide according to claim 1, further comprising an artificial spacer separating the ShK domains.
5. The polypeptide according to claim 4, wherein the spacer comprises poly-L-lysine.
6. The polypeptide according to claim 1, wherein the at least one polypeptide sharing at least 70 percent identity with said at least one ShK domain is from a filarial nematode selected from group consisting of: L. sigmodontis, D. immitis, Wuchereria bancrofti, Brugia malayi, Brugia timori, Onchocerca volvulus, and Loa loa.
7. A nucleic acid, comprising: at least one nucleic acid encoding at least one polypeptide comprising at least one ShK domain of L. sigmoidontis protein nLs_04059 according to SEQ ID NO:I; and/or at least one nucleic acid encoding at least one polypeptide sharing at least 70 percent identity with said at least one Shk domain, wherein the at least one polypeptide retains 6 cysteine residues with characteristic spacing of a ShK domain; wherein the at least one polypeptide is a chimeric polypeptide comprising an additional antigen from a source other than the filarial nematode from which the Shk domain was obtained.
8. The nucleic acid according to claim 7, wherein said nucleic acid is part of an expression vector.
9. The nucleic acid according to claim 8, wherein said expression vector is for expression in E. coli.
10. The nucleic acid according to claim 7, wherein the at least one polypeptide sharing at least 70 percent identity with said at least one ShK domain is from a filarial nematode selected from group consisting of: L. sigmodontis, D. immitis, Wuchereria bancrofti, Brugia malayi, Brugia timori, Onchocerca volvulus, and Loa loa.
11. A method of treating or preventing a filarial nematode infection, comprising the steps of: providing to a subject a therapeutically effective amount of at least one polypeptide comprising at least one ShK domain of L. sigmoidontis protein nLs_04059 according to SEQ ID NO:I and/or at least one polypeptide sharing at least 70 percent identity with said at least one ShK domain, wherein the at least one polypeptide retains 6 cysteine residues with characteristic spacing of a ShK domain wherein the at least one polypeptide is a chimeric polypeptide comprising an additional antigen from a source other than the filarial nematode from which the Shk domain was obtained.
12. The method according to claim 11, further comprising the step of: administering the therapeutically effective amount of the at least one polypeptide.
13. The method according to claim 11, wherein the subject is a human or an animal.
14. The method according to claim 11, wherein the filarial nematode infection includes a disease selected from the group consisting of: lymphatic filariasis, onchocerciasis, and loiasis.
15. The method according to claim 11, wherein the subject is an animal and the filarial nematode infection is heartworm.
16. The method according to claim 11, wherein the at least one polypeptide sharing at least 70 percent identity with said at least one ShK domain is from a filarial nematode selected from group consisting of: L. sigmodontis, D. immitis, Wuchereria bancrofti, Brugia malayi, Brugia timori, Onchocerca volvulus, and Loa loa.
17. The method according to claim 11, wherein the at least one ShK domain and/or the at least one polypeptide sharing at least 70 percent identity with said at least one ShK domain is formulated as a pharmaceutical composition.
18. The method according to claim 11, further comprising the step of: administering a nucleic acid encoding the therapeutically effective amount of the at least one polypeptide.
19. The method according to claim 11, wherein the at least one polypeptide comprises a plurality of the same or different ShK domains.
20. The method according to claim 11, wherein the at least one polypeptide further comprises an artificial spacer separating the ShK domains.
21. The method according to claim 11, wherein the at least one polypeptide further comprises an additional vaccine antigen.
Description
(1) The invention will now be further described with reference to the following Experimental Results and Figures in which:
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EXPERIMENTAL RESULTS
(17) Study 1
(18) The ability of polypeptides comprising ShK domains of filarial nematode proteins to as vaccines conferring protective immunity in respect of filarial nematode infection, and the suitability of nucleic acids encoding such polypeptides to serve as vaccines, was demonstrated by the following study. The L. sigmodontis ShK domain containing protein used as an exemplary vaccine was designated LsShK for the purposes of this study.
(19) Immunisations and infections were performed with female BALB/c mice, starting at ages of 6-7 weeks, with five animals per experimental group. Mice were housed in individually ventilated cages and infected subcutaneously with 30 or 40 L. sigmodontis infective larvae (iL3). Na?ve, uninfected animals were maintained and sampled in parallel as controls for the immunological readouts.
(20) All cloning was carried out following the recommendations of the pcDNA 3.1 Directional TOPO Expression Kit (Invitrogen). LsShK (gene ID nLs.2.1.2.t04059-RA) was amplified from a cDNA preparation of adult L. sigmodontis using specific primers. Fusion constructs containing single-chain anti-DEC205 antibody (DEC) upstream of the LsShK sequence were produced from ready-made constructs kindly provided by Dr. Ralph Steinman. Briefly, PCR products of genes of interest were digested with NotI and XbaI (Neb laboratory, UK), then ligated into an NotI and XbaI-digested anti-mouse dec-205 single chain antibody-ovalbumin construct (DEC-OVA) or antibody control Ig-OVA to replace the fragment of OVA gene, respectively. All plasmids were sequenced to confirm identity.
(21) Plasmids were injected in the tibialis anterior muscle of the left leg with a 27G needle, immediately followed by electroporation with an ECM 830 generator+Tweezertrodes (BTX Harvard Apparatus) using as settings 8 pulses, 200 V/cm, 40 ms duration, 460 ms interval. Each mouse was immunised twice separated by 2 weeks interval with 40 ?g of DNA total made up by equal quantities of each plasmid species, delivered in 50 ?l PBS. As a consequence, the quantity of each individual plasmid was reduced as the number of different plasmids incorporated into the inoculums increased. However, the quantity of each one remained in excess of the minimal efficient dose.
(22) Parasite survival was determined at experiment endpoint. Adult filariae were isolated from the pleural cavity lavage fluid in 10 ml cold PBS, fixed in hot 70% ethanol and counted. Protection was calculated as:
(mean burden in primary infected animals?mean burden of vaccinated animals)/mean burden in primary infected animals.
(23) Microfilariae were counted in 30 ?l of blood after fixation in 570 ?l of BD FACS lysing solution (BD Biosciences) under an inverted microscope.
(24) Generalised linear models were used to compare the effects of different vaccine formulations on parasitological parameters as they allow more flexibility in specifying the distribution of response variables and better model fitting through Maximum Likelihood estimation.
(25) The results of this study are illustrated in
(26) The inventors found that at day 60 post-infection, the LsShK vaccine had a modest effect on adult worm burden (?40% reduction), though this was of borderline statistical significance (p=0.07). However, the vaccinated group had no microfilariae detected in the blood, whereas the primary infection group (which received empty plasmid vector only) exhibited a median microfilaraemia of ?830 parasites per ml (p=0.005). This suggests that the medical use of ShK domains as vaccines achieves its therapeutic use through sterilising the adult female worms, or by killing migrating microfilariae before they can reach the bloodstream.
(27) Study 2
(28) The invention may further be understood by the skilled person on consideration of the following details of a study undertaking quantitative secretome analysis of a model filarial nematode (Litomosoides sigmodontis) across the parasite life cycle.
(29) 2.1 Summary
(30) Filarial nematodes (superfamily Filarioidea) are responsible for an annual global health burden of approximately 6.3 million disability-adjusted life-years, which represents the greatest single component of morbidity attributable to helminths affecting humans. No vaccine exists for the major filarial diseases, lymphatic filariasis and onchocerciasis; in part because research on protective immunity against filariae has been constrained because the human-parasitic species cannot complete their lifecycles in laboratory mice. However, the rodent filaria Litomosoides sigmodontis has become a popular experimental model, as BALB/c mice are fully permissive for its development and reproduction. Here, we provide a comprehensive analysis of excretory-secretory products from L. sigmodontis across five lifecycle stages. Applying intensity-based quantification, we determined the abundance of 302 unique excretory-secretory proteins, of which 64.6% were present in quantifiable amounts only from gravid adult female nematodes. This lifecycle stage, together with immature first-stage larvae (microfilariae), released four proteins that have not previously been evaluated as vaccine candidates: a predicted 28.5 kDa filaria-specific protein, a zonadhesin and SCO-spondin-like protein, a vitellogenin, and a protein containing six metridin-like ShK toxin domains. Female nematodes also released two proteins derived from the obligate Wolbachia symbiont. Notably, excretory-secretory products from all parasite stages contained several uncharacterised members of the transthyretin-like protein family. Furthermore, biotin labelling revealed that redox proteins and enzymes involved in purinergic signalling were enriched on the adult nematode cuticle. Comparison of the L. sigmodontis adult secretome with that of the human-infective filarial nematode Brugia malayi (reported previously in three independent published studies) identified differences that suggest a considerable underlying diversity of potential immunomodulators. The molecules identified in L. sigmodontis excretory-secretory products show promise not only for vaccination against filarial infections, but for the amelioration of allergy and autoimmune diseases.
(31) 2.2 Introduction
(32) Filarial nematodes are the most important helminth parasites of humans in terms of overall impact on public health, with an annual global burden of ?6.3 million disability-adjusted life-years (1). Lymphatic filariasis (LF) or elephantiasis, which affects populations across Africa, South Asia, the Pacific, Latin America and the Caribbean, accounts for 92% of this toll. The remainder is caused by onchocerciasis or river blindness, primarily in sub-Saharan Africa. The major human filarial pathogens are Wuchereria bancrofti (responsible for 90% of LF cases), Brugia malayi and Brugia timori (geographically restricted causes of LF), and Onchocerca volvulus (the sole agent of human onchocerciasis). In addition, Loa loa affects ?13 million people in West and Central Africa. This parasite usually induces a relatively mild disease, but has been associated with severe and sometimes fatal adverse events following anthelmintic chemotherapy (2). Filarial parasites are primarily drivers of chronic morbidity, which manifests as disabling swelling of the legs, genitals and breasts in LF; or visual impairment and severe dermatitis in onchocerciasis. The filariae are also a major problem in small animal veterinary medicine, with ?0.5 million dogs in the USA alone infected with Dirofilaria immitis (3), the cause of potentially fatal heartworm disease. However, in domesticated ungulates, filarial infections are generally quite benign (4).
(33) Currently, control of human filarial diseases is almost entirely dependent on three drugs (ivermectin, diethylcarbamazine and albendazole). Prevention of heartworm also relies on prophylactic treatment of dogs and cats with ivermectin or other macrocyclic lactones. Reports of possible ivermectin resistance in O. volvulus (5) and D. immitis (6) have highlighted the importance of maintaining research efforts in vaccine development against filarial nematodes. However, rational vaccine design has been constrained for several decades (7) by the intrinsic complexity of these metazoan parasites and their multistage lifecycle. Moreover, many filarial species carry obligate bacterial endosymbionts (Wolbachia), which may also stimulate the immune response during infection (8). As part of global efforts to improve prevention and treatment of these diseases, large-scale projects have been undertaken, including sequencing of the nematodes (9-11) and their Wolbachia (10, 12, 13), and proteomic analyses of both whole organisms and excretory-secretory products (ESP) (14, 15). Additionally, two studies (both on B. malayi) have examined lifecycle stage-specific secretomes (16, 17). In the context of vaccine design, the identification of ESP proteins and determination of their expression in each major lifecycle stage can facilitate the prioritisation of candidates for efficacy screening in animal models.
(34) One barrier to the progression of research in the filarial field is our inability to maintain the full lifecycle of the human parasites in genetically tractable, inbred hosts. The filarial lifecycle involves uptake of the first-stage larvae (microfilariae, Mf) by a haematophagous arthropod, two moults in this vector, followed by transmission of third-stage larvae (L3) to a new vertebrate host. Two further moults occur in the definitive host before the nematodes mature as dioecious adults in a species-specific, parenteral predilection site. However, the complete lifecycle of the New World filaria Litomosoides sigmodontis can be maintained in laboratory rodents, including inbred mice (18). This species was first studied in its natural host (the cotton rat, Sigmodon hispidus) (19) [the previous designation of these isolates as L. carinii is taxonomically incorrect (20)]. Drawing on the full power of murine immunology, including defined knockout strains, this model has been address questions regarding the fundamental immunomodulatory mechanisms employed by filarial parasites (21), their susceptibility to different modes of vaccination, their ability to mitigate proinflammatory pathology and autoimmune disease (22), and the impact of various vaccine strategies on adult nematode burden and fecundity (23) (24). The L. sigmodontis model has also been central in defining the role of T-regulatory cells in filarial immune evasion (25).
(35) Using the resource of a newly-determined genome sequence, coupled with a derivative of intensity-based absolute quantification (iBAQ) proteomics, we have examined the stage-specific secretome of L. sigmodontis in vector-derived L3 (vL3), adult males (AM), pre-gravid adult females (PAF), gravid adult females (GAF), and immature Mf (iMf). In addition to identifying dynamic changes in the ESP profile through the lifecycle, we show important differences in the adult secretomes of L. sigmodontis and B. malayi, especially in the abundance of two novel proteins released by female L. sigmodontis that lack orthologues in B. malayi. As has been observed in other parasitic nematodes, we find transthyretin-like family (TTL) proteins to be particularly dominant in the ESP. Leakage of uterine fluid may account for the remarkable diversity of proteins that we detect in GAF ESP, and we highlight several novel proteins that warrant evaluation in vaccine trials and as anti-inflammatory mediators.
(36) 2.3 Experimental Procedures
(37) Ethical Considerations
(38) All experimental procedures on the animals required for vL3 production at the Museum National d'Histoire Naturelle were approved by the ethical committee Cuvier (n? 68-002) and carried out in strict accordance with EU Directive 2010/63/UE and the relevant national legislation (French D?cret n? 2013-118, 1 Feb. 2013). All other parasite stages were harvested from animals maintained at the University of Edinburgh in compliance with a UK Home Office Animals (Scientific Procedures Act) 1986 project licence and the recommendations of the local ethical review committee.
(39) Parasites and Protein Preparations
(40) The life cycle of L. sigmodontis was maintained in jirds (Meriones unguiculatus) infected with vL3 harvested from the mite vector Ornithonyssus bacoti. After 70-90 days, GAF and AM were recovered from the pleural cavity by lavage with serum-free RPMI 1640 medium (Life Technologies), whereas PAF were recovered 32 days post-challenge. To harvest iMf liberated in vitro, GAF culture medium was removed after 24 h and centrifuged at 1,900 g for 20 minutes (4? C.). Blood-derived microfilariae (bMf) were obtained by overlay of blood (from cardiac puncture of jirds >75 days post-infection) onto a 25% Percoll suspension, centrifugation at 1,900 g for 20 minutes (4? C.), and passage of the bMf fraction through a PD-10 desalting column (GE Healthcare) prior to culture. The vL3 larvae were dissected directly from the mite vector and washed three times in RPMI 1640 before transfer to culture vessels.
(41) To determine the relative abundance of proteins in the secretome of each parasite stage, ESP and whole body extracts (WBE) were extracted and analysed separately. All parasite stages were incubated in serum-free RPMI 1640 supplemented with 100 U/ml penicillin, 100 ?g/ml streptomycin and 1% glucose at 37? C. (5% CO.sub.2) in ultra-low attachment flasks (Corning), and were confirmed to be viable during incubation by microscopic examination. The medium was replaced every 24 h, and spent media recovered at 24 h and 48 h were centrifuged at 1,900 g for 20 minutes (4? C.) in low protein-binding Oak Ridge tubes (polypropylene copolymer; Thermo Scientific Nalgene) to remove debris. To purify proteins from the supernatant, hydroxylated silica slurry (StrataClean Resin, Agilent Technologies) was added at 30 ?l/ml and vortex-mixed at high speed for 2 min. Resin used for each 24 h incubation sample was reused for the respective 48 h sample to concentrate ESP prior to storage at ?80? C. Initial experiments using soluble WBE (used as a proxy for ESP, as limited amounts of the latter were available) displayed no visible differences in protein profiles by SDS-PAGE using resin-bound protein compared to equivalent unbound material (data not shown). Analyses were performed with separate ESP batches in quadruplicate for GAF, triplicate for AM, and duplicate for PAF, iMf and vL3.
(42) Soluble WBE was prepared by homogenisation in 25 mM ammonium bicarbonate, 1% RapiGest SF surfactant (Waters) and cOmplete Protease Inhibitor Cocktail (Roche) using a mini-pestle in a microcentrifuge tube. This was followed by 10 cycles of sonication on ice using a Vibra-Cell VCX130PB sonicator (Sonics & Materials, Inc.) with microprobe (10 sec sonication alternating with 30 sec incubation on ice). Homogenised samples were centrifuged at 13,000 g for 20 minutes (4? C.) and the supernatant retained. The WBE preparations were obtained from single pools of parasites for all stages except GAF and AM, where two biological replicates were available. Protein concentrations were determined using the Pierce Coomassie Plus (Bradford) Protein Assay (Thermo Scientific).
(43) Surface Biotinylation of Live Worms
(44) Samples of 10 adult male and five female nematodes were washed three times with pre-chilled PBS buffer and incubated for 30 min with 1 mM EZ-link Sulfo-NHS-SS-Biotin (Thermo scientific), or PBS only (negative control), at 4? C. with gentle agitation. The biotinylating solution was removed, and the reaction quenched with 100 mM glycine in PBS before washing the nematodes three times in PBS-glycine. Labelled nematodes were stored at ?80? C. Surface proteins were extracted by sequential incubations in PBS buffer alone, 1.5% octyl ?-D-glucopyranoside (Sigma), 0.5% SDS and then 4 M urea (all in PBS) for 1 h each (room temperature). Proteins released at each step were incubated with 30 ?l of high-capacity streptavidin-agarose beads (Thermo Scientific) for 2 h at room temperature with rotary mixing. To recover bound biotinylated proteins, the supernatant was removed and the beads were washed three times in PBS and three times in 25 mM ammonium bicarbonate prior to incubation in 50 mM DTT (Sigma), 25 mM ammonium bicarbonate at 50? C. for 30 min. The supernatant was removed and the DTT diluted tenfold before digestion with 0.2 ?g proteomic-grade trypsin (Sigma) overnight at 37? C. The resultant peptides were concentrated using C.sub.18 reverse-phase spin filters (Thermo Scientific) according to the manufacturer's instructions prior to MS analysis.
(45) To confirm efficient and specific labelling of the parasite surface, AM and GAF were fixed in 70% hot ethanol after subjection to biotin and control labelling as above. Paraffin-embedded sections (4 ?m) were deparaffinised, rehydrated and blocked in 1% BSA and 0.3% Triton X-100 in PBS (blocking buffer) for 1 h (room temperature), followed by two 5-min washes in PBS with gentle agitation. The sections were incubated with streptavidin-FITC (Sigma) at a 1/1,000 dilution in blocking buffer for 1 hr (room temperature), washed three times, and mounted with ProLong Gold anti-fade reagent (Life Technologies). Images were obtained on an Axio Imager.M2 fluorescence microscope (Zeiss) using Zen 2012 software (Zeiss), combining the FITC channel with brightfield illumination.
(46) Sample Preparation for Proteomics
(47) StrataClean Resin containing bound ESP was washed twice with 25 mM ammonium bicarbonate before suspension in 0.1% RapiGest SF, 25 mM ammonium bicarbonate. The resin samples were heated at 80? C. for 10 min, reduced with 3 mM DTT at 60? C. for 10 min, cooled, then alkylated with 9 mM iodoacetamide (Sigma) for 30 min (room temperature) protected from light. All steps were performed with intermittent vortex-mixing. The samples were then digested using 0.2 ?g proteomic-grade trypsin at 37? C. overnight with rotation, centrifuged at 13,000 g for 5 min, and the supernatant removed. The resin was washed twice with 0.1% RapiGest SF, 25 mM ammonium bicarbonate and the supernatants pooled. To remove RapiGest SF, the samples were precipitated using TFA (final concentration, 1%) at 37? C. for 2 h and centrifuged at 12,000 g for 1 hr (4? C.). The peptide supernatant was concentrated using C.sub.18 reverse-phase spin filters according to the manufacturer's instructions. The WBE samples were reduced and alkylated as above, digested with trypsin at a protein:trypsin ratio of 50:1 at 37? C. overnight, and precipitated to remove RapiGest SF as for the ESP preparations.
(48) NanoLC MS ESI MS/MS Analysis
(49) Peptide solutions (2 ?l) were analysed by on-line nanoflow LC using the nanoACQUITY-nLC system (Waters) coupled to an LTQ-Orbitrap Velos (Thermo Scientific) MS as previously described (13, 26). Thermo RAW files were imported into Progenesis LC-MS (version 4.1, Nonlinear Dynamics) and spectral data were transformed to MGF files prior to export for peptide identification using the Mascot (version 2.3.02, Matrix Science) search engine as detailed previously (26). Tandem MS data were searched against the protein predictions from the L. sigmodontis genome and its Wolbachia symbiont, wLs [obtained from the online nematode genome of Litomosoides sigmodontis release nLs 2.1.2, 10,246 protein sequences (M. Blaxter, S. Kumar, G. Koutsovoulos; unpublished); and release wLs 2.0, 1,042 protein sequences (27)], together with predicted proteomes for the rodent host (Mus musculus, Uniprot release 2012_08, 16,626 protein sequences; and Meriones unguiculatus, Uniprot release 2012_08, 223 protein sequences) and a general contaminant database (GPMDB, cRAP version 2012.01.01, 115 protein sequences). Search parameters, allowable modifications and the false discovery rate were defined as reported previously (13, 26). Mascot search results were imported into Progenesis LC-MS as XML files and analysed according to the following criteria: at least two unique peptides were required for reporting protein identifications, and an individual protein had to be present in ?2 biological replicates to be included in the ESP dataset. Protein abundance was calculated by the iBAQ method; i.e., the sum of all peak intensities from the Progenesis output was divided by the number of theoretically observable tryptic peptides (28). For ESP and WBE, protein abundance was normalised by dividing the protein iBAQ value by the summed iBAQ values for the corresponding sample, and the reported abundance is the mean of the biological replicates. Normalised peptide intensities rather than iBAQ values were used to calculate fold-changes between control and biotinylated worm surface preparations. Mass spectrometric data have been deposited in the ProteomeXchange Consortium database via the PRIDE partner repository (29) with the dataset identifier XXXXXXXXX.
(50) In Silico Analyses of Proteins
(51) The domain content of proteins identified in the ESP assessed using Pfam (v. 27.0) with the gathering threshold as a cut-off. A hypergeometric test for enrichment of Pfam domains in ESP proteins compared with the complete predicted proteome of L. sigmodontis was performed using the phyper toolkit within the R programming environment (30). The Benjamini & Hochberg step-up FDR-controlling procedure was applied to the calculated, adjusted P-values (31). Structural homologues of abundant uncharacterised proteins were identified through comparison to the National Center for Biotechnology Information non-redundant protein database (
(52) ShK domains were identified in the complete predicted proteomes of the filariae B. malayi (9), D. immitis (10), L. sigmodontis, Onchocerca ochengi, Acanthocheilonema viteae (draft unpublished genomes available online at the nematode genome database; Blaxter et al., unpublished), W. bancrofti and L. loa (11), plus the ascaridid nematode Ascaris suum (40) (which is an outgroup for the filarial species), using the Pfam hidden Markov model for the domain and hmmer (version3.1b.1). Each domain was excised and a total of 531 distinct domains identified, which were aligned using ClustalOmega (41). Inspection of the alignment revealed that a subset of domains were misaligned (and therefore did not have the six cysteine residues in register with the others); these were corrected manually. The alignment was analysed for phylogenetic signal using MrBayes (version 3.2) (42) and two runs of four chains each were run for two million generations. The first million generations were discarded as burn-in after inspection in Tracer (version 1.5; A. Rambaut) and a consensus tree was inferred from the remaining 10,000 samples taken every 100 generations. Sequence logos were generated for all 531 ShK domains, all domains from nLs_04059 and orthologues, and each of the six distinct sets of orthologous domains, using the WebLogo server (43).
(53) 2.4 Results
(54) Distribution of Proteins in ESP Across Parasite Lifecycle Stages
(55) We searched ?120,000 MS spectra per lifecycle stage against protein sequences predicted from the L. sigmodontis and wLs genome assemblies. A total of 302 quantifiable filarial proteins (i.e., represented by ?2 unique peptides in ?2 biological replicates) were detected in ESP across the five lifecycle stages. A majority of these (195 proteins, 64.6%) were uniquely identified in GAF (
(56) We explored functional distinctness of ESP from different lifecycle stages by determining protein domain overrepresentation relative to the complete predicted proteome of L. sigmodontis. The greatest fold-enrichment scores were observed in the AM ESP, which contained three proteins with a major sperm protein (MSP) fibre protein 2b (MFP2b) domain and 10 proteins with a TTL family domain (
(57) Abundant Proteins Released by Adult Parasites
(58) The GAF ESP displayed the most complex composition, and the majority of the abundant proteins secreted by this stage were uncharacterised or contained conserved domains associated with very limited functional information (
(59) Functionally defined components of the ESP included a small cysteine protease inhibitor [CPI (48)], the omega-class glutathione S-transferases [GST (49)], the MSPs (50), and the microfilarial sheath protein (51) (
(60) There was extensive overlap in the identities of the most abundant proteins in the ESP of PAF, AM and iMf compared with that of the particularly diverse GAF. These less complex ESP mixtures nevertheless contained dominant components overrepresented in individual stages. In PAF, abundant proteins included several glycolytic enzymes and two heat-shock proteins, as well as a galectin (?-galactoside-binding protein 1) and a highly unusual protein, nLs_03350, containing both C-type lectin and acetylcholine receptor domains (
(61) Abundant Proteins Released by Larval Parasites
(62) Characterisation of ESP from the bMf stage posed special challenges. Despite the two-stage purification process and prolonged culture in vitro, 92.4% (61 of 66) of proteins robustly quantified in bMf ESP were derived from the rodent host. The dominant serum components identified were fibronectin, complement C3, serum albumin, hemopexin, plasminogen and ceruloplasmin; while lower amounts of IgM were also detected (Table 6). Of the five quantifiable parasite-derived molecules, three were TTL proteins. To obtain characterise Mf-derived ESP in more depth, we harvested iMf from GAF cultures in vitro, separated them from the female nematodes, and proceeded with in vitro incubation. This procedure increased the detection of proteins of nematode origin to 36 (
(63) Although ESP from the vL3 stage was the least diverse dataset in our study, it showed a distinctive repertoire of highly abundant proteins. Thus, vL3 ESP was composed of previously characterised filarial proteins that are known to be uniquely expressed or enriched in this stage [such as ASP-1 (71), ALT-1 (72), and cathepsin-L-like protease (73)], and other antigens that were well represented in ESP from other stages (RAL-2, CPI-2, Ov16 and ?-galactoside-binding proteins) (
(64) Phylogenetics of Novel, Filaria-Specific ESP Proteins
(65) The most abundant protein in GAF ESP, nLs_03577, is an enigmatic, uncharacterised molecule with a predicted MW of 28.5 kDa and a lack of conserved domains, with the exception of a classical N-terminal signal peptide. Downstream of the signal peptide, moderate to high levels of sequence conservation were apparent across the Filarioidea in the N-terminal portion (
(66) The ShK domain protein nLs_04059 was a particularly distinctive molecule identified in all ESP preparations except vL3. One other L. sigmodontis ShK domain protein, the astacin protease nLs_03368, was a rare component of GAF ESP only (Table 3). The ShK domain (or metridin-like toxin domain, also known as the SXC or six-cysteine domain) was first identified in cnidarian venoms, but is particularly abundant in nematode proteomes (74), where it is associated with secreted proteins. The prototypic ShK peptide (from the cnidarian Stichodactyla helianthus) is a type 1 toxin that blocks voltage-gated potassium channels, and synthetic analogues are currently under development as a therapy for autoimmune diseases, in which Kv1.3 channels expressed by effector memory T-lymphocytes are specifically targeted (75). Although nLs_04059 was not especially abundant in any ESP preparation, its presence in the secretomes of all mammalian-derived stages and its unusual domain structure (
(67) The nLs_04059 protein has the largest number of ShK domains (six) of any protein in L. sigmodontis. We identified orthologous genes in all the other filarial nematode genomes, each containing six ShK domains (
(68) Proteins Associated with the Adult Nematode Surface
(69) The nematode cuticle is the critical interface between the parasite and the immune system of its host (78). Surface-associated proteins may simply mirror ESP, perhaps by passive adsorption of released material, or comprise a distinct component of the exoproteome. Live AM and GAF nematodes were surface-labelled incubated with Sulfo-NHS-SS-Biotin and fractionated. Immunofluorescent imaging of fixed nematode sections confirmed that biotin labelling was largely confined to the cuticular layers (
(70) A striking feature of the surface-associated proteins was the presence of two ectoenzymes involved in purinergic signalling. These were an adenylate kinase predominant in AM extracts and a purine nucleoside phosphorylase found exclusively found in GAF extracts (79) (Table 2, Table 7). A homologue of complement component 1, q subcomponent-binding protein was identified in GAF surface-labelled extracts. Like the human homologue, the L. sigmodontis protein contained an N-terminal mitochondrial import signal sequence, although the former is expressed in a number of extramitochondrial locations, including on the surface of lymphocytes, endothelial cells, dendritic cells and platelets (80). These proteins may play a role in immunomodulation, as purinergic signalling is known to regulate lymphocyte trafficking (79), while the complement component 1q receptor is involved in vasodilation via the generation of bradykinin (80).
(71) Surface extracts from AM contained a homologue of the actin-binding protein, calponin, which has been localised to both striated muscle and the cuticle in adult O. volvulus (81). The GAF surface extracts contained two proteins, protein disulphide isomerase and a leucine-rich repeat family protein, both of which have previously been associated with cuticle synthesis in filariae and C. elegans (82, 83). Stress response-related proteins were also well represented on GAF (including thioredoxin peroxidase (84), aldehyde dehydrogenase, a thioredoxin-like protein and heat-shock proteins), as were several enzymes of pyruvate metabolism (Table 2 and Table 7). Notably, the endosymbiont-derived Wolbachia surface protein was found to be accessible to surface biotinylation in GAF.
(72) Comparison with the Secretome of Adult B. malayi
(73) The ESP from several lifecycle stages of B. malayi have been described previously (14, 16, 17). In these three studies, the only common stage was adult [with both sexes cultured together in (14)]. Of 297 proteins identified in adult L. sigmodontis ESP, 92.6% had an orthologue in B. malayi. However, the majority (61.6%) of these B. malayi orthologues were not observed in the B. malayi secretome (
(74) 2.5 Discussion
(75) Quantifying the Secretomes of a Model Filarial Nematode
(76) Filarial nematodes exact a significant burden of morbidity in human populations and are important pathogens of companion animals. While efficacious anti-filarial drugs exist, the spectre of the evolution of genetic resistance to these is ever-present (5, 6), and alternative routes to treatment are required. It would be preferable to be able to prevent infection as well as treat patent disease, and thus an anti-filarial vaccine would be an extremely valuable addition to medical and veterinary treatment options (85). The ESP released by parasites into their hosts have been the target of vaccine development for decades, but the understanding of these molecules in filarial nematodes is limited. Whereas previous studies have catalogued the proteins inferred to be present in filarial ESP, quantitative assessments of their abundance have not been explored previously using an intensity-based approach. Using the model rodent filarial nematode L. sigmodontis, it is possible to prepare material from across the nematode lifecycle, and thus examine the different vertebrate-parasitic stages in detail. Applying semi-quantitative MS analysis of ESP, we identified secreted proteins and determined their abundance, limiting our analysis to 302 proteins that could be robustly quantified using ?2 unique peptides.
(77) The Secretome of Adult Nematodes
(78) In L. sigmodontis, GAF was responsible for the majority of ESP proteomic diversity. The other four lifecycle stages examined contributed only 11 proteins (3.6% of the total) that were not present in GAF ESP. This finding contrasts with a qualitative analysis of B. malayi secretomes comparing adults, Mf and L3, and incorporating data obtained from single-peptide hits, which found that Mf contributed the greatest proportion of unique proteins (17). However, an earlier assessment of the B. malayi GAF, AM and Mf secretomes concluded that GAF produced the greatest number of unique hits (16), suggesting that methodological differences may underlie these contrasting results. The diversity of GAF ESP is consistent with the material containing not only somatic adult ESP, but also proteins released from the reproductive tract that derive from the processes of oogenesis, fertilisation and embryonic development in utero (all filarial pathogens are ovoviviparous).
(79) Nematode sperm are acutely sensitive to aerobic damage (86). The AM ESP contained proteins suggestive of roles in protection of sperm against oxidants and other stressors, including superoxide dismutase, a serine protease inhibitor and a glutaredoxin-like protein. Glutaredoxins are thiol-containing antioxidant proteins, and C. elegans GLRX-21 plays a key role in mitigating selenium toxicity (87). Mammalian seminal fluid accumulates selenium, which if in excess, can impede sperm motility (88). A homologue of the serine protease inhibitor is secreted by A. suum during the acquisition of motility and contributes to sperm competition by inhibiting the activation of surrounding spermatids (89). Lysis of sperm during aerobic culture may account for the high levels of MSPs observed in AM ESP and in ESP obtained from PAF, GAF and iMf. Female nematodes are fertilised some weeks before the first Mf are produced (90), and the dominance of MSPs in PAF ESP indicates that leakage of sperm from the female reproductive tract occurs before parturition.
(80) Several unique antioxidant proteins (nucleoredoxin-like protein-2, glutathione reductase and translationally-controlled tumour protein) were found in PAF ESP, suggesting an enhanced requirement for protection during this stage. In B. malayi, homologues of the nucleoredoxin-like proteins, which resemble large thioredoxins (91), are present in ESP but do not exhibit stage-specific expression (92). Two unique cuticle biosynthesis related proteins were also released by PAF, suggesting that cuticular remodelling occurs during their final stages of growth. This may result in increased susceptibility to immune-driven oxidative stress or damage during copulation (93). Heat-shock proteins, which were overrepresented in PAF ESP, were detected previously in B. malayi adult nematode ESP (94).
(81) The Mature Microfilarial Secretome is Dominated by Host Proteins
(82) In many filarial nematodes, microfilariae are enclosed in a proteinaceous sheath comprising an inner layer that originates from the eggshell and an outer layer that is produced by secretions in the distal portion of the uterus. Five major structural proteins have been identified in the L. sigmodontis sheath, some of which are synthesised in the developing embryo and others in the uterine epithelium (51), but none of these were found in iMf ESP, indicating that they are stable components. Many host serum proteins were released from bMf in culture. These are likely to derive from specific interactions with the parasite surface, perhaps reflecting a tension between the nematode exploiting the host and the host immune system recognising the parasite. The finding of host at the Mf surface is not new, as five serum components were only proteins released by SDS extraction of L. sigmodontis Mf sheaths (95), and human serum albumin has been detected on the sheath surface of W. bancrofti Mf (96), but is generally not found on Brugia spp. Mf (97). The L. sigmodontis sheath is permeable to molecules of up to 70 kDa (98), and therefore might retain some host proteins after transfer to culture. However, several abundant serum proteins that we detected in bMf ESP are considerably larger than this (for example, ceruloplasmin and fibronectin) and thus must be either adsorbed onto the sheath surface or proteolytically processed prior to uptake. Hemopexin and ceruloplasmin have roles in heme and copper transport (99), respectively; hence, they may be exploited as a source of essential cofactors by the parasite.
(83) Several parasite-derived products were identified as secreted by iMf, including Ls110 [a protein localised in the uterine lumen and variably present on iMf, but absent from bMf (67)] and two possible proteoglycan core proteins. Accordingly, large glycoproteins (?200 kDa) have been described from B. malayi ESP (100). The closest C. elegans homologue of the perlecan-like proteoglycan, UNC-52, is a major component of the basement membrane of contractile tissues, including the pharynx and anus in developing embryos and subsequent stages (68). The L. sigmodontis iMf-derived CPG-like protein is predicted to have chitin-binding domains and may function in eggshell and sheath development. In C. elegans, CPGs form an inner layer that binds to the central chitinous layer of the eggshell, maintaining the perivitelline space around the embryo (101) forming a barrier to prevent polyspermy (102). In L. sigmodontis, chitin has been detected in the oocytes and zygotes, although it is absent from the iMf sheath (103). The degradation of chitin during Mf sheath development in utero may release the underlying CPG, which is highly soluble (101), into the surrounding milieu. The origin and roles two of the other novel proteins that were enriched in iMf ESP is less clear. The closest homologue in C. elegans of the PAN domain protein is SRAP-1, which is expressed in the hypodermis, central nervous system and vulva of developing larvae and is secreted onto the cuticle surface during moulting (104). In C. elegans, peroxidasin PXN-2 is located in the extracellular matrix and is required for late embryonic elongation, muscle attachment, and motoneuron axon guidance choice (105).
(84) The Abundant Uncharacterised Proteins Released by Gravid Adult Female Nematodes
(85) We identified four abundantly secreted or excreted proteins, found predominantly in GAF and iMf ESP, that had not been reported previously. Two have only marginal similarity to other proteins: nLs_03577, which displayed a significant match to a P-type ATPase (but lacked an ATPase domain), and nLs_08836, which showed some similarity to zonadhesin, a VWD protein located in the head of mammalian sperm (106). However, we note that nLs_08836 is not an orthologue of the C. elegans zonadhesin-domain protein, DEX-1 (107). The third novel protein, nLs_07321, is a vitellogenin. In C. elegans, vitellogenins are expressed exclusively in the intestine, where they bind cholesterol and transport it via the body cavity to the gonad (108).
(86) Subsequently, oocytes internalise the protein and its lipid cargo by receptor-mediated endocytosis and store it in yolk granules (108). Several vitellogenins have also been identified in ESP derived from adults of the oviparous gastrointestinal nematode, Heligmosomoides polygyrus (109). The fourth protein, the ShK domain protein nLs_04059, was distinct from other proteins containing this motif in nematodes, both in the number of domains and their specific sequence. Its relative abundance, distinctiveness and presence in all the filarial species surveyed suggest that it may be a viable vaccine candidate for both human filarial diseases and canine heartworm. Its role in vivo may be to interfere with the development of acquired immunity by inhibiting the Kv1 channels of memory T-cells in a manner analogous to the activity of cnidarian ShK toxins (75).
(87) The enigmatic TTL family has emerged as one of the most typical and widespread findings in ESP from both zoo- and phytoparasitic nematodes (110). In C. elegans, there are 63 transthyretin genes, many of which are secreted and apparently upregulated in response to infectious challenge, but only TTR-52 has been ascribed a physiological function [phagocytosis of apoptotic cells (111)]. In the phytoparasite Radopholus similis, Rs-ttl-2, which is closely similar to one of the most abundant L. sigmodontis TTL proteins (nLs_07576; found in ESP from all stages except vL3 in our study), was localised to the ventral nerve cord (112). A second R. similis TTL family member, Rs-ttl-1, was expressed only in the vulval region (112), and a homologue of this molecule (nLs_07332) was detected in iMf WBE only. Furthermore, in the ruminant parasite Ostertagia ostertagi, a TTL family (Oo-TTL-1) was a major component of ESP and could be immunolocalised to the pseudocoelomic fluid of adult worms (113). In our study, a L. sigmodontis homologue of Oo-TTL-1 (nLs_09750) was abundant in all ESP preparations except those of vL3.
(88) Uterine Fluid as a Source of Nematode and Endosymbiont Products
(89) Proteins excreted or secreted from filarial nematodes could be derived from a number of routes. In addition to oral secretions from the oesophageal glands and release of faecal material from the anus, nematodes also secrete material from the anterior sensory glands (amphids) (114) and the secretory pore, and may also void material from the genital openings during copulation and release of Mf. Proteins can also be released from the hypodermis through transcuticular secretion (115), especially during moulting, and exosome release may also be important (116). From our data, we suggest that vulval excretion is the main source of ESP proteins in GAF and PAF, and that the iMf are coated with proteins secreted by the uterine epithelium. This interpretation is supported not only by the abundance of MSPs and vitellogenin in GAF and PAF ESP, but by the presence of omega-class secreted GST isoforms exclusively in GAF ESP, which in O. volvulus are only produced by embryos at the morula stage (49). Similarly, ESP proteins in the male nematode probably originate primarily from seminal fluid. Immune sera from rodents infected with A. viteae react most strongly with male and female gonad tissues, including the fluid channels between developing embryos and on sperm in both the spermatheca and seminal vesicle (117).
(90) The role of the Wolbachia endosymbionts of filariae remains unclear: are they nutritional commensals, supporting the nematode through provision of energy or cofactors, or part of the immunological avoidance mechanisms of the parasite, or both (13)? It has been proposed that Wolbachia may be present in uterine fluid (118), inside degenerating embryos (119), or exit via the secretory pore (120). Additionally, they may secrete proteins into structures that lack bacterial cells, such as the cuticle (121). Wolbachia-derived proteins were present in very low amounts in B. malayi secreted products (17). We identified Wolbachia GroELS components in ESP of PAF and GAF, but not in other lifecycle stages. GroEL is the most abundant protein in Wolbachia (13, 15), and its detection in ESP may be through release of whole bacterial cells, for example in the female uterus from degenerating oocytes or embryos, or through secretion. GroEL, as a chaperonin, would be expected to be confined to the cytosol, although GroEL homologues have been reported to moonlight on the surface of some bacterial species (122). We also detected Wolbachia surface protein by surface labelling of adult L. sigmodontis, as has been reported in B. malayi (121). This protein is a putative ligand of Toll-like receptors 2 and 4 (119), and these findings support the hypothesis that Wolbachia modifies and perhaps misdirects the immune response to filariae (123). Whether Wolbachia GroEL also stimulates proinflammatory Toll-like receptors has not been evaluated, but a precedent exists in other bacteria (124), and antibodies against this protein are associated with pathology in LF (125).
(91) The L. sigmodontis Secretome and Vaccine Development for Filariases
(92) For several decades, vaccine development for human and veterinary filariases has focused on the L3 stage because irradiated L3 are highly efficacious at inducing protective immunity (23, 126) and strong anti-L3 immunity may block parasite establishment. Litomosoides is an excellent model for L3 vaccine research, as the L3 expresses a very similar repertoire of genes to the human and veterinary pathogens (127). Analyses of ESP from L3 of L. sigmodontis aid in defining a stereotypical secretomic profile for this stage. However, no defined parasite antigens (whether alone or in combination) have reproducibly attained an equivalent level of protection to irradiated L3 in any filarial system (7). Furthermore, since a single pair of adult nematodes can generate a patent infection, vaccines directed solely against L3 face a potentially insurmountable challenge.
(93) Targeting of Mf has the potential to block transmission, and in the case of onchocerciasis, to reduce disease pathology. Moreover, the Mf stage has been shown to be more vulnerable to protective immune responses than L3 in several vaccination trials (128-130). Vaccination with a combination of ALT-1 and CPI-2 delivered as a DNA vaccine reduced circulating Mf levels by up to 90% in L. sigmodontis. Importantly, this protection was only achieved if immunomodulatory domains of the antigens were ablated (by mutation or deletion of the coding sequence) and was maintained even when the adult nematode burden was not significantly reduced. This phenomenon was probably to be due to the immunomodulatory effects of the native (active) proteins, as transplantation of a single adult female worm is sufficient to prevent clearance of injected Mf in na?ve hosts (131). We suggest that it is likely that many of the other abundant molecules secreted by GAF may similarly have roles in facilitating Mf survival and could be targeted in an anti-fecundity vaccination strategy. Furthermore, the proteins identified by surface labelling of the GAF cuticle may also participate in generating a permissive environment (79, 80); thus, vaccination against these molecules, if sufficiently divergent from host homologues, might impede parasite establishment.
(94) 2.6 Conclusions
(95) We have shown that L. sigmodontis, especially the GAF stage, releases a remarkable diversity of proteins into the external milieu and the majority of these molecules are uncharacterised. Although many of these proteins may be involved in fundamental aspects of embryogenesis, a subset are likely to be active immunomodulatory agents that protect the nematodes (and especially the circulating Mf) from the host immune response. The abundant ESP protein, CPI, may represent an archetype for this dual functionality, as it plays fundamental roles in oogenesis and fertilisation not only in parasitic nematodes but also in C. elegans (132). This suggests that its immunomodulatory properties are an example of secondary adaptation to a radically different environment. Thus, the pharmacopeia released by GAF may provide the ideal set of molecule(s) to target for immunoprophylaxis and chemotherapy of filariases; moreover, it could provide new compounds to tackle proinflammatory and autoimmune diseases (22)
(96) TABLE-US-00001 TABLE 1 Proteins unique to the excretory-secretory products of individual lifecycle stages of Litomosoides sigmodontis Parasite stage ESP.sup.a Locus tag Annotation PAF nLs_02441 Epicuticlin nLs_07093 Nucleoredoxin-like protein-2 nLs_03968 Nematode cuticle collagen N-terminal domain containing protein nLs_06052 Translationally controlled tumor protein nLs_00526 Glutathione reductase AM nLs_07249 Glutaredoxin-like protein vL3 nLs_06400 Activation-associated secreted protein-1 nLs_09374 Abundant larval transcript-1 protein nLs_03087 Cathepsin L-like precursor nLs_06524 Calmodulin iMf nLs_02254 MSP domain-containing protein .sup.aData for excretory-secretory products unique to gravid adult females are not shown due to the large number of proteins (195) in this category (see Table 3). ESP, excretory-secretory products; PAF, pre-gravid adult female; AM, adult male; vL3, vector-derived third-stage larvae; iMf, immature microfilariae.
(97) TABLE-US-00002 TABLE 2 Putative surface-associated proteins detected in biotin-labelled adult worm whole body extracts that were absent from unlabelled controls Peptides Parasite used for Confidence Presence stage Treatment quantitation score Locus tag Annotation in ESP AM OG 7 857.06 nLs_06907 Adenylate No kinase isoenzyme 1 OG 4 417.27 nLs_09715 Major sperm Yes protein OG 2 186.68 nLs_01742 Filarial antigen No Av33 OG 2 297.13 nLs_08458 Filarial antigen Yes Ov16 SDS 2 308.12 nLs_07359 Calponin actin- No binding domain containing protein GAF OG 2 233.80 nLs_09095 Protein No disulphide isomerase SDS 2 86.22 nLs_08755 Leucine-rich No repeat family protein SDS 3 118.56 nLs_09715 Major sperm Yes protein SDS 2 99.43 nLs_02353 Complement No component 1, q subcomponent- binding, mitochondrial- like SDS 2 61.97 nLs_01344 Thioredoxin No peroxidase 1 SDS 2 108.87 nLs_07321 Vitellogenin Yes PBS 2 309.24 nLs_00851 DNA repair Yes protein Rad4- containing protein PBS 2 309.24 nLs_07061 Heat shock 70 kDa Yes protein PBS 2 309.24 nLs_09360 FMN-binding No domain protein PBS 3 463.79 nLs_01364 Transthyretin- Yes like protein, partial PBS 2 309.24 nLs_03263 Thioredoxin Yes domain- containing protein ESP, excretory-secretory products; AM, adult male; OG, octyl ?-D-glucopyranoside; GAF, gravid adult female; FMN, flavin mononucleotide.
(98) TABLE-US-00003 TABLE 3 Protein predictions from the WLS genome Normalised iBAQ values wLs acc Description GAF ESP GAF WBE PAF ES PAF WBE AM ESP wLs_340 co-chaperonin GroES 0.001135899 0.006317773 0.004180529 0.008366027 wLs_2830 molecular chaperone GroEL 0.000349505 0.001423155 0.000279378 0.013718142 wLs_3910 Outer surface protein Wsp 0.001218499 0.005119482 wLs_4630 hypothetical protein 0.000583035 0.001471599 ws_5240 hypothetical protein Wbm0603 0.000387749 0.001641741 wLs_1920 50S ribosomal protein L7/L12 0.000380102 wLs_9920 thioredoxin 0.000269733 wLs_930 Outer membrane protein, pal-like 0.000133493 0.000208532 wLs_4010 molecular chaperone DnaK 4.7978E?05 0.000214183 wLs_1320 hypothetical protein Wbm0010 3.3069E?05 0.000286269 wLs_5680 isoprenoid biosynthesis protein 9.91181E?06 0.000233577 with amidotransferase-like domain wLs_9580 elongation factor Tu 0.000538185 wLs_8490 hypothetical protein Wbm0655 0.000190391 wLs_5270 superoxide dismutase, SodA 0.000182807 wLs_1650 nucleoid DNA-binding protein 9.97899E?05 wLs_5000 heat shock protein 90 1.99158E?05 Normalised iBAQ values wLs acc Description AM WBE vL3 ESP vL3 WBE IMF ESP IMF WBE wLs_340 co-chaperonin GroES 0.000987231 0.004898528 0.007004044 wLs_2830 molecular chaperone GroEL 0.000305902 0.001431935 0.005708963 wLs_3910 Outer surface protein Wsp 0.000169721 0.003337764 0.002241786 wLs_4630 hypothetical protein 0.000104917 ws_5240 hypothetical protein Wbm0603 2.49886E?05 0.00097545 0.004069358 wLs_1920 50S ribosomal protein L7/L12 wLs_9920 thioredoxin wLs_930 Outer membrane protein, pal-like 3.65116E?05 0.000291644 wLs_4010 molecular chaperone DnaK 0.000236783 wLs_1320 hypothetical protein Wbm0010 1.90627E?06 1.60884E?05 0.000626075 wLs_5680 isoprenoid biosynthesis protein with amidotransferase-like domain wLs_9580 elongation factor Tu wLs_8490 hypothetical protein Wbm0655 wLs_5270 superoxide dismutase, SodA wLs_1650 nucleoid DNA-binding protein 0.00025721 wLs_5000 heat shock protein 90
(99) TABLE-US-00004 TABLE 4 Homologues of abundant Litomosoides sigmodontis excretory-secretory proteins identified by DELTA-BLAST (National Centre for Biotechnology Information) Identity Query cover Query Filter.sup.a Top annotated hit.sup.b [species] and accession Max. score (%) (%) E-value nLs_00113 AT PAN domain containing protein [Brugia malayi] XP_001900239.1 652 37 77 0.0 FE Flagellin [Salmonella enterica] WP_023208887.1 134 15 12 2.sup.?28 CO Protein SRAP-1, isoform a [C. elegans] NP_495398.3 114 26 56 3.sup.?24 nLs_01398 AT Protein UNC-52, isoform m [Caenorhabditis elegans] NP_001254444.1 1848 52 97 0.0 nLs_02001 AT KH domain-containing protein [Loa loa] EFO27012.2 513 75 58 2.sup.?174 FE Far upstream element-binding protein 1-like [Setaria italica] XP_004972470.1 97.4 18 65 5.sup.?18 CO RNA helicase GLH-2 [C. elegans] AAB03337.1 72.0 25 33 2.sup.?12 nLs_03577 AT Hypothetical protein Bm1_38495 [Brugia malayi] XP_001899152.1 128 60 100 2.sup.?31 FE Heavy metal translocating P-type ATPase [Dorea sp. 5-2] WP_016217557.1 63.5 29 74 2.sup.?08 CO Protein THOC-2 [C. elegans] NP_498392.2 42.0 27 55 1.sup.?03 nLs_04059 AT Hypothetical protein LOAG_17826 [Loa loa] EJD74931.1 262 51 87 7.sup.?80 FE A disintegrin and metalloproteinase with thrombospondin motifs 3-like 52.0 29 68 7.sup.?04 [Aplysia californica] XP_005091919.1 CO .sup.c nLs_05850 AT Hypothetical protein LOAG_04060 [Loa loa] XP_003139645.1 269 54 94 1.sup.?80 FE Chondroltin proteoglycan 2 [Ascaris suum] ERG86992.1 247 25 98 1.sup.?68 CO CBR-CPG-2 protein [C. briggsae] XP_002633936.1 218 20 93 8.sup.?63 nLs_08836 AT Apolipophorin [Ascaris suum] ERG86007.1 1535 42 99 0.0 FE Zonadhesin-like [Saccoglossus kowalevskii] XP_002738323.1 256 19 44 4.sup.?65 CO Protein VIT-4 [C. elegans] NP_508612.1 97.1 21 8 9.sup.?20 nLs_01626 AT Animal heme peroxidase [Loa loa] XP_003141164.1 1367 84 98 0.0 FE Peroxidasin-like protein [Ascaris suum] ERG87495.1 1308 72 98 0.0 CO CBR-PXN-2 protein [C. briggsae] XP_002644069.1 1093 47 99 0.0 AT, all taxa; FE, Filarloidea excluded; CO, Caenorhabditis only. .sup.aFilters were applied only where the top hit was to taxa other than Caenorhabditis spp. .sup.bOnly annotated hits are shown for non-filarial proteins. .sup.cThe only hits were to hypothetical proteins containing ShK domains.
(100) TABLE-US-00005 TABLE 5 Homologues of abundant Litomosoides sigmodontis excretory-secretory proteins identified by PSI-BLAST (Phyre.sup.2) Normalised UniRef50 identity Query Top annotated hit [species] ID Bits [%] E-value nLs_02001 Transcription elongation factor SPT5 P0CR70 135 14.9 3.sup.?30 [Crytococcus neoformans var. neoformans serotype D] nLs_04059 Sortilin-related receptor [Homo sapiens] Q92673 210 10.0 1.sup.?52 nLs_08836 SCO-spondin [Danio rerio] B3LF39 351 10.4 1.sup.?94
(101) TABLE-US-00006 Quantifiable proteins present in the excretory-secretory products of blood-derived microfilariae Peptides used for Confidence Normalised Accession | Gene name quantification score Description (species) iBAQ Q91X72|HEMO_MOUSE 7 898.19 Hemopexin (Mus musculus) 1.44.sup.?03 Q8VCM7|FIBG_MOUSE 8 1040.62 Fibrinogen ? chain (Mus musculus) 1.15.sup.?01 Q8K0E8|FIBB_MOUSE 9 1594.25 Fibrinogen ? chain (Mus musculus) 9.56.sup.?02 O35090|ALBU_MERUN 29 5013.66 Serum albumin (Meriones unguiculatus) 6.09.sup.?02 P70274||SEPP1_MOUSE 3 203.64 Selenoprotein P (Mus musculus) 5.76.sup.?02 Q61147|CERU_MOUSE 12 2215.19 Ceruloplasmin (Mus musculus) 5.56.sup.?02 P29788|VTNC_MOUSE 6 1035.62 Vitronectin (Mus musculus) 5.43.sup.?02 Q61702|ITIH1_MOUSE 7 1236.34 Inter-?-trypsin inhibitor heavy chain H1 (Mus musculus) 5.32.sup.?02 P11276|FINC_MOUSE 49 8184.33 Fibronectin (Mus musculus) 3.80.sup.?02 P01027|CO3_MOUSE 28 3368.39 Complement C3 (Mus musculus) 3.63.sup.?02 P01942|HBA_MOUSE 2 149.76 Hemoglobin subunit ? (Mus musculus) 3.60.sup.?02 P97515|FETUA_MERUN 6 665.57 ?-2-HS-glycoprotein (Meriones unguiculatus) 3.24.sup.?02 P20918|PLMN_MOUSE 13 2062.8 Plasminogen (Mus musculus) 3.13.sup.?02 P13020|GEL5_MOUSE 5 1587.22 Gelsolin (Mus musculus) 2.83.sup.?02 Q62577|AMBP_MERUN 6 1120.89 Protein AMBP (Meriones unguiculatus) 2.33.sup.?02 P01029|CO4B_MOUSE 11 1814.14 Complement C4-B (Mus musculus) 1.52.sup.?02 P05367|SAA2_MOUSE 4 820.91 Serum amyloid A-2 protein (Mus musculus) 1.34.sup.?02 Q61703|ITIH2_MOUSE 7 1223.42 Inter-?-trypsin inhibitor heavy chain H2 (Mus musculus) 9.20.sup.?03 nLs.2.1.2.t10069-RA 4 264.21 Transthyretin-like protein, partial (Litomosoides 9.11.sup.?03 sigmodontis) P04186|CFAB_MOUSE 6 361.74 Complement factor B (Mus musculus) 8.08.sup.?03 P52430|PON1_MOUSE 2 166.19 Serum paraoxonase/arylesterase 1 (Mus musculus) 7.36.sup.?03 Q02105|C1QC_MOUSE 2 292.88 Complement C1q subcomponent subunit C (Mus musculus) 6.31.sup.?03 P06909|CFAH_MOUSE 2 156.16 Complement factor H (Mus musculus) 5.81.sup.?03 P05017|GF1_MOUSE 2 458.52 Insulin-like growth factor I (Mus musculus) 5.69.sup.?03 P14105|C1QB_MOUSE 2 62.86 Complement C1q subcomponent subunit B (Mus musculus) 3.94.sup.?03 A6X935|ITIH4_MOUSE 4 301.36 Inter-?-trypsin inhibitor, heavy chain 4 (Mus musculus) 3.94.sup.?03 E7D4P4|E7D4P4_MERUN 9 1172.7 Apolipoprotein E (Meriones unguiculatus) 3.78.sup.?03 P97298|PEDF_MOUSE 5 420.63 Pigment epithelium-derived factor (Mus musculus) 3.31.sup.?03 P47878|IBP3_MOUSE 5 520.36 Insulin-like growth factor-binding protein 3 (Mus musculus) 3.10.sup.?03 P46412|GPX3_MOUSE 3 275.46 Glutathione peroxidase 3 (Mus musculus) 2.99.sup.?03 Q88H35|CO8B_MOUSE 4 586.84 Complement component C8 ? chain (Mus musculus) 2.99.sup.?03 Q64118|A1AT_MERUN 3 183.98 ?-1-antitrypsin (Meriones unguiculatus) 2.89.sup.?03 Q06890|CLUS_MOUSE 5 427.41 Clusterin (Mus musculus) 1.91.sup.?03 P70389|ALS_MOUSE 3 471.58 Insulin-like growth factor-binding protein complex acid 1.88.sup.?03 labile subunit (Mus musculus) P35441|TSP1_MOUSE 8 853.9 Thrombospondin-1 (Mus musculus) 1.81.sup.?03 P68033|ACTC_MOUSE 2 978.66 Actin, ? cardiac muscle 1 (Mus musculus) 1.64.sup.?03 Q00724|RET4_MOUSE 4 335.66 Retinol-binding protein 4 (Mus musculus) 1.63.sup.?03 P26262|KLKB1_MOUSE 5 625.92 Plasma kallikrein (Mus musculus) 1.52.sup.?03 Q61704|ITIH3_MOUSE 4 422.55 Inter-?-trypsin inhibitor heavy chain H3 (Mus musculus) 1.52.sup.?03 P19221|THRB_MOUSE 7 587.54 Prothrombin (Mus musculus) 1.45.sup.?03 P33434|MMP2_MOUSE 3 283.7 72 kDa type IV collagenase (Mus musculus) 1.42.sup.?03 Q9JHH6|CBPB2_MOUSE 3 367.6 Carboxypeptidase B2 (Mus musculus) 1.38.sup.?03 P32261|ANT3_MOUSE 2 306.33 Antithrombin-III (Mus musculus) 1.30.sup.?03 nLs.2.1.2.t03443-RA 3 366.35 Hypothetical protein, Bm1_50630 homolog (Litomosoides 1.18.sup.?03 sigmodontis) nLs.2.1.2.t01366-RA 2 269.78 Transthyretin-like protein, partial (Litomosoides 9.56.sup.?04 sigmodontis) P11680|PROP_MOUSE 2 36.41 Properdin (Mus musculus) 9.27.sup.?04 Q61645|HPT_MOUSE 3 236.91 Haptoglobin (Mus musculus) 8.65.sup.?04 Q9JM99|PRG4_MOUSE 5 683.96 Proteoglycan 4 (Mus musculus) 8.57.sup.?04 Q92111|TRFE_MOUSE 4 728.55 Serotransferrin (Mus musculus) 8.09.sup.?04 P28798|GRN_MOUSE 2 300.7 Granulins (Mus musculus) 7.86.sup.?04 P26928|HGFL_MOUSE 4 375.93 Hepatocyte growth factor-like protein (Mus musculus) 7.43.sup.?04 Q9UN5|CBPN_MOUSE 3 219.53 Carboxypeptidase N catalytic chain (Mus musculus) 7.01.sup.?04 P47879|IBP4_MOUSE 2 211.08 Insulin-like growth factor-binding protein 4 (Mus musculus) 6.96.sup.?04 Q07968|F13B_MOUSE 3 410.68 Coagulation factor XIII B chain (Mus musculus) 6.89.sup.?04 Q9DBD0|ICA_MOUSE 6 787.37 Inhibitor of carbonic anhydrase (Mus musculus) 5.99.sup.?04 P97290|IC1_MOUSE 4 387.11 Plasma protease C1 inhibitor (Mus musculus) 5.94.sup.?04 Q8K182|CO8A_MOUSE 2 332.91 Complement component C8 ? chain (Mus musculus) 5.78.sup.?04 Q70362|PHLD_MOUSE 2 100.85 Phosphatidylinositol-glycan-specific phospholipase D 4.92.sup.?04 (Mus musculus) P01872|IGHM_MOUSE 2 174.92 Ig ? chain C region secreted from (Mus musculus) 4.48.sup.?04 P06684|COS_MOUSE 2 338.69 Complement CS (Mus musculus) 4.07.sup.?04 Q8K0D2|HABP2_MOUSE 2 60.74 Hyaluronan-binding protein 2 (Mus musculus) 3.53.sup.?04 nLs.2.1.2.t01870-RA 2 196.87 ML domain-containing protein (Litomosoides sigmodontis) 3.44.sup.?04 nLs.2.1.2.t01365-RA 2 188.68 Transthyretin-like protein, partial (Litomosoides 2.74.sup.?04 sigmodontis) P28665|MUG1_MOUSE 3 312.94 Murinoglobulin-1 (Mus musculus) 2.05.sup.?04 Q08879|FBLN1_MOUSE 2 305.68 Fibulin-1 (Mus musculus) 1.90.sup.?04 Q8CG16|C1RA_MOUSE 2 102.86 Complement C1r-A subcomponent (Mus musculus) 1.04.sup.?04 IBAQ, intensity-based absolute quantification; AMBP, ?-1-microglobulin/bikunin precursor.
(102) TABLE-US-00007 TABLE 7 Putative surface-associated proteins exhibiting >50-fold enrichment in biotin-labelled adult worm whole body extracts relative to unlabelled controls Parasite Peptides used for Confidence Presence stage Treatment quantification score Fold-difference Locus tag Annotation in ESP AM SDS 4 316.19 1,769.5 nLs_09715 Major sperm protein Yes SDS 2 249.88 341.7 nLs_01747 Filarial antigen RAL-2 Yes SDS 6 873.95 62.2 nLs_06907 Adenylate kinase isoenzyme 1 No PBS 4 172.54 50.6 nLs_09625 Transthyretin-like protein 5 Yes GAF Urea 2 306.56 430.9 nLs_02969 Cysteine protease inhibitor-2 Yes Urea 2 180.49 149.4 nLs_08458 Filarial antigen Ov16 Yes Urea 2 302.26 65.2 nLs_09625 Transthyretin-like protein 5 Yes OG 2 233.80 60,617.8 nLs_09890 Purine nucleoside phosphorylase Yes OG 2 183.22 336.5 nLs_00852 Proliferating cell nuclear antigen domain protein No OG 2 224.62 271.9 nLs_04749 60S ribosomal protein L18 No OG 2 191.67 168.3 nLs_01364 Transthyretin-like protein, partial Yes OG 3 194.25 156.3 nLs_02023 Tetratricopeptide-repeat domain protein Yes OG 2 159.32 139.9 nLs_02001 KH domain-containing protein Yes OG 2 188.56 118.1 nLs_08084 Type I inositol-trisphosphate 5-phosphatase Yes OG 3 367.83 79.9 nLs_02969 Cysteine protease inhibitor-2 Yes OG 3 367.26 66.6 nLs_02463 FKBP-type peptidyl-prolyl cis-trans isomerase Yes OG 3 220.00 65.6 nLs_00523 KH domain containing protein Yes OG 3 376.34 59.0 wLs_3910 Wolbachia surface protein No OG 2 258.28 52.2 nLs_05241 Tetratricopeptide-repeat domain protein No SDS 2 50.67 1,059.7 nLs_07759 Cyclophilin Ovcyp-2 homologue Yes SDS 6 306.80 328.9 nLs_08458 Filarial antigen Ov16 Yes SDS 7 488.42 304.1 nLs_01747 Filarial antigen RAL-2 Yes SDS 4 156.74 262.8 nLs_05279 HSP20/?-crystallin family protein No SDS 2 144.16 242.7 nLs_08696 Lysozyme protein 8, partial Yes SDS 2 87.95 235.6 nLs_09890 Purine nucleoside phosphorylase Yes SDS 7 432.37 216.4 nLs_09625 Transthyretin-like protein 5 Yes SDS 2 82.06 202.7 nLs_2001 KH domain-containing protein Yes SDS 6 332.26 193.2 nLs_08148 Papilin Yes SDS 2 122.31 162.4 nLs_06907 Adenylate kinase isoenzyme 1 Yes SDS 2 44.96 162.1 nLs_00117 L-lactate dehydrogenase Yes SDS 3 144.30 148.4 nLs_05914 Pyruvate dehydrogenase E1 component, ?- Yes subunit SDS 2 77.92 115.1 nLs_09750 Transthyretin-like protein 45 Yes SDS 7 329.76 86.5 nLs_03034 p27 heat shock protein homologue Yes SDS 5 166.70 82.3 nLs_08836 von Willebrand factor type-d domain protein Yes SDS 3 138.70 71.7 nLs_03328 Myosin No SDS 2 89.16 67.5 nLs_01364 Transthyretin-like protein, partial Yes SDS 6 560.89 52.8 nLs_08415 Enolase Yes PBS 5 631.96 111.4 nLs_02378 Aldo/keto reductase family protein No PBS 2 309.24 104.0 nLs_03070 Atypical RIO/RIO2 protein kinase Yes PBS 3 372.59 93.5 nLs_00473 Aldehyde dehydrogenase 11 Yes PBS 6 1058.57 89.2 nLs_06488 Acid phosphatase Yes PBS 9 1570.87 69.9 nLs_01747 Filarial antigen RAL-2 Yes PBS 4 647.70 62.5 nLs_03174 Nematode secreted protein 22U Yes ESP, excretory-secretory products; AM, adult male; OG, octyl ?-D-glucopyranoside; GAF, gravid adult female; FKBP, FK506-binding protein; HSP, heat-shock protein
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(104) TABLE-US-00008 Sequenceinformationandcomparisons
(105) Illustrated above provides a sequence alignment comparison between ShK domain-containing proteins nLs.2.1.2.t04059-RA (from Litomosoides sigmodontis) of SEQ ID NO.1 and nDi.2.2.2.t03402-RA (from Dirofilaria immitis) of SEQ ID NO.2.
(106) While overall identity between the two sequences is only 52.8%, it can be seen that the identity shared between the ShK domains (highlighted) of these two filarial nematode proteins is considerably higher.