Background to the Invention The present invention relates to assays for detecting whether or not an animal is or has been infected with Trypanosoma vivax. In addition the invention relates to specific antigens for use in the assays of the invention, as well a specific types of assays and assay devices which employ said specific antigens. Introduction

Trypanosoma vivax is a protozoan parasite of the genus trypansomatidae spread primarily by biting insects. Together with T brucei and T congolense, it is a causative agent of African Animal Trypanosomosis (AAT) in cattle. T vivax causes a severe version of AAT, often characterised by hemorraghic fever as well as the more typical weight loss, fatigue and anaemia .

As T vivax does not require midgut gestation within the vector it can be transmitted mechanically by body fluid contamination and hematophagous flies. This has allowed the spread of the disease in South America, an area previously free from T vivax. Over eleven million cattle are estimated to be at risk in this region in addition to the 46 million cattle at risk in sub-Saharan Africa.

Diagnostics are limited for this parasite, relying principally on microscopy, specific antibody detection using whole parasite lysates as target antigen [1] or PCR that requires specialised equipment [2, 3]. In most cases, farmers rely upon symptom- based diagnosis, which is complicated by the numerous other diseases with similar manifestations in the endemic regions. The present invention is based on the development of a low-cost pen-side diagnostic test for T vivax infection in cattle using lateral flow text (LFT) technology. The inventors developed the approach of identifying parasite antigens selectively recognised by cattle infection sera by proteomics, followed by recombinant protein expression in E. coli and antigen assessment by ELISA to select an antigen for LFT prototyping. Summary of Invention

The inventors of the present invention have developed a kit for diagnosis of Trypanosoma vivax infection, which exhibits excellent specificity and sensitivity and is capable of discriminating infection with Trypanosoma vivax and other Trypanosome species, and/or identifying mixed Trypanosoma vivax and other Trypanosome species infection, with ease and economically. Further, the inventors developed a diagnostic kit including purified antigens from Trypanosoma vivax for use in detecting whether or not an animal has been infected with Trypanosoma vivax.

In a first aspect there is provided a method of detecting whether or not an animal has been infected with Trypanosoma vivax, the method comprising: a) contacting a fluid sample from the animal with an isolated antigen comprising a sequence selected from SEQ ID No: 1 and 2 and antigenic fragments thereof; and

b) detecting any complexes formed between any antibodies present in the fluid sample from the animal and the isolated antigen, wherein detection of any complexes indicates that the animal is and/or has been infected with Trypanosoma vivax.

The term "isolated" is understood to mean that the antigen has been removed or otherwise purified, such that it is substantially free from other Trypanosoma vivax material and/or proteins, or material and/or proteins from a host harbouring nucleic acid which is capable of expressing the antigen.

In one embodiment of the method of the present invention, the detection of any complexes being formed between the purified antigen and any antibodies present in the fluid sample, is carried out using a detection agent which is coloured and can optionally be detected, when present in sufficient quantities, by the naked eye.

In a further aspect, there is provided a purified antigen comprising a sequence selected from SEQ ID NO: 1 and 2 or antigenic fragment thereof for use in a method of detecting whether or not an animal is and/or has been infected with Trypanosoma vivax. At least a portion of the purified antigen may comprise, consist essentially of, or consist of the sequence identified in SEQ ID No: 1 or 2, or antigenic fragment thereof. Thus, the purified antigen may be identical to the identified sequences or antigenic fragment thereof, or may comprise a sequence or portion of sequence which is at least 95%, 98%, 99%, 99.5% identical to the identified sequences or antigenic fragment thereof. Optionally, the purified antigen may be larger in size and may comprise additional sequence at the 5' and/or 3' ends of the identified sequences. The identified sequences include 3 amino acids at the 5' end which remain following cleavage of a TEV sequence. The sequences of the present invention may exclude these three amino acids.

In one embodiment at least a portion of the purified antigen sequence comprises, consists essentially of, or consists of the sequence identified in SEQ ID No: 1 , or variants as described above, for use in a method of detection as described herein.

The antigens of the present invention were selected on the basis of (a) being identified by proteomics as belonging to a small group of antigens present in a total detergent lysate of T. vivax parasites that bind specifically and selectively to the immunoglobulin G (IgG) fraction of calves experimentally infected with T.vivax and (b) being similar to invariant surface glycoproteins previously known in the art. Such proteins comprise a signal peptide, an extracellular domain, a transmembrane domain and internal domain. The invention is directed in particular to the antigenic properties of the extracellular domain and SEQ ID No: 1 and 2 correspond to extracellular domain sequences of 2 proteins which were identified as having an infection:control intensity response ratio >10.

The antigens of the present invention have been shown not to cross-react with serum obtained from animals infected with T. congolense. Thus, the antigens of the present invention may be considered as being specific for T. vivax and hence can be used for specifically identifying T. vivax as the causative pathogen for any animal identified as suffering from AAT. The antigens of the present invention may be used alone in any method or device etc according to the present invention, or may be used in conjunction with another antigen or antigens, such as GM6 antigen which is/are intended to simply identify if an animal is infected with any trypanosome species.

The methods of the present invention are conventionally known as immunoassays. However, the present invention is not limited to a particular type of immunoassay and the immunoassay may take a variety of forms. Thus, certain embodiments of the present invention include an Enzyme-Linked immunosorbent Assay (EL!SA), dipstick immunoassay, sandwich assay, competitive assay, immunochromatographic assay radioimmunoassay, flow-through immunoassay, etc. In certain embodiments, the present invention is in the form of a immunochromatographic assay and may be a lateral flow type assay known in the art.

Immunochromatographic assays (iCA), also referred to as a rapid test due to its convenient and rapid features, is based on a principle in which an antibody in a fluid sample reacts with a tracer antigen bound to a coloured particle and then combines with a capture antigen or antibody located on the inner surface of pores of a nitrocellulose membrane to form a coloured band while transferring through the pores by a capillary phenomenon, thereby identifying positivity or negativity with the naked eye.

As mentioned in certain embodiments, the ICA takes the form of a Lateral flow assay. Lateral flow assays are typically based on the use of a series of capillary beds, such as pieces of porous paper or sintered polymer. Each of these elements has the capacity to transport a fluid (e.g., blood or urine) spontaneously. The first element (the sample pad) acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid migrates to the second element (conjugate pad) in which there is stored a so-called conjugate, a dried format of bio-active particles (see below) in a salt-sugar matrix that contains everything to guarantee an optimized chemical reaction between the antigen and an antibody specifically reactive with the antigen that has been immobilized on the particle's surface. The conjugate pad may further comprise control particles, which are designed simply to confirm to a user that a particular assay has worked appropriately. While the sample fluid dissolves the salt- sugar matrix, it also dissolves the particles and in one combined transport action the sample and conjugate mix while flowing through the porous structure. In this way, any antibodies present in the fluid sample bind to the particles while migrating further through the third capillary bed. This material has one or more areas, or stripes where a third molecule has been immobilized. By the time the sample-conjugate mix reaches these areas/stripes, antibodies have been bound to the particles and the third 'capture' molecule binds the complex. After a while, when more and more fluid has passed the stripes, particles accumulate and the stripe-area changes color. Typically there are at least two stripes: one (the control) that captures any particle and thereby shows that reaction conditions and technology worked appropriately, the second contains a specific capture molecule and only captures those particles onto which a desired antibody molecule has been complexed. After passing these reaction zones the fluid enters the final porous material, the wick, which simply acts as a waste container. Lateral Flow assay can operate as either competitive or sandwich assays. Thus in a further aspect the present invention further relates to a lateral flow assay device for detecting the antibodies to Trypanosoma vivax present in a fluid test sample, said lateral flow assay device comprising a porous membrane in liquid communication with a conjugate pad and a wicking pad: said conjugate pad located upstream from a detection zone, said conjugate pad having antigen-conjugates comprising an isolated antigen comprising a sequence selected from SEQ ID No: 1 and 2 or antigenic fragment thereof immobilised on a nanopartic!e and; said detection zone having an immobilized first capture reagent, said first capture reagent being configured to bind to at least a portion of any antibody-antigen conjugates formed, so as to generate a detection signal; and a control zone located downstream from said detection zone, wherein a second capture reagent is immobilized within said control zone, said second capture reagent being configured to bind control antigen-conjugate or antibody-conjugate complexes to generate a second detection signal. Typically the first and second detection signals are visualised as a colour. Conveniently the second signal may be lower than the first signal, that is the first signal is visualised more intensely as compared to the second signal

In one embodiment the control takes the form of nanoparticies which have been modified to have an antibody conjugated to the surface of the nanoparficie. These nanoparticies may be captured by antibodies which are capable of binding the antibody which is conjugated to the surface of the nanopartide. The nanoparticies may comprise a chicken IgY antibody conjugated to the surface of the nanoparticies and this may be captured by, for example, a goat, or other mammal anti-chicken IgY antibody. In the methods and kits or devices for use in the present invention, an antigen- antibody complex is detected by a colour particle coupling method, in which examples of coloured particles include a colloidal gold or silver particle, colored glass, or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Conveniently the particles may be gold-nanoparticles and the signal is a visible colour signal which is observed through accumulation of the gold nanoparticies at the sites of the respective capture agents

Many types and styles of lateral flow assays are known in the art and may be envisaged by the skilled reader. General methods and devices are described for example in US6485982 and Geertruida et al (Antibodies Applications and New Developments, 2012, 175-183) to which the skilled reader is directed and the contents of which are hereby incorporated herein by way of reference.

The methods of the present invention may be used to detect any antibodies which are capable of binding to the purified antigen(s) in any sample which is suspected of, or is capable of, harbouring such antibodies. Typically, samples for use in the methods of the invention are ex vivo samples taken from the body of an animal to be tested. Such samples may be tested either on site or taken remotely and transferred to a testing facility for assay.

In a preferred embodiment, the sample is taken from a mammal, such as a pig or a wild or domesticated ruminant (e.g. cattle, buffalo, sheep, goat, camel and deer). The skilled person will appreciate that any suitable type of sample from such animals may be used. For example, suitable samples include vesicular epithelium, vesicular fluid, blood samples, probang samples (collection of fluid from the throat), cardiac muscle (such as whole heart from young pigs or lambs), semen, saliva and milk. In some circumstances, it may be beneficial to culture cells from such a sample, and to perform the diagnostic methods of the invention on the cultured cells. This latter approach can be particularly advantageous if optimal assay sensitivity is desired. However, more often or not the sample is a sample of blood or serum. The antigens of the present invention may be purified by any known method. For example, the antigens may be extracted from Trypanosoma vivax obtained from an infected animal, or grown in vitro. Alternatively the antigens may be obtained by recombinant means e.g. from a culture of cells which are transformed to express the antigen. The sequences of the nucleic acid molecules encoding the antigen can be obtained through cloning techniques known in the art. The nucleotide sequences can be amplified from Trypanosoma vivax nucleic acid using PCR and related techniques known to the skilled addressee, or synthesised de novo and cloned into a suitable expression vector. If appropriate the nucleic acid can be modified to facilitate its expression in a particular host organism, by altering one or more codons of the nucleotide sequence. The sequence may also be modified to include a cleavable 5' or 3' tag sequence, such as a His tag sequence known in the art, which is designed to facilitate purification of the antigen. The sequence may be modified to include a tobacco etch virus (TEV) protease cleavage site between an N-terminal tag sequence, such as a hexahistidine affinity tag sequence. The expression vector which harbours the nucleotide sequences capable of expressing the antigen of the present invention is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention. Such methods of expressing proteins in recombinant cells lines are widely known in the art (for example, see Sambrook & Russell, 2000, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor, New York).

In addition to using the naturally occurring sequence, it will be appreciated that variants of such antigens may be used. By "a variant" we include a polypeptide comprising the amino acid sequence of the naturally occurring antigen, wherein there have been amino acid insertions, deletions or substitutions, either conservative or non-conservative, such that the changes do not substantially reduce the ability of the variant to bind to said antibodies, compared to the binding ability of the naturally occurring antigen. For example, the variant may have increased affinity for said antibodies compared to that of the naturally occurring 'parent' antigen. Alternatively, the variant may have increased stability, e.g. tolerance to proteases, pH and/or thermal stability. Such variant antigens may be made using methods of protein engineering and site-directed mutagenesis commonly known in the art (for example, see Sambrook & Russell, 2000, supra).

The present invention also provides vectors (such as expression vectors), host cells (such as a bacterial (e.g. E.coli) host cells) for expressing the antigens of the invention and methods for making such antigens comprising culturing a host cell or vector according to the invention.

The present invention will now be further described by way of example and with reference to the Figures which show: Figure 1 shows a bar graph of ELISA data of recombinant proteins TvY486_0045500 and TvY486_0019690 against pooled T. vivax sera from animals pre-infection (day- 7) and post-infection (day+28). Figure 2. shows examples of the TvY486_0045500 prototype lateral flow tests developed with control and infection sera, as indicated. Top band control line, bottom band TvY486_0045500 diagnostic line.

Materials and Methods

Sera

All sera were provided by GALVmed, Edinburgh, UK. The sera used for the antigen identification by immunoprecipitation and proteomics were from four animals obtained commercially in Burkina Faso and treated prophylactically for 7 vivax infections before experimental infection with 7 vivax. Further sera, including 113 samples for ELISA and LFT blind testing, were obtained from Burkina Faso, Mozambique and ClinVet in South Africa, the latter were all from cattle raised under fly-nets. Of the 1 13 sera for blind testing: The Burkina Faso samples (21 sera) were from 4 calves and consisted of 4 pre-infection and 17 7. vivax post-infection sera. The Mozambique samples (20 sera) were from 2 calves and consisted of 20 7 vivax post-infection sera. The ClinVet samples (72 sera) were from 31 calves and consisted of 27 pre-infection sera and 32 7 vivax post-infection sera and 13 7 congolense post-infection sera. Strains used to infect cattle (one isolate per calf) were: In Mozambique, Y486 and IL700. In Burkina Faso, Sokoroni 19, Napie22, Komborodougou and Gondo Bengaly. At ClinVet , ILRAD560.

IgG purification from pre- and post-infection sera

Sera were collected in Burkina Faso from four calves before and 28 days after experimental infection with 7 vivax. Aliquots (250 μΙ) of the pre- and post-infection sera were pooled and IgG fractions were purified on protein-G Sepharose, as previously described [5, 7]. Purified IgG was coupled to CNBr-activated Sepharose 4B (GE Healthcare) at 4mg IgG per milliliter of packed gel, according to the manufacturer's instructions.

Preparation T. vivax parasite lysate Three BALB/c mice were injected with one stabilate of 7. vivax ILRAD V34. After five days, infected mouse blood was harvested with citrate anticoagulant, adjusted to 5x10 ⁴ parasites per ml with phosphate-buffered saline (PBS) and aliquots of 0.2 ml were injected into the peritoneal cavity of 45 NMR1 mice. The mouse blood was harvested after 7 days and the parasites were purified by centrifugation, to yield a buffy coat enriched in trypanosomes, followed by DE52 ion exchange chromatography to remove white blood cells and residual erythrocytes, as described in [5, 7]. The purified trypanosomes were dissolved to 1 x 10 ⁹ cells. mL ^"1 in 50 mM sodium phosphate buffer, pH 7.2, 2% n-octyl^-D-glucopyranoside (nOG) detergent, 1 mM phenylmethylsulfonyl fluride (PMSF), 0.1 mM /V-p-tosyl-L-lysine chloromethyl ketone (TLCK), I mg.mL ^"1 leupeptin and aprotinin and 1x Roche protease cocktail minus EDTA. The lysate was incubated for 30 min on ice and then centrifuged at 100,000 g for i h at 4°C.

Immunoprecipitation Aliquots of T. vivax detergent lysate (10 ml) were incubated with 0.75 ml packed volume of each of the 4 mg.ml ^"1 Sepharose-lgG (infection and non-infection/control) gels, rotating for 3 h at 4°C. The gels were then packed into disposable 10 ml columns and washed with 10 ml of 10 mM Na2P04, pH 7.2, 200mM NaCI, 1 % nOG, followed by 10 ml of 5 mM Na2P04 pH 7.2, 1 % nOG. The trypanosome proteins were eluted with 500 μΙ of 50 mM sodium citrate, pH 2.8, 1 % nOG into tubes containing 100 μΙ of 1 M Tris pH 8.5 for neutralization. The eluates were further concentrated to 270 μΙ using a centrifugal concentrator (Millipore, 0.5 ml capacity with 10 kDa MW cut off membrane). The concentrates containing the trypanosome proteins were then transferred to low binding Eppendorf tubes and the proteins precipitated by adding 1 ml ice-cold ethanol and incubation for 24 h at -20°C.

Proteomic protein identification

Following ethanol precipitation, the proteins eluted from the post-infection IgG and pre-infection IgG columns were dissolved in SDS sample buffer, reduced with DTT and run on a precast 4-12% Bis-Tris gradient SDS-PAGE (Invitrogen) using the MES running system. The gel was stained with colloidal Coomassie blue and equivalent regions of the infection and control lanes were cut out, reduced and alkylated with iodoacetamide and digested in-gel with trypsin. The tryptic peptides were analysed by LC-MS/MS on a Thermo Orbitrap Velos system and MaxQuant 1.4 software was used to match peptides to the predicted trypanosome protein databases (GeneDB). Where possible, annotated gene names, or the names of homologues identified using BLAST [8] or the protein fold/family identified with Pfam [9], were used (Table 1). The program MaxQuant 1.4 was also used to obtain relative intensity data of the peptides recovered from the post-infection and pre-infection (control) IgG columns.

Table 1 : Quantitative proteomics results

The antigens are ordered by their infection: control LC-MS/MS intensity ratios and colour coded according to their absolute LC-MS/MS intensities: black bold >1000; black > 500; grey >10

Control:inf

Protein ID ected ratio intensity 10 ⁶ Putative name/protein family

TvY486_0806350 74.33 37.734

TvY486_0045500 32.15 2875.40 ISG

REV TvY486 10137

80 31.28 93.62 Arm

TvY486_0019690 29.46 1030.10 ISG

TvY486_0806260 24.79 83.81 guanine deaminase

TvY486_0040570 23.77 30.10

TvY486_0304300 16.15 74.97 5-histidyl sulfoxide synthase

TvY486 0704280

;TvY486_0704300 14.02 52.13 adomet mtase, fge sulfatase

TvY486 0040800

;TvY486_0013960

;TvY486_0040500

;TvY486 0027530

;TvY486 0010260

;TvY486_0027540 13.75 42.68 VSG

TvY486_0040090

;TvY486 0034340

;TvY486_0022920

;TvY486_0034330

;TvY486_0002180

;TvY486 0045260

;TvY486 0000TvY48

6 0019690 13.04 32.17 TvY486_l 003730 12.32 610.93 proteasome activator protein pa26

TvY486_l 106220 9.71 60.63 ribosome binding ubiquitin-conjugating

TvY486_l 103390 9.55 49.96 Enzyme

TvY486_0906400 9.54 132.31 Syntaxin

TvY486_0009580

;TvY486_0018880 9.29 65.29 VSG

TvY486_0023330 9.25 76.89

TvY486_0603490 8.25 150.23 kinase/hydrolase

TvY486_0007180 7.59 99.96

TvY486_0702240 6.68 42.57 tyrosyl-tRNA synthetase,

TvY486_0041380

;TvY486_0038160 6.56 12.50

TvY486_0040150 6.47 16.71

TvY486_1106950 6.46 852.61 M17 aminopeptidase

_0019690 6.32 9.46

TvY486_1109080

;TvY486_1109070 5.14 410.26 ribonuclease Il-like protein

Cloning

A DNA construct encoding residues 42-363 of TvY486_0019690 was amplified from T. vivax (strain ILRAD V34) genomic DNA using the forward and reverse primers 5' -CATATGGAGAATGAGATTGCTCGGG-3' and 5'-

GGATCCAATGCTGAGTTTGCTATTGTTAGCTGA-3', respectively, where the underlined bases are the Nde1 and BamH1 cloning restriction sites. The gene TvY486_0045500, which is very similar in sequence to TvY486_0019690, could not be selectively amplified and a construct encoding residues 40-363 was instead synthesised by GenScript and optimised to avoid rare codon combinations in E. coli, unfavourable mRNA structures for protein expression and cis elements. The gene was obtained in a pUC vector with restriction sites (Nde1 and BamH1) in place for downstream cloning. Both constructs were ligated into pCR2.1-TOPO using the TOPO ® TA Cloning ® Kit (Invitrogen) and then inserted into a pET15b-derived plasmid (Novagen) modified to include a tobacco etch virus (TEV) protease cleavage site between the N-terminal hexahistidine affinity tag and the protein sequence of interest. Recombinant gene expression of the TvY486_0045500 construct was achieved with E. coli BL21- CodonPlus (DE3) RIPL cells (Stratagene) in autoinduction medium [10] containing 50 μg ml_ ^"1 ampicillin and 12 μg ml_ ^"1 chloramphenicol. The construct for TvY486_0019690 was expressed in BL21 (DE3) Gold cells (Stratagene) in autoinduction medium containing containing 50 μg ml_ ^"1 ampicillian. Cells were cultured for 24 h at 22°C before harvesting by centrifugation (3,500 x g, 30 min, 4 °C) the bacterial pellet was resuspended in buffer A (50 mM Tris-HCI, pH 7.5, 250 mM NaCI) containing an EDTA-free protease inhibitor cocktail (Roche).

Protein purification of recombinant proteins Purification was achieved using the methods described in [1 1]. Briefly, E. coli cells were mechanically lysed in the presence of DNAse than clarified by centrifugation (4 °C , 40 min, 30,000 g). The proteins were captured using a 5 ml immobilised metal affinity chromatography (IMAC) (HisTrap GE Healthcare) and eluted with an imidazole gradient. Affinity tags were removed, via proteolytic cleavage (1 mg TEV protease per 20 mg protein, 4 °C, 16 h) and the protein dialysed into buffer A (50 mM Tris-HCI, pH 7.5, 250 mM NaCI). The protease, uncleaved protein and affinity tag contaminants were removed with a further subtractive IMAC step. Final purification was achieved by size exclusion chromatography (Superdex 200 26/60) eluted with buffer A. Finally, proteins were dialysed into PBS and adjusted to at least 1 mg.ml ^"1 using 10 kDa cut-off centrifugal concentrators. All proteins were >95% pure, as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and Coomassie blue staining. Enzyme linked immunosorbent assay (ELISA)

White polystyrene Costar untreated 96 well plates were coated with 50 μΙ per well of target protein at a concentration of 2μg ml_ ^"1 in plating buffer (0.05 M NaHCOs , pH 9.6). Then blocked with 200 μΙ of PBS containing 5% bovine serum albumin (BSA) and 0.1 % Tween-20 to block non-specific sites overnight at 4°C. Calf sera were diluted 1 :2500 in PBS containing 5% BSA, 0.1 % Tween- 20 and transferred in triplicate by a liquid handling device (Bio-Tek, Precision) to the ELISA plates and incubated for 1 h at room temperature. After 1 h the diluted sera were aspirated and the wells were washed with PBS containing 0.1 % BSA with the liquid handling device. This wash cycle was repeated 5 times. Biotinylated anti-bovine-lgG (Jackson labs) was added at dilution of 1 :4000 (50 μΙ per well) and incubated for 1 h. Excess anti-bovine-lgG antibody was washed away (as described before) and 50 μΙ per well of ExtrAvidin-Horse Radish Peroxidase (HRP) at a dilution of 1 :4000 was added to the plates and incubated for 1 h. The solution was aspirated and the wash steps were repeated. Finally, chemiluminescent super signal Femto substrate (Pierce) diluted 1 :5 (i.e. , 0.5 ml solution A, 0.5 ml solution B with 4 ml PBS) was applied to the wells at 50 μΙ per well and plates were read using an Envision plate reader within 5 minutes of addition of the substrate.

Prototype lateral flow test manufacture and use

Purified recombinant TvY486_0045500 residues 40-363 (7.5 mg) were provided to BBI-Solutions (Dundee, UK) an immunoassay development and manufacturing company that has completed more than 250 lateral flow projects over the last 25 years, with manufacturing sites in Europe, USA and South Africa, and 2400 prototype LFT devices were manufactured. Triplicate aliquots of 5 μΙ of calf sera diluted with 15 μΙ of PBS were added to the LFTs followed by an 80 μΙ of chase-buffer (PBS containing 0.05% Tween 20). Tests were discarded if upper control line was not clearly visible. After 30 min, scoring of the test bands was performed by visual comparison of freshly completed tests with a scoring card [12] and the consensus score from three devices for each serum was recorded. After reading, the nitrocellulose test strips were taken out their cases for photography.

Results

Selection of candidate diagnostic antigens for T. vivax An immunoprecipitation experiment was carried out to identify candidate diagnostic antigens for T. vivax. Pooled pre-infection (day -7) and post-infection (day +28) calf sera from four animals from Burkina Faso were used to generate IgG antibody columns. Detergent lysate of bloodstream form T. vivax cells was generated from parasites recovered from mice infected with T. vivax strain ILRAD V34. Identical amounts of parasite detergent lysate were mixed with the pre-infection and post-infection Sepharose-lgG beads and proteins bound to the washed beads were eluted with a buffer containing high salt and low pH to break antibody-antigen interactions. The eluted samples from the pre-infection and post-infection columns were concentrated and subjected to SDS-PAGE gels for antigen separation. The pre-infection and post-infection eluates were run on separate SDS-PAGE gels to reduce potential antigen cross-contamination. The Coomassie blue stained gel lanes were cut into ten segments and each subjected to in-gel reduction and alkylation and trypsin digestion. The peptides from each gel slice were separately analysed by LC-MS/MS and the data concatenated for the pre-infection and post-infection eluate samples, respectively. These concatenated data sets were used to search the predicted protein database for T. vivax (Y486) using MaxQuant 1.4.

Many proteins (>1300) were identified in the combined data from each of the Sepharose-lgG eluates. However, the protein identification lists were sorted to select for proteins found either uniquely in the post-infection Sepharose-lgG eluate or that were >10 fold enriched in the post-infection Sepharose-lgG column eluate, as judged using the label-free quantification function in MaxQuant 1.4 [13], (Table 1). Of the twenty-three proteins unique to the post -infection IgG eluate, all had low LC-MS/MS intensities suggesting that the immune response to these antigens, while specific, was low [4, 5]. Eleven proteins had infection : control intensity ratios >10. Two of these stood out as possible immunodiagnostic antigens, TvY486_0045500 and TvY486_0019690. These are two closely related proteins sharing 91 % and 80% amino acid sequence identity and similarity, respectively. These proteins have a typical ISG domain structure, consisting of an N-terminal signal peptide, an ISG domain, a transmembrane domain and a small intracellular domain [14]. We chose to investigate these antigens because ISGs have previously proved to be good diagnostics antigens for T. brucei, T. congolonse and T. evansi [4-6].

Expression of and evaluation of recombinant T. vivax ISG antigens.

Similar segments of TvY486_0045500 (amino acid residues 40-363) and TvY486_0019690 (amino acid residues 42-363), avoiding the N-terminal signal peptides and C-terminal transmembrane domains, were cloned and expressed with cleavable hexa-histidine tags in E. coli. Following nickel affinity purification and proteolytic cleavage of the hexa-histidine tags, monodisperse forms of both proteins collected from a subsequent gel-filtration purification step (and separated from aggregated material appearing at the void volume) with a final yield of 0.6 mg.L ^"1 for TvY486_0019690 and 4 mg.L ^"1 for TvY486_0045500. The predicted amino acid sequences of the two recombinant proteins are identified in the attached sequence listing

The two purified recombinant proteins were used to coat ELISA plates and tested with the sera of 14 calves that had been collected pre- (day -7) and post- (day +28) experimental infection with T. vivax at the ClinVet site. These data (Figure 1) indicated that both recombinant TvY486_0045500 and TvY486_0019690 coated ELISA plates could discriminate infected from uninfected sera. TvY486_0045500 (residues 40-363) was selected for the manufacture of a prototype lateral flow device, because of its relative ease of protein expression, and was supplied to BBI Solutions (Dundee, UK)

Both the TvY486_0045500 and TvY486_0019690 coated ELISA plates, and the TvY486_0045500 LFT prototype, were tested in triplicate with 1 13 randomised cattle sera provided by GALVmed. After data collection, the sera codes were broken and the data are collated. Sera were classified as being either from uninfected animals or from animals that had been exposed to experimental T. vivax infection. Based on this classification, there were 69 infection (positive) and 44 control (negative) sera. Cut-off values for maximum positive/negative discrimination were determined (600,000 and 1 , 100,000 units for the TvY486_0045500 and TvY486_0019690 ELISA plates, respectively, and >=2 units for the LFT) and, using these cut-offs, the sensitivity and specificity data were determined (Table 2). Representative LFT data are shown in (Figure 2) and the results of all 113 tests are shown in.

Table 2. Sensitivity and specificity data for T. vivax ISG ELISAs and prototype LFT.

Discussion

In this study, we used quantitative proteomics to identify candidate diagnostic antigens, i.e., those proteins in whole T. vivax detergent lysate that bound selectively (>10 fold more) to the immobilised IgG from calves experimentally infected for 28 days with T. vivax versus immobilised IgG from the same animals collected 7 days prior to experimental infection. In total, we found 34 candidate protein groups but rejected most of these based on the low intensities of their combined peptide ions in the LC-MS/MS analyses (we interpret low peptide intensities as an indication that only a small proportion of infection- specific IgG is directed towards their parent antigens [4, 5]). The two proteins that produced the most intense peptides, and significant (around 30-fold) enrichment, were two related ISGs. We therefore focussed on these proteins and made recombinant versions in E. coli that lack the predicted cleavable N- terminal signal peptides and the predicted C-terminal transmembrane and short cytoplasmic domains. Both recombinant proteins were immunoreactive with pooled T. vivax infection sera and both antigens were subsequently tested in ELISA format against 1 13 randomised calf sera and found to have identical overall performance in terms of sensitivity and specificity (Table 1). Further, the Pearson coefficient between the two ELISA data sets was 0.9876, indicating that there is nothing to choose between the two antigens with respect to immunodiagnostic potential. The antigen that expressed most efficiently in E. coli (TvY486_0045500) was used to make a prototype LFT device and this was also tested blind with the same 1 13 randomised sera. Upon breaking the code, it became clear that we could obtain maximum discrimination between T. vivax positive and negative sera by setting the visual score cut-off at >=2 (Figure 2) and, using these criteria, the sensitivity and specificity of the prototype LFT was similar to that of the ELISA (Table 2). These are promising performance results considering that the prototype LFT was not optimised with respect to antigen density on the test strip, antigen-gold conjugation or chase-buffer composition which, individually or collectively, should allow a reduction in background (false positive) scores of 0.5 and 1 for some sera.

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