TpUB05, a Homologue of the Immunodominant Plasmodium falciparum Protein UB05, Is a Marker of Protective Immune Responses in Cattle Experimentally Vaccinated against East Coast Fever

Introduction East Coast fever, a devastating disease of cattle, can be controlled partially by vaccination with live T. parva sporozoites. The antigens responsible for conferring immunity are not fully characterized. Recently it was shown that the P. falciparum immunodominant protein UB05 is highly conserved in T. parva, the causative agent of East Coast fever. The aim of the present investigation was to determine the role of the homologue TpUB05 in protective immunity to East Coast fever. Methods The cloning, sequencing and expression of TpUB05 were done according to standard protocols. Bioinformatics analysis of TpUB05 gene was carried out using algorithms found in the public domain. Polyclonal antiserum against recombinant TpUB05 were raised in rabbits and used for further analysis by Western blotting, ELISA, immunolocalization and in vitro infection neutralization assay. The ability of recombinant TpUB05 (r-TpUB05) to stimulate bovine PBMCs ex-vivo to produce IFN-γ or to proliferate was tested using ELISpot and [3H]-thymidine incorporation assays, respectively. Results All the 20 cattle immunised by the infection and treatment method (ITM) developed significantly higher levels of TpUB05 specific antibodies (p<0.0001) compared to the non-vaccinated ones. Similarly, r-TpUB05 highly stimulated bovine PMBCs from 8/12 (67%) of ITM-immunized cattle tested to produce IFN-γ and proliferate (p< 0.029) as compared to the 04 naїve cattle included as controls. Polyclonal TpUB05 antiserum raised against r-TpUB05 also marginally inhibited infection (p < 0.046) of bovine PBMCs by T. parva sporozoites. In further experiments RT-PCR showed that the TpUB05 gene is expressed by the parasite. This was confirmed by immunolocalization studies which revealed TpUB05 expression by schizonts and piroplasms. Bioinformatics analysis also revealed that this antigen possesses two transmembrane domains, a N-glycosylation site and several O-glycosylation sites. Conclusion It was concluded that TpUB05 is a potential marker of protective immunity in ECF worth investigating further.


Introduction
East Coast fever (ECF), a tick-borne disease caused by Theileria parva results in severe economic loss to livestock production, and kills 1 million animals each year [1]. The disease is present in 11 countries in eastern, central and southern Africa [2] and is spreading due to a wider distribution of the tick vector and uncontrolled livestock movement [3]. A well established, safe and affordable vaccine that can confer full and long lasting protection against all parasite populations is still awaited. A live parasite-based vaccine, which is used to immunize the animals by an infection and treatment method (ITM), is available [4,5] but has serious disadvantages. Its laborious production in addition to the requirement of a liquid nitrogen cold chain for delivery and oxytetracycline co-treatment makes it expensive, whereas animals vaccinated by the ITM protocol remain life-long carriers of the parasite, which poses risk for spread of the disease [6].
Current control methods for East Coast fever include the use of acaricides and immunization by the ITM [7]. The strong immune response engendered by using the ITM vaccine indicates the feasibility of a successful recombinant vaccine against ECF. This implies there are parasite antigens that can induce protection, which could be included in the development of subunit vaccines [8]. Recent advances made in the development of a subunit vaccine against T. parva based on the p67 sporozoite surface antigen, with induction of immunity to ECF in 50% of vaccinated cattle, shows that more protective antigens need to be identified [9] as it has been suggested for malaria [10].
A study on the apicomplexan parasite that causes malaria in humans, Plasmodium falciparum, identified an immunodominant antigen, UB05 (GenBank Accession Number DQ235690; [11]), which was shown to be a potential marker of protective immunity to malaria [11,12].
The principle of the conservation of gene function suggests that most orthologous gene products play similar roles in closely related organisms [13]. Conceivably, T. parva orthologue of UB05 could play a similar role in immunity to ECF. The aim of the present investigation was to clone the T. parva homologue of UB05 designated as TpUB05 (GenBank accession number: KF875450) and to investigate its role in the host-parasite interplay in ECF.

Ethical Statement
The study reported here was carried out in strict accordance with the recommendations in the standard operating procedures of the International Livestock Research Institute Institutional Animal Care and Use Committee (ILRI IACUC). The ILRI' Experimental Animal Request Form and Protocol for blood collection and polyclonal antibody production was approved by the ILRI IACUC (IACUC ref no. 2013.05).

Molecular Biology reagents and bacterial strains
The plasmid vectors pGEM-T Easy (Promega) and pET32a+ (Novagen) were used for cloning and protein over-expression. Escherichia coli strains JM109 (Promega) and Rosetta (DE3) pLysS (Novagen) were used for bacterial overexpresson. Ni-NTA agarose was from Qiagen GmbH. Restriction enzymes and HisProbe-HRP were purchased from Thermo Fisher Scientific, Inc. (USA). The reagents for PCR amplification, IPTG, agarose and reagents for protein purification were purchased from Applied Biosciences (USA) and Sigma (USA). Oligonucleotides were purchased from Bioneer Corporation (Korea). Animals used in the study were raised at the ILRI Farm Tables 1, 2 and 3.

Construction of overexpressing Theileria parva TpUB05 clone
Total RNA isolated from in vitro culture of the T. parva-infected bovine lymphocyte cell line F100 TpM 3087 [14] was used to generate a TpUB05 cDNA clone. Briefly, RT-PCR was performed with 5 μg of total RNA in 50 μl of reaction using the OneStep RT-PCR (Qiagen) Kit as recommended by the manufacturer. The TpUB05 ORF 5' primer sequence was 5'-GGGATC CGCAGATCTCACCAAACGCAAAC-3' and the 3'primer was 5'-CAAGCTTTTAGTTTGATC TCCTGAAAC-3'. The first 7 nucleotides being extrinsic to the TpUB05 gene, introducing the underlined unique BamHI and HindIII sites at the 5' and 3' ends of the amplified DNA fragment, respectively, and creating the deletion of the ATG initiation codon while conserving the stop codon. The PCR product was purified from agarose gel and cloned into pGEM-T Easy vector using JM109 (Promega). The cloned fragment was excised with BamHI and HindIII from the recombinant plasmid and directionally subcloned into pET32a+ expression plasmid vector. The pET32a+ expression vector possesses six consecutive histidine residues (6xHis tag) for efficient purification of recombinant proteins. The resulting recombinant plasmid was sequenced to verify the TpUB05 sequence and then transformed into E. coli Rosetta (DE3)pLysS for overexpression and affinity purification.
Tp5 (GenBank Acc. No. XP_765334), a T. parva CD8 + T-cell antigen encoded by a gene containing 4 exons, was used as an internal positive control as well for positive amplification (it should be noted that Tp5 is a different protein from TpUB05, which is studied in this paper). Tp5 forward fusion primer 5'-CGTATCGCCTCCCTCGCGCCATCAGACGAGTGCGTGTATGC TCGGTAATGGCAG-3' with the Tp5 specific sequenced underlined and reverse primer 5'-TTATAAATCATCGATATCGAAATC-3' were used to amplify Tp5 DNA from the third to the fourth exons for 35 PCR cycles.

Cattle sera
The cow antisera used in this study were from animals of the Friesian and Ayshire breed Tables 1 and 2 reared in the ILRI experimental farm.

Production of polyclonal anti-sera in rabbits
All animal experimentation procedures were reviewed and approved by the International Livestock Research Institute (ILRI) Institutional Animal Care and Use Committee. Two New Zealand rabbits Table 3 were used for the production of polyclonal antibodies against recombinant TpUB05. The rabbits were fed on pellets (Unga Feeds Limited) and water ad libitum. Polyclonal antibody production was done as earlier described [16] with some modifications. Briefly, rabbits were inoculated intramuscularly with 75 μg of recombinant TpUB05 antigen. Rabbits were boosted on days 14 and 28 by intramuscular injection with the same amount of antigen in Titermax adjuvant (Sigma-Aldrich). After the antibody titre reached 1:1000 as tested by ELISA, the final boost of neat antigen was given intraveneously. Then rabbits were sacrificed and blood was collected and used to prepare polyclonal antibodies. The blood was incubated at room temperature for 60 min to allow clotting and then centrifuged at 1500 x g for 10min. The serum supernatant was transferred into a fresh tube and heated in a water bath at 56°C for 60 min to inactivate serum complement that may affect neutralizing antibody assays. Inactivated serum was mixed briefly and subjected to ammonium sulfate precipitation. In ammonium sulfate precipitation, saturated ammonium sulfate was prepared by dissolving 1000 gram ammonium sulfate in 1000ml of water (distilled). This was added slowly to the serum sample while stirring, to a final saturation of 50%, for 30 minutes. The homogenate was span at 4000 rpm for 30 minutes. The pellet was resuspended in ammonium sulfate to 50%, original volume, and span at 4000 rpm/30mins. The supernatant was discarded and the pellet resuspended in PBS as required (small volume). The polyclonal antibody preparation was dialysed extensively three times in PBS. Aliquots were stored at-20°C until use.

Overexpression and purification of recombinant TpUB05 protein
Overexpression and purification of recombinant TpUB05 (r-TpUB05) was performed as earlier described [17] with the following modifications. A colony of Rosetta (DE3)pLysS strain transformed with TpUB05/pET32a+ plasmid was grown overnight at 37°C in 50 ml LB supplemented with 100 μg/ml Carbenicillin and 34 μg/ml chloramphenicol. The following day, 500 ml LB containing the respective antibiotics was inoculated with the overnight culture and let to grow to an OD of 1.0 at 600 nm. IPTG was then added to a final concentration of 2 mM. Culture was allowed to proceed for 4 hours and harvested by centrifugation in a Beckmann Table Top centrifuge at 2100 x g for 10 min. The pellet was resuspended in 40 ml of denaturing lysis buffer (8M urea, 0.1 M NaH 2 PO 4, 20 mM Tris-HCl, 30 mM imidazole, pH 8.0, 0.01 M phenylmethylsulfonyl fluoride (PMSF)). Cells were disrupted by sonication (60W, 70% pulser, 15 min). Insoluble bacterial components were removed by centrifugation (2100 x g, 10 min).
Batch purification was carried out by mixing the clarified supernatant with 1.5 ml of equilibrated Ni-NTA resin slurry for 30 min on a rotator (Denley Spiramix 5). The bound resins were collected by centrifugation (400 x g, 5 min) and washed five times with 20 ml Wash buffer (40 mM Imidazole, 100 mM NaH 2 PO 4, 300 mM NaCl, 10% glycerol, 0.05% NaN 3 , 0.1% Triton-X-100). The recombinant protein was eluted with 0.5 M imidazole in PBS. The concentration and purity of the protein was measured using a spectrophotometer. 50 mM of L-arginine, 50 mM L-glutamic acid and 0.1 M PMSF were added for solubility and stability to the purified protein samples and dialyzed against PBS and stored at-20°C until use.

ELISA for the antibody responses to TpUB05
The presence of antibodies to TpUB05 in bovine serum was determined by the enzyme linked immunosorbent assay (ELISA) as decribed [17] with modifications. Microtitre plates (Polysorp Nunc, Roskilde, Denmark) were coated with 150 μμl per well of optimized concentration of 10 μg/ml antigen in PBS at 37°C, 2 hours. They were washed thrice with Wash buffer (0.05% Tween 20, 0.25% BSA, 0.02% NaN 3, in PBS) followed by blocking with 0.2% casein in PBS containing 0.05% Tween 20. The blocking buffer was removed and antisera added at 1:200 dilution in Wash buffer containing 1% skimmed milk. It was incubated for 30 min at 37°C in an Insel incubator/shaker (Insel Instruments # 1S89). Plates were then washed three times with Wash buffer. 150 μl of anti-bovine IgG-HRP conjugate (Sigma) diluted at 1: 20,000 in Wash buffer containing 1% skimmed milk was added to each well and incubated for 30 min at 37°C with gentle shaking and then washed as above. The reaction was revealed using the substrate 2,2'azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt (ABTS). In addition to the positive (Polymorphic Immunodominant Molecule) and negative controls, the fusion partner (Tag only) of the expression vector was also included as another negative control. Optical densities were read at 405nm on a microplate reader (Labsystems Multiskan MCC 340, Helsinki, Finland). A serum is considered positive if the mean OD 405 of the test wells is greater than the baseline; the baseline being mean control OD 405 plus three standard deviation (mean control OD 405 + 3 SD).

Isolation of bovine PBMC, CD4 + and CD8 + T cells
Blood (30 ml) was collected from the jugular vein into a syringe containing an equal volume of Alsever's solution and mixed gently. Then the blood was layered onto Ficoll Paque solution (Pharmacia LKB, Sweden) at a ratio of 3:2 and centrifuged at 900 x g for 30 min at room temperature without brakes. Using a sterile pipette the PBMCs were aspirated from the interface, transfered to a sterile tube and topped up with PBS solution. This was centrifuged at 500 x g for 10 min at room temperature. The pelleted PBMCs were washed three times in Alsever's solution by centrifugation at 500 x g at room temperature, to remove platelets. 5 ml of Red Blood Cell lysis solution per 10 ml of original blood volume was added to the pelleted PBMCs and left for 5 min at room temperature. 25 ml of PBS was added and the solution centrifuged at 300 x g for 15 min to pellet the PBMCs. CD4+ and CD8+ T cells were purified from peripheral blood mononuclear cells by magnetic cell sorting as described previously [18]. Briefly, CD4+ or CD8+ cells were labeled with MACS Microbeads carrying either anti-CD4 antibody or anti-CD8 antibody, respectively. The sample was then placed in a MACS column and then in a MACS Separator. The flow-through fraction was collected as the negative fraction depleted of the labeled cells. The column is removed from the separator and the retained cells are eluted as the enriched, positively selected cell fraction.

Bovine ELISpot assay
The enzyme-linked immunospot (ELISpot) assay was performed with isolated PBMCs, purified CD4+ T-cells or purified CD8+ T-cells using a bovine ELISpot assay kit (MABTECH, ELI-SPOT) and according to the manufacturer's instructions. Briefly, plates were coated overnight with the coating antibody provided. 250,000 PBMCs/well and TpUB05 at 10 μg/ml were added to each well on the plates and incubated for 20 hours at 37°C, 5% CO 2 . The plate was washed and the biotinylated detection antibody added, followed by streptavidin-HRP conjugate. The spots were revealed using 3, 3', 5, 5'-Tetramethylbenzidine (TMB) substrate. Concanavalin A (Con A) was used as the positive control antigen at 5 μg/ml and no antigen was added in the negative control wells. The developed spots were counted using an ELISpot reader. A reaction was considered positive if the stimulation index (SI) was greater than two. The SI was obtained by dividing the mean test spot counts by the mean negative control spot counts.

T-cell proliferation assay
Isolated PBMCs were adjusted to 2.5 x10 6 cells/ml of complete RPMI. 100 μl of the cell suspension was added into each well in triplicates in a 96-well plate (Corning, Costar). To each well was added 100 μl complete RPMI containing TpUB05 antigen at 10 μg/ml (test wells), Con A at 5 μg/ml (positive control wells) or no antigen (for negative control wells). The plate was incubated at 37°C in a humified incubator with 5% CO 2 . The cells were pulsed with [3H]-thymidine on day 4. The plate was incubated again overnight for isotope uptake. Using a Harvester, cells were harvested and the filter matts dried and read on a counter. A reaction was considered positive if its stimulation index (SI) was greater than two.

Immunolocalization assay
The generated anti-TpUB05 antiserum was used to determine which of the stages of T parva Mugaga (piroplasm, schizont and sporozoite) did express TpUB05. The cells were spun unto a glass slide using Cytospin (Cytospin 2, Shandon Southern Products Ltd., Cheshire, U.K.), airdried, and fixed in 4% paraformaldehyde for 30 min at room temperature. The fixed cells were permeabilized using 0.5% Triton X-100 in PBS for 15 min at room temperature. Free sites were blocked with blocking solution (3% BSA, 10% fetal calf serum, in TTBS (0.3 M NaCl, 20 mM TrisÁCl. pH8.0, 0.1% Tween 20. 0.01% NaN 3 )) for 30 min at room temperature. Antisera (neat or at 1:100 dilution) or monoclonal antibody (1:100 dilution) were added to the slide and incubated for 2 hours at room temperature. Washing was done three times for 15 min each, in TTBS. Secondary antibody ((anti-rabbit FITC (Sigma, F-0382) or anti-mouse FITC (Sigma, F-0257)) was added at room temperature for 2 hours in the dark and washed twice with TTBS.
Hoechst staining (1:200) was performed for 30 min at room temperature and washed once with TTBS for 5 min. Mounting oil (10mg ρ-phenylenediamine, 1ml PBS, 9ml glycerol) was added to the slide and covered with a cover slip. Nail polish was used to seal cover slip. Slides were observed under a fluorescent microscope (Axio Imager D1 Upright Microscope). Negative control consisted of staining schizonts with pre-immunization serum while positive control involved the staining of schizonts with monoclonal anti-schizont antibody.

In vitro neutralization of T. parva sporozoites infection
The antibody neutralization of sporozoite infection tests were performed following some modifications of the method earlier described [19]. Briefly, 5 x 10 4 sporozoites in RPMI 1640 medium with 10% fetal bovine serum and 1:2 dilution of anti-TpUB05 antisera were mixed in each well of a 24-well microtiter plate. After a 30 min incubation at 37°C, 5 x 10 6 bovine PBMCs from two ITM-immunized and two naive cattle that were randomly selected, were added to each well and the plates were kept at 37°C for 72 hours. Giemsa-stained cytospin smears prepared from each well were examined for the presence of schizonts. Four hundred cells were counted and the percentage bearing schizonts was determined.

Bioinformatics analyses
TpUB05 recombinant gene construct was sequenced by pyrosequencing using the 454 GS FLX Sequencer (Roche) and analyzed with VecSreen (National Centre for Biotechnology Information; NCBI) to remove vector sequences. The TpUB05 sequence was then analysed with BLAST of the GenBank non-redundant nucleotide and protein databases (NCBI) using the family of algorithms with default parameters [20]. The open reading frame was determined by open reading frame (ORF)-Finder and conserved-domain search was performed using NCBI conserved domain search [21]. The significant match with TP04_0076 was then selected for further analyses. Predictions of the physicochemical and structural properties of the derived protein sequence were performed using the protein analysis software available at Expasy.

B-cell and T-cell epitope prediction
The antibody epitopes predictions were made on 30 th November 2014 using the IEDB analysis resource Consensus tool [22][23][24].
Prediction of T-cell epitopes on TpUB05 that binds MHC Class I haplotypes was done using NetMHCplan version 2.8 on the 30 th November 2014 [25]. Strong binding peptides have a rank threshold below 0.5 while weak binding peptides have a rank threshold between 0.6 and 2.0.

Statistical analysis
The non parametric analysis of variance (ANOVA), Mann-Whitney, Kruskal-Wallis and Friedman tests performed for the evaluation of fixed effects on different traits by using SAS software, Release 8.2 (SAS Institute Inc., Cary, NC) and SPSS Statistics 17.0 Release 17.0.0 (Aug, 23 2008) respectively. A 95% confidence limit was used. A p-value less than 0.05 was considered statistically significant.

Molecular Cloning and expression of TpUB05
Using TpUB05 specific primers, a 291 bp DNA fragment encoding a 96 amino acid long protein of 11kDa was amplified from schizont derived RNA Fig 1A. Subsequent cloning and expression in pET32a+ vector yielded a 28 KDa recombinant fusion protein as shown by SDS-PAGE. The increase in size was fully accounted for by the presence of 6xHis tag, a S Tag domain and a 109 amino acid thioredoxin fusion protein partner.

Expression and immunolocalization of TpUB05
Semi-quantitative PCR experiments demonstrated that TpUB05 was transcribed in all the developmental stages of T. parva examined. When compared with the house keeping gene GAPDH, the relative abundance of TpUB05 RNA increases as the sporozoite form of the parasites differentiate into the main pathogenic stage of ECF, the schizonts Fig 1B. In addition, the TpUB05 expression profile differed from that of the control antigen p104, that show the highest and lowest levels of transcription in the sporozoite and schizont forms of the parasite, respectively.
To examine the spatial distribution and determine what parasite stage(s) expressed TpUB05, fixed parasites and T. parva-infected cells were stained with antibodies against TpUB05. The antibodies specifically stained the schizonts inside the infected cells, with no preferred localizsation in any specific parasite compartment Fig 2A. The antibodies also detected TpUB05 in the piroplasms of T. parva Muguga Fig 2B but the intensity of the stain was stronger in the schizonts than in the piroplasms. However, there was no obvious expression of TpUB05 by the sporozoites under the conditions employed. The positive control also showed a strong reaction. This result is consistent with the transcription data that showed high, medium

Western blot analysis of recombinant TpUB05 protein
To determine the antibody responses to recombinant TpUB05 (r-TpUB05) western blot analyses were conducted with sera from ITM-immunized and control cattle. It can be seen from Fig 1C, lane 2 that the r-TpUB05 protein was recognized by rabbit anti-r-TpUB05 antiserum. Antiserum from an ITM-immunized cattle also recognized r-TpUB05 Fig 1C, lane 3. But antiserum from an un-immunized cattle (infected but not treated) did not recognize r-TpUB05 Fig 1C,   TpUB05, a Marker of Protective Immunity to East Coast Fever

Antibody responses to TpUB05
Using ELISA, the immunogenicity of antibodies from ITM-immunized cattle against TpUB05 was compared with that of sera from infected/but not treated animals. An antiserum was considered positive if the mean OD 405 of the test wells was greater than the baseline (baseline = mean control OD 405 + 3 Standard Deviations). The result showed that 25 of the 28 (89.3%) cattle studied possess antibodies against TpUB05. All the twenty ITM-immunized cattle recognized TpUB05 and produced significantly higher anti-TpUB05 antibody titers compared to the infected/but not treated cattle (p<0.0001) Fig 3. The pre-immunisation serum from one naїve cattle included in this study did not have antibodies against TpUB05 and served as the negative control. The fusion partner (Tag only) gave OD values that were comparable to the naїve serum (data not shown). This study strongly indicated that TpUB05 is an immunogenic antigen in cattle and probably induced specific antibodies in the ITM vaccinated cattle.

PBMCs from immunized animals recognized TpUB05
Having probed the antibody response in the section 3.4 above, the next series of experiments was designed to probe the T-cell response to TpUB05. The T-cell response to TpUB05 was determined using ELISpot assay. Isolated PBMCs from cattle were stimulated ex-vivo with r-TpUB05 for the production of interferon-gamma (IFN-γ) as measured by bovine ELISpot assay. Eight out of the twelve (67%) ITM-immunized animals of different major histocompatibility complex (MHC) class I genotypes tested produced IFN-γ in a manner significantly higher than the naїve animals studied (p < 0.0332) Fig 4. Most responders were cattle expressing MHC class I of the alleles A18 (5 animals), A15 (3 animals) and A12 (2 animals). However, one of the two naive cattle was shown to release significant amounts of IFN-γ. CD8+ T cell target antigen (Tp1) which is well characterizsed to be the target of CD8 T-cell responses of T. parva immune cattle and which is restricted by the MHC class I allele A18 was included in the study as an internal positive control Fig 4, Tp1 yellow bar.
T-cell proliferation induced ex-vivo by r-TpUB05 was also measured by [3H]-thymidine incorporation. The PBMCs from ITM-immunized cattle proliferated significantly (p <0.029) as compared to the naїve cattle with mean SI values of 3.28 and 0.98, respectively. When the different subsets of T-cells (CD4+ and CD8+) were sorted from three animals (BB007, BH055, BA219) and stimulated with r-TpUB05, CD4+ cells of BH055 and B219 produced IFN-γ in a manner that was significantly higher (p = 0.013) compared to their CD8+ T- ELISpot assay. Using bovine ELISpot assay, PBMCs from twelve ITM immunized cattle and two naïve cattle were stimulated with TpUB05 for the production of interferon-Gamma. The SI of the ITM immunized cattle were significantly higher than those of the naive cattle (p<0.0332). Eight out of the 12 ITM immunized cattle recognized TpUB05 and produce IFN-γ. One of the two naïve cattle also recognized TpUB05. The well-characterized potential subunit vaccine candidate was included in the assay as an internal positive control. An animal is considered positive if two conditions are met; (i) sample SI is greater than the mean+3SD of the control, (ii) the SI of the sample is at least twice that of the control. Experiment was run twice in triplicates.

Infection inhibition assays using anti-TpUB05 antibodies
We used the neutralization of in vitro infectivity assay to test the ability of polyclonal anti-TpUB05 antiserum to block the infection of bovine PBMCs by T. parva Muguga sporozoites 3087. The efficiency of the sporozoite infectivity varied among the 4 animals used. The lowest being with BH053 and the highest was observed with BH033 Fig 7. Antibody against TpUB05 was able to reduce by 30%, 50% and 70% the sporozoites infection of PBMCs from the cattle BH058, BH053 and BJ059, respectively Fig 7. In contrast, the anti-TpUB05 antiserum did not significantly block the infection of PBMCs from cattle BH033 Fig 7. The reason for this inactivity was not explored further. Generally speaking, the blocking of PBMCs infection by anti-TpUB05 antiserum was marginally significant (p < 0.046) using Friedman test.

Bioinformatics characterization of TpUB05
Bioinformatics analysis did not identify a signal peptide in the TpUB05 antigen. The software Protter version 1.0 [26] predicted two transmembrane domains spanning amino acid residues 20-41 and 73-92 that flanked a hypothetical surface exposed domain Fig 8 and lead to the exposure of both the N-and the C-terminals of TpUB05 in the cytosol. The unique N- Fig 6. ELISpot assay to assess subset of PBMCs stimulated by TpUB05 to produce interferongamma. CD4+ and CD8+ T-cells were separated from three (3) animals and stimulated with r-TpUB05 using bovine ELISpot assay. The CD4+ subpopulations of BH055 and BA219 appear to be significantly stimulated by TpUB05 (p = 0.013) compared to their CD8+ subpopulations. The CD4+ T-cells of BB007 did not significantly produce a higher amount of IFN-γ compared to its CD8+ T-cells subset. Comparing the response of all three animals tested, CD4+ T-cells produced a higher amount of IFN-γ (p = 0.046) compared to the CD8 + T-cells. An SI>2 is considered positive. The experiment was run in triplicates.  TpUB05, a Marker of Protective Immunity to East Coast Fever glycosylation consensus sequence Asn-Leu-Thr at residues 76-78 was localized in the second transmembrane domain. TpUB05 has 8 and 7 serine and threonine residues, respectively, which constitute potential O-glycosylation sites. None of the identified glycosylation sites was found in the predicted surface exposed domain (aa 42-72).
BLAST analysis indicated that antigen UB05 has 43.3% identity and 67% similarity with TpUB05 Fig 9. In silico analysis using prediction tools predicted five antibody epitopes in TpUB05 that span nearly the whole sequence Table 4. Algorithms that predict T-cell epitopes indicated the presence of these epitopes in TpUB05 that can bind strongly to most of the common BoLA types in the world Table 5. The phylogenetic relationship between TpUB05 and its orthologues in apicomplexans was predicted using tools freely available at the NCBI. The analyses revealed a close ancestral relationship between TpUB05 and UB05 hence having a common ancestor Fig 10.

Discussion
The present investigation shows for the first time that the antigen TpUB05 is an important marker of protective immunity against East Coast fever. This view is supported by the development of significantly higher antibody and T-cell responses in ITM vaccinated as compared to control animals, Further more, the observation that specific rabbit antibodies raised against r-TpUB05 were shown to block the invasion of cattle PBMCs by T. parva in vitro, further suggest that TpUB05 contributed to the protective immunity in the ITM-vaccinated cattle.
The titers of antibodies to TpUB05 were consistently higher in animals immunized against ECF than in naїve ones, even though in in vitro infection blocking assay, the antibody neutralizing activity was demonstrated with only 50% of the animals tested. The lack of complete inhibition of infection may be due to (i) a low level of expression of TpUB05 in the sporozoites as compared to the schizonts as revealed by RT-PCR or (ii) native TpUB05 may exist in a conformation that does not permit optimal binding with the anti-r-TpUB05 antiserum. A low transcriptional level of TpUB05 could also lead to its low copy number on the sporozoites surface that might have reduced the efficiency of the parasite neutralizing mechanism(s). Another explanation for this observation could be that the avidity and/or affinity of the antibody are weak. Also different animals might respond differently when inoculated with the same antigen [26].
IFN-γ has been shown to be an important cytokine of the Th-1 response, which plays an important role in the induction of protective immunity against many intracellular parasitic   [27][28][29]. In our study, the TpUB05 antigen induced IFN-γ production as well as Tcell proliferation in 8 out of 12 ITM-immunized animals. This indicates that ITM vaccinated animals have TpUB05 memory T-cells that expand and produce cytokines upon re-stimulation with TpUB05. Hence, TpUB05 possesses T-cell epitopes that are presented by bovine antigen presenting cells in association with their corresponding restricting MHC class I. It is expected that only PBMCs from animals with the appropriate MHC class I haplotypes for presenting TpUB05 epitopes were stimulated to produce and release IFN-γ. The fact that animals of different MHC class I background were stimulated suggests the presence of either several epitopes or a promiscuous T-cell target epitope in TpUB05. Further analyses such as cytotoxic assay and epitopes characterization need to be done. CD4+ T cells are known to play a crucial role in the induction of naive CD8+ T cell responses, it may therefore be important to include antigens that contain CD4+ helper T cell epitopes in a CTL-targeted vaccine.
The observed higher SI values from the T-cell proliferation as compared to the ELISpot assays could be explained by the targeting of whole PBMCs in the former and only CD4+ and CD8+ in the latter. Innate effector T-cells have been shown to respond to antigen without prior exposure. This subset of cells are primarily located in the thymus but are also found in circulation [27,29] and also possess rapid effector function. One such innate effector, the invariant natural killer (NK) T cells (iNKT) does express a semi-variant T-cell receptor and can recognize "new" antigen and become activated to produce a variety of cytokines including IFN-γ [28,29]. This could explain why one of the naive animals in this study produced IFN-γ significantly upon stimulation with TpUB05. This also strengthens the point that TpUB05 could as well be a potential marker of protective immunity for ECF.
The demonstrating of cross reactivity of antibody raised to the two antigens, TpUB05 and UB05 is consistent with the extensive sequence homology; 43.3% identity and 67% similarity observed the two antigens. Consistent expression of the TpUB05 in the life cycle stages of T. parva lents support to the view that a plays a key function in the parasite.
The present investigation shows for the first time that TpUB05 (GenBank accession number: KF875450), a homologue of a P. falciparum immunodominant antigen is strongly recognized by antibodies and PMBCs from cattle successfully vaccinated against East Coast fever (T. parva infection) using the infection followed by treatment (ITM) protocol. This implies that TpUB05 is involved in the immune response to cattle immunized against ECF. Our study also suggests the interesting possibilty that conserved homologuous antigens from phylogenetically related pathogens may be better stimulators of protective immunity. This is consistent with the strategy in vaccinology to use closely-related pathogens to create effective vaccines, for example, the cow pox virus to protect against small pox virus in the classical Jenner vaccine [30]; the successful use of Mycobacterium bovis to develop the well-known BCG vaccine to protect humans against Mycobacterium tuberculosis infection [31]. Also other investigators have shown that there is cross protection against Onchocerca volvulus infection by exposure to O. ochengi [32]. Thus, the potential role of TpUB05 in inducing immune protection against East Coast fever merits further investigation.
Supporting Information S1 Fig. cDNA and predicted amino acid sequence of the TpUB5 from Theileria parva. TpUB05 amplicon was purified from agarose gel, cloned into pGEM-T Easy vector (Promega) TpUB05, a Marker of Protective Immunity to East Coast Fever then excised with BamHI and HindIII restriction enzymes digestion and subcloned into pET32a+ (Novagen) digested with BamHI and HindIII. The recombinant pET32/TpUB05 plasmid was sequenced using the vector T7 promoter and T7 terminator primers. The nucleotide sequence of the 300 bpTpUB05 amplicon shown is flanked by forward and reverse PCR primers regions double-underlined with the respective 5' BamHI and 3' HindIII directional cloning sites shadowed. The open reading frame starts with the nucleotide number 1 of the introduced BamHI site and ends with the 3' TAA stop codon shown in bold italics. The first two amino acid residues of the 97 single letter peptide encoded by the cDNA correspond to the inframe BamHI cloning site. The asterisk ( Ã ) indicates the stop codon.  Table. Intensity fraction. A: Using GelQuant.NET Version 1.8.2 (BiochemLAbSolutions. com), the intensity fractions, shows that, the expression of the house-keeping GAPDH is almost the same for all the stages while that of TpUB05 changes significantly between life cycle stages. B: Normalizing the intensity of TpUB05 with respect to GAPDH in the various major life cycle stages. (DOC) S2 Table. ELISA using Tag-only antigen. Tag only was tested in ELISA against the serum of all test animals. The OD values obtained was comparable to that of the naïve cattle.