A multi-antigen diagnostic immunoassay and vaccine against babesiosis

Abstract

A method for detecting Babesia microti in a biological sample by detecting antibodies in the sample that bind to a set of Babesia microti antigens that includes BmSAl-PLl and one or more of BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2. Also provided is a vaccine for ameliorating Babesia microti infection in which the vaccine includes one or more polypeptides having the amino acid sequences of BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2. Further disclosed are a composition and a kit for detecting Babesia microti, and a method for treating Babesia microti infection.

Description

A MULTI- ANTIGEN DIAGNOSTICIMMUNOASSAY AND VACCINE AGAINST BABESIOSISCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Application Serial No. 63 / 650,363, filed May 21, 2024, and U.S. Provisional Application Serial No. 63 / 667,289, filed July 3, 2024, the contents of which are hereby incorporated by reference in their entireties.GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant HL060961 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Human babesiosis is a malaria-like illness caused by protozoan parasites of the genus Babesia. Babesia microti is responsible for most cases of human babesiosis in the United States, particularly in the Northeast and the upper Midwest.

[0004] Babesiosis has a wide spectrum of clinical presentation that is largely governed by the immune status of the host. While the majority of naturally acquired infections are asymptomatic or mild and self-limiting, Babesia microti infection in the immunocompromised host may develop into a severe and even fatal disease (1-4).

[0005] Existing diagnostic assays include serological detection of antibodies, direct measurement of parasite DNA by PCR, detection of parasite-infected erythrocytes by microscopy, and inoculation of small mammals with human blood.

[0006] At present, a positive serology evaluation is not sufficient for diagnosis because the indirect immunofluorescent assay (IFA), which uses whole parasite antigen, cannot distinguish between active and recent infection. There are no US Food and Drug Administration (FDA) approved immunoassays currently in use for the detection of Babesia microti infection.

[0007] The downside of immunoassays has always been that the early window-period infections will be missed because antibodies have not yet been generated. Moreover, individuals with resolved infections are likely to be detected as positive and could be barred indefinitely from blood donation.

[0008] The need exists to develop rapid, sensitive, and specific serological assays to detect Babesia microti infection and to develop effective treatments for babesiosis.SUMMARY

[0009] To meet the need discussed above, a method for detecting Babesia microti in a biological sample is provided. The method includes the steps of obtaining a biological sample containing antibodies; contacting the biological sample with a set of Babesia microti antigens; and determining that antibodies in the biological sample bind specifically to the set of Babesia microti antigens, thereby detecting Babesia microti. The set of Babesia microti antigens includes: a polypeptide consisting of the amino acid sequence of BmSAl-PEl or an amino acid sequence having at least 85% amino acid identity to BmSAl-PEl and one or more polypeptides consisting of the amino acid sequences selected from BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2 or amino acid sequences having at least 85% amino acid identity to BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2.

[0010] Also within the scope of the invention is a vaccine for ameliorating Babesia microti infection in a subject in which the vaccine comprises one or more polypeptides consisting of the amino acid sequences of BmSAl-PEl, BmSAl-RBD,BMN1-20-PC1, and BM4.12-4PC2 or one or more vectors that express the one or more polypeptides.

[0011] Further disclosed is a composition for detecting Babesia microti in which the composition includes polypeptides consisting of the amino acid sequence of BmSAl-PLl and one or more of BmSAl-RBD, BMN1-20-PC1 and BM4.12-4PC2 or polypeptides consisting of amino acid sequences having at least 85% amino acid identity to BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2.

[0012] The invention also encompasses a kit for detecting Babesia microti, the kit containing a solid support having immobilized thereon polypeptides consisting of the amino acid sequence of BmSAl-PLl and one or more of BmSAl-RBD, BMN1- 20-PC1, and BM4.12-4PC2 or polypeptides consisting of amino acid sequences having at least 85% amino acid identity to BmSAl-PLl, BmSAl-RBD, BMN1-20- PC1, and BM4.12-4PC2; and reagents for determining specific binding of antibodies to the immobilized polypeptides.

[0013] Also disclosed herein is a method for treating Babesia microti infection in a subject, the method including the steps of obtaining a biological sample from the subject; contacting the biological sample with a set of Babesia microti antigens; determining that antibodies in the biological sample bind specifically to the set of Babesia microti antigens, thereby detecting Babesia microti; and treating the subject with an antimicrobial compound effective against Babesia microti. The set of Babesia microti antigens includes polypeptides consisting of the amino acid sequence of BmSAl-PLl and one or more of BmSAl-RBD, BMN1-20-PC1, and BM4.12- 4PC2 or polypeptides consisting of amino acid sequences having at least 85% amino acid identity to BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2.

[0014] Further disclosed herein are methods of therapeutically targeting the unique immunogenic extracellular domain in BMN1-20. Derivatives of Neocuproine, a known copper chelator, are generated as therapeutic inhibitors of a PCI segment in BMN1-20. These derivatives may alter the copper homeostasis in Babesia microti and related parasites thus revealing new methods for inhibiting proliferation.

[0015] The details of one or more embodiments are set forth in the description and the examples below. Other features, objects, and advantages will be apparent from the detailed description, from the drawings, and also from the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0017] FIGS 1A-1J demonstrate characterization of Babesia microti BmSAl antigen (BMN1-9; BmGPI12).

[0018] FIG. 1A is a diagram showing the erythrocyte-binding domain (RBD) and immunoreactive PL1 segment in Babesia microti BmSAl antigen (BMN1-9; BmGPI12) identified by phage display cDNA screens. BmSAl consists of 326 amino acids including a signal peptide at its N-terminus and a glycosylphosphatidylinositol (GPI) motif at the C-terminus.

[0019] FIG. IB shows the amino acid sequence of BmSAl. The 68-amino acid erythrocyte-binding domain (RBD) and 32-amino acid immunoreactive PL1 segment are shown in red and purple, respectively.

[0020] FIG. 1C shows Coomassie-stained SDS-PAGE (Tris-Glycine 4-20%) of thioredoxin (Trx) and recombinant Trx-BmSAl (RBD) protein.

[0021] FIG. ID shows Western blots using anti-Trx mAb showing binding of Trx-BmSAl (RBD) protein to human and mouse erythrocytes or RBCs, respectively. Trx control (1.0 pM); Trx-BmSAl (RBD) (1.0 pM).

[0022] FIG. IE shows AlphaFold predicted BmSAl structure showing the location of RBD (red) and PK1 (purple) regions of BmSAl.

[0023] FIG. IF shows Western blots using anti-Trx mAb. Loading controls of Trx and Trx-BmSAl (RBD) proteins (lanes 1 and 2). RBCs with no RBD protein (lane 3) and with RBD protein (lane 4). No effect of enzyme treatments on BmSAl (RBD) binding to RBCs (lanes 5-7).

[0024] FIG. 1G shows efficacy of enzyme treatments. Binding of Trx control (1.0 pM) and Trx-EBL1-F2i (1.0 pM) to human RBCs. Neuraminidase and chymotrypsin treatments diminished the binding of EBL1-F2i to RBCs. No effect of EBL1-F2i binding on trypsin-treated RBCs.

[0025] FIG. 1H shows induction of reticulocytes in C57BL / 6 mice after sequential injections with phenylhydrazine (PHZ). New methylene blue stained bloodsmears and purification of reticulocytes by the gradient density centrifugation from three independent experiments.

[0026] FIG. II is a bar graph showing the quantification of the data shown in FIG. 1H, plotted as percent reticulocytes / RBC versus control or PHZ treatment.

[0027] FIG. 1 J shows Western blotting using anti-Trx mAb showing binding of BmSAl (RBD) protein (1.0 pM) to mouse RBCs and reticulocytes from 3 separate experiments. Semi-quantitative measurements indicated -20% reduction of BmSAl (RBD) to reticulocytes as compared to mature erythrocytes.

[0028] FIGS. 2A-2E demonstrate identification of an immunodominant region in BMN1-20 (BMN1-17; Bm32).

[0029] FIG. 2A provides a schematic location of 213-amino acids immunodominant segment (PCI; shown in yellow) in BMN1-20 putative copper transporter identified by multiple phage display cDNA clones, aligned with copper transporters from other parasites and human.

[0030] FIG. 2B shows the PCI segment is located within the extracellular domain of BMN1-20, a putative Babesia microti copper transport protein. Notably, the predicted extracellular domain of BMN1-20 is relatively longer (427 amino acids) as compared to copper transporters of malaria parasite Plasmodium falciparum (112 amino acids), a tickbome protozoan Theileria orientalis (69 amino acids), and human CTR1 (62 amino acids). The immunodominant PCI segment of BMN1-20 does not overlap with any other putative copper transporters.

[0031] FIGS. 2C-2D show the PCI segment consists of several color-coded repeat regions including five NKS repeats. These repeat regions were identified by multiple phage clones termed PC2, PC3, and PC4. Several predicted glycosylated asparagine sites (NLSI, NKSE, NKSG, NGSN) were also identified in the PCI segment. The PCI segment partially overlaps with a previously reported sero-reactive antigen region of BMN1-20.

[0032] FIG. 2D shows the amino acid sequences of the PCI, PC2, and PC3 regions of BMN1-20.

[0033] FIG. 2E shows the PCI region includes two short alpha helices and a large flexible region predicted by the AlphaFold algorithm.

[0034] FIGS. 3A-3D demonstrate identification of an immunodominant region in BM4.12 (Nl-15).

[0035] FIG. 3A shows phage display plaque-lift screens identified an immunodominant segment (amino acids 1438-1579) in BM4.12 / N1-15 antigen using patients' plasma / serum samples. The 142-amino acids segment (4PC2) is shown in red within the full length Nl-15 protein sequence.

[0036] FIG. 3B provides a sequence alignment of the C-terminal half of 4PC2 segment (shown in red) indicated 100% identify with BM4.12. The N-terminal half of 4PC2 (shown in black) indicated partial sequence alignment with Nl-15.

[0037] FIG. 3C provides a partial sequence alignment of 4PC2 segment with Nl-15 antigen.

[0038] FIG. 3D shows a 4PC2 segment is composed of multiple alpha-helices (red color) in the structure of Nl-15 protein predicted by the Alpha-Fold algorithm.

[0039] FIGS. 4A-4D demonstrate the development of a diagnostic assay for human babesiosis.

[0040] FIG. 4A shows the result of a screen of plasma samples from 4 patients with babesiosis for the presence of antibodies against four recombinant Babesia microti antigens [BM4.12, BMN1-20, BmSAl (PL1), BmSAl (RBD)]. Each antigen was purified and coated individually on Immulon 2HB ELISA plates as described in the Methods section. Secondary antibody-only blank (2°), Trx protein antigen negative control, and healthy human plasma (Hl or H2) negative controls were included to calculate absorbance (450 nm) reactivity cutoffs. The positive samples with error bars represent mean ± standard deviations of 3 independent replicates.

[0041] FIG. 4B shows the result of a screen of 9 additional patient plasma samples for the B. microti antigens as described for FIG. 4A.

[0042] FIG. 4C shows the result of a screen of 9 additional patient plasma samples for the B. microti antigens as described for FIG. 4A.

[0043] FIG. 4D shows the result of a screen of 9 additional patient plasma samples for the B. microti antigens as described for FIG. 4A.

[0044] FIGS. 5A-5C demonstrate the detection of babesiosis using pooled Babesia microti antigens. All plasma samples that recognized BmSAl (RBD) segment also tested positive with BmSAl (PL1). Based on these data, BmSAl (PL1)segment was combined with BMN1-20 (PCI) and BM4.12 (4PC2) segments and ELISA plates were coated with the pooled antigens.

[0045] FIG. 5A shows the results of screening 56 plasma samples from a blood bank repository in a pooled ELISA to detect antibodies against BmSAl (PL1), BMN1-20 (PCI), and BM4.12 (4PC2) segments, with an absorbance reactivity cutoff of 0.0822. The pooled ELISA performed in a blind manner confirmed all positive plasma samples that tested positive by conventional IFA screen. Plasma samples #7, 9, 25, and 56 that did not test positive by conventional IFA screen showed low intensity positive signals by the pooled antigen ELISA. Error bars represent mean ± standard deviations of 3 independent replicates.

[0046] FIG. 5B shows 12 additional plasma samples screened as above using newly-coated ELISA plates. The samples included a secondary antibody-only (2°) negative control (8 replicates), two positive babesiosis plasma samples identified from previous screens (B1019 and 19-08, in 4 replicates), and four malaria-infected patient plasma samples (M301, M325, M355, M365, in 5 replicates). The error bars of all 12 plasma samples tested represent mean ± standard deviations of 5 independent replicates..

[0047] FIG. 5C shows the same samples shown in FIG. 5B tested with ELISA plates coated and stored for two years.

[0048] FIG. 6 shows quantification of plasma dilution factor for ELISA. Selected patient plasma samples 17-47 were evaluated for the optimal dilution factor for subsequent ELISA screens. Immulon 2HB 96-well plates were coated with 50 pL of full length, purified recombinant Trx-BmSAl (50 ng / well) protein in PBS to test plasma reactivity at dilutions of 1:5,000, 1:10,000, and 1:20,000. Human serum (Hl) was used as a negative control to calculate an absorbance reactivity cutoff of 0.14000713. Error bars represent mean ± standard deviations of 2 independent replicates.

[0049] FIG. 7 provides a flow chart protocol to reduce the non-specific reactivity in Phage display cDNA library screens.

[0050] FIG. 8 is a bar graph showing antibody titers in mice immunized with TRX-BMN1-20 (AA 134-346) fusion proteins or TRX protein.

[0051] FIG. 9 is a plot of percent parasitemia (%) versus days after inoculation with TRX or TRX-BMN1-2O(AA 134-346).

[0052] FIG. 10 shows a model for a functional role of Babesia microti BMN1- 20 during rupture, egress, and invasion into erythrocytes. The model depicts B. microti with a functionally active BMN1-20 on the parasite surface. Upon rupture, the extracellular domain of BMN1-20 interacts with a cognate receptor on the RBC surface facilitating invasion. Panel on right shows a Giemsa- stained blood smear from an infected mouse immunized with BMN1-20 (AA 134-346) segment showing inhibition of invasion and parasite clearance in immuno-competent mice.

[0053] FIGs. 11A-11D shows localization of BMN1-20 in Babesia microti infected mouse erythrocytes.

[0054] FIG. 11A shows Immunofluorescence microscopy (IF) of BMN1-20. iRBCs were stained with mouse antiserum against BMN1-20. Arrows show the fluorescence signals concentrated inside iRBCs.

[0055] FIG. 11B shows DAPI stain (blue).

[0056] FIG. 11C is a confocal image of iRBCs stained for BMN1-20 (green). Arrows show BMN1-20 signals in the cytoplasm of iRBCs, coinciding with the staining of parasite DNA. No staining was detected with the non-immune serum (data not shown).

[0057] FIG. 1 ID shows an overlay of green and blue of FIGS. 1 IB and 11C Specific staining of BMN1-20 in B. microti.DETAILED DESCRIPTION

[0058] Human babesiosis is a malaria-like illness caused by protozoan parasites of the genus Babesia. Babesia microti is responsible for most cases of human babesiosis in the United States, particularly in the Northeast and the upper Midwest. Babesia microti is primarily transmitted to humans through the bite of infected deer ticks, but also through transfusion of blood components, particularly red blood cells. There is a high risk of severe and even fatal disease in immunocompromised patients. To date, serology testing relies on an indirect immunofluorescent assay that uses the whole Babesia microti antigen. Described herein is the construction of phage display cDNA libraries from Babesia microti-infected erythrocytes as well as human reticulocytes obtained from donors with hereditary hemochromatosis. Plasma samples were obtained from patients who were or had been infected with Babesia microti. The non-specific antibody reactivity of these plasma samples was minimized by pre-exposure to the human reticulocyte library. Immunoreactive segments were identified in three Babesia microti antigens termed BmSAl (also called BMN1-9; BmGPI12), BMN1-20 (BMN1-17; Bm32), and BM4.12 (Nl-15). The findings described herein indicate that the major immunoreactive segment of BmSAl does not overlap with the segment that mediates BmSAl binding to mature erythrocytes. When used in combination, the three immunoreactive segments form the basis of a sensitive and comprehensive diagnostic immunoassay for human babesiosis with implications for vaccine development.

[0059] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art. The following references provide one of skill with a general definition of many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0060] Standard techniques may be used for recombinant DNA, tissue culture and transformation, protein purification, etc. Enzymatic reactions and purification techniques may be performed according to the manufacturer's specifications or as commonly accomplished in the art or as described herein. The following procedures and techniques may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the specification. See, e.g., Sambrook et al., 2001, Molecular Cloning: A Laboratory Manuel, 3.sup.rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclature used in connection with, and the laboratory procedures and techniques of, analytic chemistry, organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques maybe used for chemical synthesis, chemical analyses, pharmaceutical preparation, formulation, and delivery and treatment of patients.

[0061] Disclosed herein are methods for detecting Babesia microti in a biological sample. The methods may include, among other steps, obtaining a biological sample containing antibodies, contacting the biological sample with a set of Babesia microti antigens, and determining that antibodies in the biological sample bind specifically to the set of Babesia microti antigens.

[0062] In some embodiments, the determining step is carried out by one or more of an enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, flow cytometry, magnetic levitation assay (see Andersen et al., Lab Chip, 2017, Oct l l;17(20):3462-3473), Western blot assay, and Lateral flow assay.

[0063] A biological sample may include, but is not limited to, whole blood, blood serum, blood plasma, and skin graft tissue (e.g., a skin graft may be analyzed for changes in metabolites such as copper). In a specific detecting method, the biological sample is blood plasma.

[0064] In some embodiments, the set of Babesia microti antigens includes a polypeptide having the amino acid sequence of BmSAl-PLl. In some embodiments, the polypeptide has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% amino acid identity to BmSAl-PLl. The set of Babesia microti antigens may further include one or more polypeptides having the amino acid sequences selected from BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2. In some embodiments, the one or more polypeptides have the amino acid sequences having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% amino acid identity to BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2. In one embodiments, the set of Babesia microti antigens includes polypeptides consisting of the amino acid sequences of BmSAl-PLl, BMN1-20-PC1, and BM4.12-4PC2.

[0065] Also disclosed herein is a vaccine, e.g., for ameliorating babesiosis or Babesia microti infection. The vaccine may include one or more polypeptides consisting of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, BMN1-20- PC1, and / or BM4.12-4PC2. In some embodiments, the vaccine includes one, two, three, or four polypeptides. An exemplary vaccine can include one or more vectors,e.g., DNA, RNA, mRNA, and viral vectors, which express the one or more polypeptides. In some embodiments, the vaccine includes the polypeptides consisting of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, and BMN1-20-PC1. In some embodiments, the vaccine includes the polypeptides consisting of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, and BM4.12-4PC2. In some embodiments, the vaccine includes the polypeptide consisting of the amino acid sequences of BmSAl-PLl, BMN1-20-PC1, and BM4.12-4PC2. In some embodiments, the vaccine includes the polypeptide consisting of the amino acid sequences of BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2. In one embodiment, the vaccine includes the polypeptides consisting of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2.

[0066] In some embodiments, the vaccine includes the one or more vectors expressing the polypeptides consisting of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, and BMN1-20-PC1. In some embodiments, the vaccine includes the one or more vectors expressing the polypeptides consisting of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, and BM4.12-4PC2. In some embodiments, the vaccine includes the one or more vectors expressing the polypeptide consisting of the amino acid sequences of BmSAl-PLl, BMN1-20-PC1, and BM4.12-4PC2. In some embodiments, the vaccine includes the one or more vectors expressing the polypeptide consisting of the amino acid sequences of BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2. In one embodiment, the vaccine includes the one or more vectors expressing the polypeptides consisting of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2.

[0067] Adjuvants and dose delivery protocols known in the art can be used to vaccinate an individual with the vaccine above comprising one or more viral vectors that express recombinant peptides,. For mRNA based vaccines, standard nanoparticle delivery vehicles can be used. Alternatively, stabilized extracellular vesicles containing mRNAs and microRNAs can be used.

[0068] Vaccination frequency can vary from once every 3 months, once every 6 months, once every 9 months, and once every 12 months. Preferably, vaccination is performed annually. Immuno-protection against conserved epitopes of Babesia microti or related parasites are expected to provide long-term immunity.

[0069] Also disclosed herein is a composition for detecting Babesia microti. The composition may include polypeptides consisting of the amino acid sequences of one or more of BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1 and BM4.12-4PC2. In some embodiments, the polypeptides consist of the amino acid sequences of BmSAl- PLl, BmSAl-RBD, and BMN1-20-PC1. In some embodiments, the polypeptides consist of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, and BM4.12- 4PC2. In some embodiments, the polypeptides consist of the amino acid sequences of BmSAl-PLl, BMN1-20-PC1 and BM4.12-4PC2. In some embodiments, the polypeptides consist of the amino acid sequences of BmSAl-RBD, BMN1-20-PC1 and BM4.12-4PC2. In one embodiment, the polypeptides consist of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1 and BM4.12-4PC2. In some aspects, the composition comprises polypeptides consisting of the amino acid sequences having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% amino acid identity to BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and / or BM4.12-4PC2.

[0070] Described herein is a kit. The kit may include a solid support with polypeptides immobilized on it and reagents. The solid support may be, but is not limited to, plastic, glass, nitrocellulose paper, and magnetic beads. In some aspects, gold particles may be utilized for visualization. In some embodiments, the immobilized polypeptides consist of the amino acid sequences of one or more of BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2. In some embodiments, the polypeptides consist of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, and BMN1-20-PC1. In some embodiments, the polypeptides consist of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, and BM4.12-4PC2. In some embodiments, the polypeptides consist of the amino acid sequences of BmSAl- PLl, BMN1-20-PC1 and BM4.12-4PC2. In some embodiments, the polypeptides consist of the amino acid sequences of BmSAl-RBD, BMN1-20-PC1 and BM4.12- 4PC2. In one embodiment, the polypeptides consist of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1 and BM4.12-4PC2. In some aspects, the composition comprises polypeptides consisting of the amino acid sequences having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% amino acid identity to BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and / or BM4.12-4PC2.

[0071] The kit may further include reagents for determining specific binding of antibodies to the immobilized polypeptides. For example, the reagents can include a detection antibody, e.g., an anti-human IgG antibody. In some embodiments, the anti-human IgG antibody is conjugated to a detection means, such as an enzyme, a fluorescent moiety, or a detectable moiety. The enzyme conjugated to the detection antibody can be, for example, horseradish peroxidase and alkaline phosphatase. A detectable moiety can be, but is not limited to, gold particles and biotin.

[0072] Some embodiments of the present invention relate to methods for treating babesiosis, e.g., in a subject. In some embodiments, the method for treatment comprises obtaining a biological sample from a subject, contacting the biological sample with a set of Babesia microti antigens, determining that antibodies in the biological sample bind specifically to the set of Babesia microti antigens, thereby detecting babesiosis, and treating the subject with an antimicrobial compound.

[0073] Treating babesiosis is understood to include alleviating symptoms thereof, for example, flu like symptoms including fever, chills, sweats, headache, body aches, loss of appetite, nausea (the feeling of sickness in the stomach), or fatigue.

[0074] The antimicrobial compounds used for treating babesiosis can be coadministered with anti-malarial drugs known in the art. In particular treatments, the drugs are administered orally.

[0075] In some embodiments, the set of Babesia microti antigens includes one or more polypeptides consisting of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2. In some embodiments, the polypeptides consist of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, and BMN1-20-PC1. In some embodiments, the polypeptides consist of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, and BM4.12-4PC2. In some embodiments, the polypeptides consist of the amino acid sequences of BmSAl-PLl, BMN1-20-PC1 and BM4.12-4PC2. In some embodiments, the polypeptides consist of the amino acid sequences of BmSAl-RBD, BMN1-20-PC1 and BM4.12-4PC2. In one embodiment, the polypeptides consist of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1 and BM4.12-4PC2. In some aspects, the set of Babesia microti antigens includes one or more polypeptides consisting of the amino acid sequenceshaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% amino acid identity to BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and / or BM4.12- 4PC2.

[0076] In some embodiments, determining that antibodies in the biological sample bind specifically to the set of Babesia microti antigens occurs by one or more of an enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, flow cytometry, magnetic levitation assay, Western blot assay, and Lateral flow assay.

[0077] In some embodiments, the biological sample can be, e.g., blood serum, blood plasma, and whole blood.

[0078] In some embodiments, the antimicrobial compound for treating the subject is effective against babesiosis and / or Babesia microti. The antimicrobial compound can be, but is not limited to, atovaquone, azithromycin, quinine, tafenoquine, and clindamycin. In one embodiment, the antimicrobial compound is tafenoquine and the subject to be treated is an immunocompromised individual suffering from relapsing babesiosis.

[0079] As used herein, the terms "treat" and "treating" refers to a treatment / therapy from which a subject receives a beneficial effect, such as the reduction, decrease, attenuation, diminishment, stabilization, suppression, inhibition or arrest of the development or progression of a condition or disease (e.g., an infection), or a symptom thereof. “Treating” a condition or disease refers to curing as well as ameliorating at least one symptom of the condition or disease, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject in need relative to a subject which does not receive the composition. "Treatment" as used herein covers any treatment of a disease or condition of a mammal, particularly a human, and includes: (a) preventing symptoms of the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet begun experiencing symptoms; (b) inhibiting the disease or condition (e.g., arresting its development); or (c) relieving the disease or condition (e.g., causing regression of the disease or condition, providing improvement in one or more symptoms).

[0080] As used herein, a “subject” means a human or animal. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal.Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “patient” and “subject” are used interchangeably herein. Preferably, the patient is a human (Homo sapiens). The subject may be of any gender.

[0081] For administration to a subject, an antimicrobial compound, alone or in combination with other agents, can be administered to a subject, for example in pharmaceutically acceptable compositions. These pharmaceutically acceptable compositions comprise a therapeutically effective amount of a population of the compound(s) as described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents.

[0082] As described in detail below, the pharmaceutical compositions of the present invention can be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), lozenges, dragees, capsules, pills, tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained- release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8) transmucosally; or (9) nasally. Additionally, compounds can be implanted into a patient or injected using a drug delivery system. See, for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. “Controlled Release of Pesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S. Pat. No. 3,773,919; and U.S. Pat. No. 35 3,270,960.

[0083] As used here, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0084] As used here, the term “pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound or agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or poly anhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (24) C2- C12 alcohols, such as ethanol; and (25) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms suchas “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.

[0085] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The details of the description and the examples herein are representative of certain embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0086] The articles “a” and “an” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consistessentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.

[0087] Where the claims or description relate to a composition of matter, e.g., a cell or gene trap vector it is to be understood that methods of making or using the composition of matter according to any of the methods disclosed herein, and methods of using the composition of matter for any of the purposes disclosed herein are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where the claims or description relate to a method, e.g., a method of using a cell or gene trap vector, it is to be understood that the cell or gene trap vector, and methods of using it, are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0088] Where ranges are given herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, the invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum. Numerical values, as used herein, include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by “about” or “approximately”, the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by “about” or“approximately”, the invention includes an embodiment in which the value is prefaced by “about” or “approximately”. “Approximately” or “about” generally includes numbers that fall within a range of 1% or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value). It should be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. It should also be understood that any product or composition of the invention may be “isolated”, e.g., separated from at least some of the components with which it is usually associated in nature; prepared or purified by a process that involves the hand of man; and / or not occurring in nature.

[0089] Without further elaboration, it is believed that one skilled in the art can, based on the disclosure herein, utilize the present disclosure to its fullest extent. The following specific examples are, therefore, to be construed as merely descriptive, and not limitative of the remainder of the disclosure in any way whatsoever. All documents cited herein are hereby incorporated by reference.EXEMPLIFICATION

[0090] Example 1 : Identification of Babesia microti immunoreactive antigens by phage display cDNA screen

[0091] Babesiosis, a malaria-like infection, has a wide spectrum of clinical presentation that is largely governed by the immune status of the host. While the majority of naturally acquired infections are asymptomatic or mild and self-limiting, Babesia microti infection in the immunocompromised host may develop into a severe and even fatal disease (1-4). This infection is gradually increasing in incidence as the geographic range of Babesia microti expands (5, 6). Diagnostic assays include serological detection of antibodies, direct measurement of parasite DNA by PCR, detection of parasite-infected erythrocytes by microscopy, and inoculation of small mammals with human blood. The Infectious Diseases Society of America (IDSA)recommends that a definitive diagnosis of babesiosis be made by microscopic examination of blood smears and / or amplification of parasite DNA in blood. At present, a positive serology evaluation is not sufficient for diagnosis because the indirect immunofluorescent assay (IFA), which uses whole parasite antigen, cannot distinguish between active and recent infection. There are no FDA approved immunoassays currently in practice for the detection of Babesia microti infection. Yet, serology is readily amenable for scale up to the level needed to screen millions of units of blood per year. The downside of immunoassays has always been that the early window-period infections will be missed because antibodies have not yet been generated. Moreover, the individuals with resolved infections are likely to be detected as positive and could be deferred indefinitely from blood donation. While the FDA approved nucleic acid based diagnostic assays are actively used for blood screening, the development of rapid, sensitive, and specific serology assays requires a comprehensive understanding of parasite proteins that are secreted and / or expressed on the surface of infected erythrocytes. A better understanding of these proteins will also help to select Babesia microti antigens for the detection of diagnostic antibodies / antigens and identify critical regions that mediate parasite binding to host erythrocytes (2, 7-9). Herein, the inventors elected to use a phage display cDNA library approach to identify such protein candidates.

[0092] Notwithstanding the high clinical significance of babesiosis, there are several aspects of Babesia microti biology that remain poorly understood. These impediments include lack of an in vitro culture system, molecular identification of critical host-parasite interactions, and a vaccine against babesiosis. The mechanism of erythrocyte invasion by Babesia shares some similarities with Plasmodium since erythrocytes are the only host cells that are invaded by Babesia during infection. The original objective was to apply the phage display cDNA technology to identify Babesia microti surface proteins (ligands) that are essential for parasite invasion in human erythrocytes. Phage display allows for the identification of peptides and protein segments that interact with their cognate receptors or substrates (8, 10-14). The overall rationale was to identify specific peptides derived from the minimumbinding regions of ligands and / or receptors as potential inhibitors of parasiteerythrocyte interactions and invasion.

[0093] Building on the experience with phage display cDNA technology in the pursuit of Plasmodium falciparum protein segments that interact with human erythrocytes (15-17), the inventors constructed state-of-the-art phage display cDNA libraries for Babesia microti and human reticulocytes. By combining “forward” and “reverse” screening strategies, the inventors aim to identify novel immunodominant segments in parasite antigens and to investigate how parasite proteins mediate parasite attachment to erythrocytes. Using a novel plaque-lift phage display strategy and plasma samples obtained from patients with babesiosis, the inventors have identified immunoreactive segments in three parasite antigens termed BmSAl, BMN1-20, and BM4.12. These recombinant antigen segments, when used in combination, allowed for a comprehensive detection of Babesia microti infection.

[0094] RESULTS

[0095] Identification of the immunodominant epitope in BmSAl / BMN 1 -9

[0096] BmSAl, also known as BMN1-9 or BmGPI12, is a major B. microti surface protein composed of 326 amino acids. It has been implicated as a potential ligand for parasite invasion in erythrocytes (8, 18) but is also detected as secreted (7, 19, 20). BmSAl is an immunoreactive antigen that may be useful for the diagnosis of human babesiosis (7, 19-21). Immunoreactivity has been mapped to several regions within BmSAl (8, 18, 22). The inventors elected to identify conformationally stable and immunodominant segments in BmSAl using the optimized phage display plaquelift screen. First, the inventors expressed the full-length BmSAl in bacteria by cloning its cDNA into pET30a plasmid containing a His-tag (FIGS. 1A-1B). Purified full- length recombinant BmSAl protein was used to detect antibodies in donor plasma samples by ELISA and Western blotting methods. Antigen-reactive plasma samples were then used to screen the B. microti cDNA library by the phage plaque-lift assay. The phage screen revealed a 32-amino acid segment of BmSAl designated here as PL1 (AA 26-57) (FIG. 1A, shown in purple). Purified recombinant PL1 segment detected antibodies in all plasma samples that were tested positive using full-length BmSAl. The primary structure of BmSAl-PLl segment is consistent with the short immunoreactive epitopes previously identified by using 12-mer linear peptides (23). Based on these observations, the inventors selected BmSAl-PLl segment for the development of a multi-antigen diagnostic assay for babesiosis.

[0097] A specific segment ofBmSAl binds to human erythrocytes

[0098] The inventors next inquired whether BmSAl-PLl segment facilitates parasite binding to RBCs. An advantage of phage display technology is the identification of relatively short and often conformationally stable segments of prey proteins as potential ligands captured by bait cells or purified receptor molecules. Using human erythrocytes as bait, the inventors identified several overlapping clones that encoded a 68-amino acid segment (AA 146 to 213). The inventors termed this segment the Red cell Binding Domain (RBD, FIG. 1A, shown in red). RBD is flanked by the N-terminal signal sequence, SS, and a C-terminal GPI motif (FIG. 1A) that likely anchors BmSAl in the membrane (18, 20). The phage display cDNA screens also identified several additional phage-bearing peptides / proteins that bound to intact erythrocytes (Table 1). These clones of unknown function were not characterized further.

[0099] Babesia microti is easily propagated in mice, indicating its unique ability to recognize host receptors on mouse as well as human erythrocytes. To evaluate whether BmSAl -RBD segment can bind to both human and mouse erythrocytes, a recombinant RBD segment was generated by cloning the RBD insert in a pET32b plasmid containing the N-terminal Trx-His tag (FIGS. 1B-1C). Sedimentation and immunoblotting assays showed direct binding of recombinant RBD segment to both human and mouse erythrocytes (FIG. ID). Of note, RBD segment is composed of two tandem alpha-helical repeats (FIG. IE, shown by red helices) as predicted by the AlphaFold algorithm (Al-system, DeepMind).

[0100] The invasion of erythrocytes by Babesia diver gens is significantly reduced by treatment of erythrocytes with neuraminidase (24, 25). Neuraminidase removes sialic acid residues from host glycophorins, suggesting their functional involvement in the invasion of Babesia diver gens in erythrocytes. A similar approach has been used to identify multiple erythrocyte receptors used by Plasmodium species for host cell invasion (26). Since BmSAl-RBD segment directly binds to human and mouse erythrocytes, the inventors tested whether this interaction is sensitive to neuraminidase, trypsin, and chymotrypsin (FIG. IF). Binding measurements showed no inhibitory effects of these enzymes on the binding of RBD segment to human erythrocytes (FIG. IF). In fact, a relatively small but consistent increase of RBDbinding was observed upon treatment of erythrocytes with chymotrypsin or neuraminidase but not with trypsin (FIG. IF). To validate this finding, the inventors included a recombinant EBL-1 protein derived from Plasmodium falciparum as a positive control. EBL-1 binds to human erythrocyte glycophorin B (27, 28) in a manner that is sensitive to neuraminidase, chymotrypsin but not trypsin (FIG. 1G). Together, these observations suggest that BmSAl-RBD segment recognizes an unknown host receptor on the surface of human erythrocytes, which remains undisturbed by neuraminidase, trypsin, or chymotrypsin.

[0101] Interaction ofBmSAl-RBD with erythrocytes and reticulocytes

[0102] The inventors evaluated the comparative binding activity of BmSAl- RBD segment to mature erythrocytes and reticulocytes. Blood was collected from C57BL / 6 mice injected with phenylhydrazine to induce anemia. Reticulocytes were isolated by Percoll density gradient centrifugation and quantified using New Methylene Blue staining and flow cytometry (FIGS. 1H- II). Binding of BmSAl-RBD to reticulocytes is -20% lower than binding to mature erythrocytes (FIG. 1 J). While this observation is consistent with prior evidence that B. microti primarily invades mature erythrocytes in mice (29), the inventors cannot rule the contribution of RBD- independent regions in BmSAl or other ligands to the tropism of B. microti for mature erythrocytes.

[0103] Evaluation of BmSAl in erythrocyte invasion

[0104] BmSAl has been investigated for the detection of B. microti infection (20, 21, 30-32). The presence of a GPI motif in BmSAl suggests that it may be incorporated into vesicles released from infected erythrocytes (33). Since the host receptor for BmSAl is not yet known, the precise mechanism of BmSAl -mediated invasion in host erythrocytes and its role in pathogenesis remains undefined. The identification of the 68-amino acid BmSAl-RBD segment prompted the inventors to test its functional role in vivo by injecting recombinant BmSAl-RBD to suppress parasitemia in a ragl- / - mouse model of babesiosis (29). Several attempts were unsuccessful to reduce parasitemia presumably due to the rapid degradation of recombinant BmSAl-RBD segment in vivo. In vitro evaluation of the invasion inhibitory activity of BmSAl-RBD will require an ex vivo culture system where B. microti can be propagated continuously. Despite several attempts (34-36), it has notbeen feasible to culture B. microti continuously permitting investigations on hostpathogen interactions using established biochemical and genetic disruption strategies. If an ex vivo culture for B. microti becomes available in the near future, it will require genetic deletion of BmSAl-RBD segment, targeted mutagenesis, and quantitative biochemical validation of BmSAl and its defined segments for RBC binding.

[0105] The extracellular immunodominant epitope ofBMNl-20 / BMNl- 17 / Bm32

[0106] While all plasma samples that reacted with full-length recombinant BmSAl protein also reacted with recombinant BmSAl -PL 1 segment, this segment showed relatively weak reactivity to some plasma samples that tested positive by IFA. These observations prompted the inventors to search for additional immunoreactive antigens. Using multiple plaque-lift assays, the inventors identified a specific region of BMN1-20 as highly immunoreactive to antibodies in plasma samples positive for babesiosis by IFA (FIG. 2). This immunoreactive segment of BMN1-20 (AA 134- 346) is designated as PCI segment (Clone #1, FIGS. 2A-2B, shown in yellow). Additional phage plaque-lift screens identified several cDNA clones encoding multiple internal repeats located within PCI segment (FIGS. 2C-2D). These phage clones encoding the internal repeat regions are designated as PC2 (AA 209-339), PC3 (AA155-176), and PC4 (AA 160-258) (FIG. 2C). The repeats identified in the screens consist of 22, 24, 49, 59, and 131 amino acids (FIG. 2D). The primary structure of these repeats is consistent with the immunoreactive peptides identified in previous screens (18, 22, 23).

[0107] The complete amino acid sequence of PCI segment, which is located within the extracellular region of BMN1-20 (FIGS. 2B and 2D), includes two short alpha helical regions near the N-terminus and an extended unfolded region as predicted by the AlphaFold Al-algorithm (FIG. 2E, shown in red). The in-silico analysis of full-length BMN1-20 (AA 535 amino acids) by TOPCONS, a software used for the prediction of membrane topology domains, identified at least three putative transmembrane domains with a signal peptide protruding out of the plasma membrane (FIG. 2B). The PCI segment contained within the extracellular domain of BMN1-20 (FIG. 2B) includes five putative NKS repeats and several predicted glycosylation sites (FIG. 2C, right panel). The sequence alignment analysis of PCIsegment indicated a partial overlap with a previously reported sero-reactive antigen originating from BMN1-20 (18).

[0108] BMN1-20 is predicted to be a putative copper transporter due to its similarity with the primary structure of copper-binding membrane proteins in Plasmodium falciparum, Theileria orientalis, and Homo sapiens (37, 38). The amino acid sequence alignment indicated that the PCI segment in BMN1-20 is unique among other copper transporters (FIG. 2B). Recombinant PCI segment was recognized by blood donors’ plasma samples by ELISA and Western blotting. Based on these observations, the PCI segment of BMN1-20 was selected as a candidate antigen for the development of a multi-antigen diagnostic assay to detect babesiosis.

[0109] The major immunoreactive segment in BM4.12 / N1-15

[0110] While specific immunoreactive segments identified in BmSAl (PL1) and BMN1-20 (PCI) were recognized by antibodies present in donor plasma samples with babesiosis, there were some plasma samples that showed reduced reactivity to these antigens. Therefore, the inventors performed additional plaque-lift screens to identify any remaining immunoreactive antigens detectable under these conditions. Five overlapping phage display clones were identified that encoded a 142 amino acid segment of BM4.12 / N1-15 antigen designated here as 4PC2 segment (FIG. 3A, shown in red). Sequence alignment analysis of 4PC2 segment indicated a 100% overlap of 78 amino acids (FIG. 3B, highlighted in red) with BM4.12 antigen (accession # AY170614.1) (7, 8, 22, 39). Recent sequence annotation indicates that BM4.12 gene is a shorter version of a larger gene termed Nl-15 (Maltese Cross- reactive antigen) that is composed of 2,396 amino acids (FIG. 3A; accession # XP012650105.1). The sequence alignment analysis of 142 amino acid 4PC2 segment showed partial sequence similarity to the Nl-15 protein (FIG. 3C). Structural prediction of 4PC2 segment by the AlphaFold algorithm shows a folded structure composed of several short alpha helices exposed on the surface of Nl-15 protein (FIG. 3D). The recombinant 4PC2 segment was found to be highly reactive with some plasma samples. Based on these observations, the inventors selected 4PC2 segment of BM4.12 / N1-15 as a candidate antigen for the development of a multi-antigen diagnostic assay for babesiosis.

[0111] Screening for minor immunoreactive antigens

[0112] The inventors reasoned that removal of antibodies against dominant antigens could reveal some minor immunoreactive antigens that were not detected by the initial phage display screens. The inventors prepared acetone powder of purified recombinant BmSAl, BMN1-20, and BM4.12 segments, respectively, using protocol as described in “Antibodies: A Laboratory Manual by Harlow and Lane.” Donor plasma samples with babesiosis showing high immunoreactivity were incubated with pooled acetone powder preparations to deplete antibodies against BmSAl, BMN1-20 and BM4.12 segments. Depleted plasma samples were then used to screen the phage display cDNA library of B. microti. The phage display screens did not capture any immunoreactive segments of BmSAl, BMN1-20 and BM4.12, thus validating the depletion strategy. Although these screens did not identify any novel immunoreactive segments originating from minor antigens, one phage screen identified a Babesia microti protein (BMRl_01G01435 / XM_012791904.1) of unknown function. This protein shows similarity to a Plasmodium falciparum protein (PF3D7_1303900) of unknown function. Because multiple phage clones of BMRl_01G01435 were not identified in the subsequent screens, the inventors did not proceed further with the characterization of this protein at this stage. The biological significance of minor surface antigens expressed by Babesia microti remains to be investigated. The experimental strategy optimized here could be broadly applicable for the detection of low-abundance antigens in future studies.

[0113] Development of a diagnostic assay for human babesiosis

[0114] The identification of specific immunoreactive segments in BmSAl, BMN1-20 and BM4.12 proteins (FIGS. 1-3) prompted the inventors to develop an ELISA to detect antibodies in donor plasma samples with babesiosis. First, the inventors identified the optimal concentrations of antigens required for coating ELISA plates. From a range of 2 to 500 ng / well, the inventors determined the optimal concentration to be 8 ng / well for each antigen. The inventors next identified the optimal plasma dilution. Plasma samples were tested at 1:5,000, 1:10,000, and 1:20,000 (FIG. 6). Although the 1:10,000 dilution was sufficient to detect a signal, the 1:5,000 dilution allowed for the capture of even low sensitivity signals in subsequent screens. Several negative controls were included to determine the signal reactivity cutoff including the Trx protein, two plasma samples from healthy subjects,and four plasma samples from malaria patients (17). The inventors coated four recombinant antigenic segments individually, namely PL1 from BmSAl, PCI from BMN1-20, 4PC2 from BM4.12, and RBD from BmSAl (FIGS. 4A-4D). Babesiosis patients #17-01, #19-01, and #19-04 had been splenectomized (Table 2, Methods). Others were immunosuppressed. Babesiosis patient #19-10 was being treated with Rituximab and Methotrexate (Mtx) for Sjogren’s syndrome and rheumatoid arthritis. Patient #19-12 was receiving Imuran (Azathioprine) and Plaquenil (Hydroxychloroquine) for systemic lupus erythematosus. All patients positive for babesiosis (Table 2) also tested positive using the four antigen-based ELISA screen (FIGS. 4A-4D). Plasma from patient #19-03, for whom babesiosis was suspected but not confirmed, reacted strongly with BmSAl (PL1). Plasma from patient #19-12, who tested negative by smear and PCR (Table 2), showed robust immunoreactivity with BmSAl-PLl segment but also with the other two antigens (FIG. 4D). Patient #17-01 who tested positive by smear but had a negative IFA test for B. microti specific IgG antibodies showed negligible immunoreactivity with each of the antigens (FIG. 4D). It is likely that this patient, who had been treated with rituximab up to the time of definitive diagnosis of babesiosis, had not developed antibodies against Babesia microti antigens when his plasma was tested.

[0115] ELISA for human babesiosis using pooled Babesia microti antigens

[0116] Guided by the differential response of patients’ plasma samples to several antigens (FIGS. 4A-4D), the inventors compared the immunoreactivity of BmSAl-PLl segment with BmSAl-RBD segment. Because the BmSAl-RBD segment was identified using intact erythrocytes as bait, it was reasoned that this segment may be less immunogenic than BmSAl-PLl segment identified using patients’ plasma samples. This speculation turned out to be the case. Therefore, the inventors elected to combine BmSAl (PL1), BMN1-20 (PCI), and BM4.12 (4PC2) segments for the development of a comprehensive diagnostic assay for human babesiosis. To validate the utility of a multi-antigen-based ELISA, the inventors tested 56 donor plasma samples obtained from the blood bank repository in a blind manner. Plasma samples #5, 22, 23, 26 and 30 tested strongly positive in the assay (FIG. 5A). These samples were also confirmed positive by the independent IFA screens with the titers of 1:1024, 1:512, 1:1024, 1:1024, and 1:1024, respectively. Thepooled antigen-based ELISA also detected lower intensity signals from blood donors #7, 9, 25, and 56 who had tested negative by the IFA screens with a cut-off of 1:64 (FIG. 5A). The precise status of plasma sample from donor #9 originating from the non-endemic area is not known.

[0117] To further validate the utility of pooled antigens-based EEISA with plasma samples of known clinical data, the inventors obtained 12 additional plasma samples from patients exposed to Babesia microti (FIG. 5B, Table 2). Of note, these data were generated by coating freshly prepared antigens. In addition, the inventors included one secondary antibody negative control, four negative controls from malaria patients, and the reactivity cutoff value shown in FIG. 5B. The results from the EEISA screen (FIG. 5B, Table 2) are consistent with the data obtained from plasma samples shown in FIG. 4. Finally, to test the stability of recombinant proteins coated on EEISA plates, the inventors screened the same EEISA plates that were used in FIG. 4B but stored for ~24 months at 4°C (see Methods section, FIG. 5C) under identical conditions. This side-by-side comparison showed that several plasma samples that were positive with the freshly coated antigens (FIG. 5B) are not detectable using the two-year-old plates (FIG. 5C). This observation suggests that one or more coated antigens may have lost their immunoreactivity after prolonged storage. In any case, a direct comparison between new and old EEISA plates suggests that a combination of all three antigens is essential for the development of a comprehensive EEISA for babesiosis. Together, these results suggest that a combination of immunoreactive segments originating from BmSAl, BMN1-20, and BM4.12 identified by the phage display cDNA screens constitute an optimal substrate for the detection of antibodies induced by Babesia microti infection in plasma samples tested in this study.DISCUSSION

[0118] Babesiosis shares similarities with malaria vis-a-vis parasite entry, growth, and egress from host erythrocytes. The understanding of molecular interactions mediating malaria pathogenesis and the ensuing immune response is considerably more advanced than for human babesiosis. One notable advantage of malaria biology is the availability of an in vitro propagation system for Plasmodium falciparum in human erythrocytes (40). At present, Babesia microti cannot bemaintained in vitro in human or mouse erythrocytes. This impediment largely contributes to the limited understanding of the ligand-receptor interactions and of the immune response in babesiosis. While the generation of an effective vaccine against human babesiosis remains a priority, the development of a simple and comprehensive diagnostic assay is of clinical significance. Several attempts have been made to identify Babesia microti antigens that are suitable for diagnostic use purpose (4, 7, 8, 14, 41-44). The emerging interest in the use of phage display cDNA technology prompted the inventors to search for novel Babesia microti proteins that are involved in RBC invasion and / or trigger an immune response with the expectation that these proteins could be harnessed for diagnostic assay and potentially vaccine development.

[0119] To identify functionally important Babesia microti ligands / antigens, the inventors generated two distinct phage display cDNA libraries from Babesia microti and used human RBCs as bait. Multiple phage display clones encoded a 68- amino acid segment of BmSAl, termed BmSAl-RBD. The inventors attempted to identify specific erythrocyte receptor(s) for BmSAl -RBD by treating human erythrocytes with various enzymes, a strategy that has been successfully used to identify host receptors for Plasmodium falciparum (27, 45). Direct interaction of BmSAl -RBD segment with erythrocytes was not affected by treatment with trypsin, chymotrypsin, or neuraminidase (FIG. IF). These observations suggest that Babesia microti utilizes unique receptor(s) to invade erythrocytes in contrast to other Babesia species (25). Since the BmSAl-RBD is the only segment identified by the phage display screens that directly interacts with intact erythrocytes, the inventors propose that this segment may be critical for the attachment and / or invasion of Babesia microti in host cells. The identification of BmSAl-RBD segment is consistent with the localization of BmSAl on erythrocyte and parasite surface, secreted vesicles, and in vivo protection from Babesia microti conferred by vaccination with BmSAl (7, 14, 19, 32, 46).

[0120] The inventors constructed a phage display cDNA library from human reticulocytes for the identification of erythrocyte receptor(s) recognized by BmSAl- RBD segment. Using purified BmSAl-RBD segment as bait, the majority of phage clones identified from the reticulocyte cDNA library encoded hemoglobin chains consistent with the highly abundant (>95%) hemoglobin mRNA in reticulocytes.

[0121] The optimization of phage display cDNA technology led the inventors to investigate whether antibodies in blood donors with babesiosis can recognize BmSAl-RBD segment as well as other sites within BmSAl. By subsequent phage screens, the inventors identified a 32-amino acid segment that is designated as BmSAl-PLl, which was relatively more immunoreactive than BmSAl-RBD segment. This observation is consistent with the flexible loop structure of BmSAl- PLl segment that protrudes out of the BmSAl protein topology as predicted by the AlphaFold. During the course of additional phage display screens, the inventors identified two additional immunodominant segments in BMN1-20 and BM4.12 proteins. A 213-amino acid segment termed BMN1-20-PC1 was substantially more immunoreactive as compared with BmSAl-PLl and BM4.12-4PC2 segments. Of note, the location and size of BMN1-20 PCI segment is unique among the family of copper transporters, suggesting a specialized biological function of copper transport in the pathogenesis of B. microti. The precise localization of BMN1-20 has not been established yet. However, a related homolog of Plasmodium falciparum has been localized to the erythrocyte and parasite membranes in trophozoite stages (37). Because of its high immunoreactivity, the inventors selected the BMN1-20 PCI segment as the second antigen for the development of a diagnostic assay. Finally, a third immunoreactive 142 amino acid segment termed BM4.12-4PC2 was identified. BM4.12-4PC2 segment appears to be exposed on the parasite surface as predicted by the AlphaFold. Although the biological function of BM4.12 / N1-15 antigen remains unknown at this stage, it has been previously identified as a secretory antigen (22).

[0122] To develop a comprehensive immunoassay for human babesiosis, the inventors screened plasma samples from babesiosis patients using the four antigenic segments identified in the phage display cDNA screens (FIGS. 4A-4D). The inventors pooled the three immunodominant segments termed BmSAl-PLl, BMN1-20-PC1, and BM4.12-4PC2 for the development of an ELISA. The pooled antigen-based ELISA detected all donor samples with 100% accuracy for human babesiosis. In fact, some of plasma samples obtained from the blood donors that were negative by the conventional serology tested positive by the pooled antigens ELISA albeit with lower signal output (FIG. 5A). Together, these results demonstrate that a combination of specific immunodominant segments identified by the phage display cDNA screens ofBabesia microti provide an optimal substrate for the development of a comprehensive diagnostic immunoassay for babesiosis.

[0123] This is the first demonstration of the identification of precise segments of Babesia microti antigens by the phage display cDNA technology where the nonspecific reactivity originating from phage and host targets can be eliminated upon pretreatment of filters with the phage display cDNA library derived from human reticulocytes. See FIG.7. This approach suggests potential applications of phage display plaque-lift strategy for other blood-stage diseases. The pre-adsorption step as outlined in this study is critical as it removes non-specific and presumably low- affinity antibodies from immune plasma samples. It conversely removes pathogen- derived peptides that are recognized non-specifically by immune plasma. The inventors also optimized the conditions for storing ELISA plates for at least two years to evaluate the stability of recombinant antigens. An initial ELISA screen of ELISA plates stored for 12 months indicated comparable immunoreactivity between old and newly coated ELISA plates. However, the same ELISA plates when stored for two- years resulted in the selective loss of immunoreactivity of plasma samples using the pooled antigens ELISA (FIG. 5C). These findings suggest that a combined pool of three antigens appear to be stable for short duration at temperatures that do not require deep freezing. Nonetheless future mapping of the immunoreactive segments may identify shorter but equally immunoreactive epitopes in BmSAl, BMN1-20, and BM4.12 that can be chemically synthesized for alternative forms of diagnostic immunoassays. While the pooled immunoreactive segments approach appears to be effective for the detection of antibodies in plasma samples with babesiosis, the four segments identified in the phage display screens could be considered suitable for future development of a multi-subunit vaccine against human babesiosis.

[0124] REFERENCES FOR EXAMPLE 1

[0125] 1. Vannier E, Krause PJ. 2012. Human babesiosis. N Engl J Med 366:2397-407.

[0126] 2. Puri A, Bajpai S, Meredith S, Aravind L, Krause PJ, Kumar S. 2021. Babesia microti: Pathogen Genomics, Genetic Variability, Immunodominant Antigens, and Pathogenesis. Front Microbiol 12:697669.

[0127] 3. Carpi G, Walter KS, Mamoun CB, Krause PJ, Kitchen A, Lepore TJ, Dwivedi A, Comillot E, Caccone A, Diuk-Wasser MA. 2016. Babesia microti from humans and ticks hold a genomic signature of strong population structure in the United States. BMC Genomics 17:888.

[0128] 4. Lobo CA, Cursino-Santos JR, Alhassan A, Rodrigues M. 2013. Babesia: an emerging infectious threat in transfusion medicine. PLoS Pathog 9:el003387.

[0129] 5. Bloch EM, Kumar S, Krause PJ. 2019. Persistence of Babesia microti Infection in Humans. Pathogens 8.

[0130] 6. Krause PJ, McKay K, Gadbaw J, Christianson D, Closter L, Lepore T, Telford SR, 3rd, Sikand V, Ryan R, Persing D, Radolf JD, Spielman A, Tick-Borne Infection Study G. 2003. Increasing health burden of human babesiosis in endemic sites. Am J Trop Med Hyg 68:431-6.

[0131] 7. Elton CM, Rodriguez M, Ben Mamoun C, Lobo CA, Wright GJ. 2019. A library of recombinant Babesia microti cell surface and secreted proteins for diagnostics discovery and reverse vaccinology. Int J Parasitol 49:115-125.

[0132] 8. Verma N, Puri A, Essuman E, Skelton R, Anantharaman V, Zheng H, White S, Gunalan K, Takeda K, Bajpai S, Lepore TJ, Krause PJ, Aravind L, Kumar S. 2020. Antigen Discovery, Bioinformatics and Biological Characterization of Novel Immunodominant Babesia microti Antigens. Sci Rep 10:9598.

[0133] 9. Meredith S, Oakley M, Kumar S. 2021. Technologies for Detection of Babesia microti: Advances and Challenges. Pathogens 10.

[0134] 10. Kay BK, Kasanov J, Yamabhai M. 2001. Screening phage- displayed combinatorial peptide libraries. Methods 24:240-6.

[0135] 11. Kokoszka ME, Kay BK. 2015. Mapping protein-protein interactions with phage-displayed combinatorial Peptide libraries and alanine scanning. Methods Mol Biol 1248:173-88.

[0136] 12. Lanzillotti R, Coetzer TL. 2008. Phage display: a useful tool for malaria research? Trends Parasitol 24:18-23.

[0137] 13. Smith GP, Petrenko VA. 1997. Phage Display. Chem Rev 97:391-410.

[0138] 14. Meredith S, Majam V, Zheng H, Verma N, Puri A, Akue A, KuKuruga M, Oakley M, Kumar S. 2023. Protective efficacy and correlates of immunity of immunodominant recombinant Babesia microti antigens. Infect Immun 91:e0016223.

[0139] 15. Li X, Chen H, Khan AA, Lauterbach SB, Lanzillotti R, Rai PR, Kane RS, Coetzer TL, Chishti AH. 2008. Receptor-based identification of an inhibitory peptide against blood stage malaria. Biochem Biophys Res Commun 376:489-93.

[0140] 16. Baldwin MR, Li X, Hanada T, Liu SC, Chishti AH. 2015. Merozoite surface protein 1 recognition of host glycophorin A mediates malaria parasite invasion of red blood cells. Blood 125:2704-11.

[0141] 17. Almukadi H, Schwake C, Kaiser MM, Mayer DCG, Schiemer J, Baldwin MR, Hegde S, Lu Y, Hanada T, Chishti AH. 2019. Human erythrocyte band 3 is a host receptor for Plasmodium falciparum glutamic acid-rich protein. Blood 133:470-480.

[0142] 18. Lodes MJ, Houghton RL, Bruinsma ES, Mohamath R, Reynolds LD, Benson DR, Krause PJ, Reed SG, Persing DH. 2000. Serological expression cloning of novel immunoreactive antigens of Babesia microti. Infect Immun 68:2783- 90.

[0143] 19. Li M, Ao Y, Guo J, Nie Z, Liu Q, Yu L, Luo X, Zhan X, Zhao Y, Wang S, An X, He L, Zhao J. 2019. Surface Antigen 1 Is a Crucial Secreted Protein That Mediates Babesia microti Invasion Into Host Cells. Front Microbiol 10:3046.

[0144] 20. Luo Y, Jia H, Terkawi MA, Goo YK, Kawano S, Ooka H, Li Y, Yu L, Cao S, Yamagishi J, Fujisaki K, Nishikawa Y, Saito-Ito A, Igarashi I, Xuan X. 2011. Identification and characterization of a novel secreted antigen 1 of Babesia microti and evaluation of its potential use in enzyme-linked immunosorbent assay and immunochromatographic test. Parasitol Int 60:119-25.

[0145] 21. Cornillot E, Dassouli A, Pachikara N, Eawres E, Renard I, Francois C, Randazzo S, Bres V, Garg A, Brancato J, Pazzi JE, Pablo J, Hung C, Teng A, Shandling AD, Huynh VT, Krause PJ, Lepore T, Delbecq S, Hermanson G, Liang X, Williams S, Molina DM, Ben Mamoun C. 2016. A targeted immunomic approachidentifies diagnostic antigens in the human pathogen Babesia microti. Transfusion (Paris) 56:2085-99.

[0146] 22. Homer MJ, Bruinsma ES, Lodes MJ, Moro MH, Telford S, 3rd, Krause PJ, Reynolds LD, Mohamath R, Benson DR, Houghton RL, Reed SG, Persing DH. 2000. A polymorphic multigene family encoding an immunodominant protein from Babesia microti. J Clin Microbiol 38:362-8.

[0147] 23. Tokarz R, Mishra N, Tagliafierro T, Sameroff S, Caciula A, Chauhan L, Patel J, Sullivan E, Gucwa A, Fallon B, Golightly M, Molins C, Schriefer M, Marques A, Briese T, Lipkin WI. 2018. A multiplex serologic platform for diagnosis of tick-bome diseases. Sci Rep 8:3158.

[0148] 24. Lobo CA. 2005. Babesia divergens and Plasmodium falciparum use common receptors, glycophorins A and B, to invade the human red blood cell. Infect Immun 73:649-51.

[0149] 25. Lobo CA, Rodriguez M, Cursino-Santos JR. 2012. Babesia and red cell invasion. Curr Opin Hematol 19:170-5.

[0150] 26. Gaur D, Mayer DC, Miller LH. 2004. Parasite ligand-host receptor interactions during invasion of erythrocytes by Plasmodium merozoites. Int J Parasitol 34:1413-29.

[0151] 27. Li X, Marinkovic M, Russo C, McKnight CJ, Coetzer TL, Chishti AH. 2012. Identification of a specific region of Plasmodium falciparum EBL-1 that binds to host receptor glycophorin B and inhibits merozoite invasion in human red blood cells. Mol Biochem Parasitol 183:23-31.

[0152] 28. Mayer DC, Cofie J, Jiang L, Hartl DL, Tracy E, Kabat J, Mendoza LH, Miller LH. 2009. Glycophorin B is the erythrocyte receptor of Plasmodium falciparum erythrocyte-binding ligand, EBL-1. Proc Natl Acad Sci U S A 106:5348- 52.

[0153] 29. Borggraefe I, Yuan J, Telford SR, 3rd, Menon S, Hunter R, Shah S, Spielman A, Gelfand JA, Wortis HH, Vannier E. 2006. Babesia microti primarily invades mature erythrocytes in mice. Infect Immun 74:3204-12.

[0154] 30. Luo Y, Terkawi MA, Jia H, Aboge GO, Goo YK, Cao S, Li Y, Yu L, Ooka H, Kamyingkird K, Masatani T, Zhang S, Nishikawa Y, Igarashi I, Xuan X. 2012. A double antibody sandwich enzyme-linked immunosorbent assay for detectionof secreted antigen 1 of Babesia microti using hamster model. Exp Parasitol 130:178- 82.

[0155] 31. Priest JW, Moss DM, Won K, Todd CW, Henderson L, Jones CC, Wilson M. 2012. Multiplex assay detection of immunoglobulin G antibodies that recognize Babesia microti antigens. Clin Vaccine Immunol 19:1539-48.

[0156] 32. Chand M, Choi JY, Pal AC, Singh P, Kumari V, Thekkiniath J, Gagnon J, Timalsina S, Gaur G, Williams S, Ledizet M, Mamoun CB. 2022. Epitope profiling of monoclonal antibodies to the immunodominant antigen BmGPI12 of the human pathogen Babesia microti. Front Cell Infect Microbiol 12:1039197.

[0157] 33. Oliva Chavez AS, Wang X, Mamin L, Archer NK, Hammond HL, Carroll EEM, Shaw DK, Tully BG, Buskirk AD, Ford SL, Butler LR, Shahi P, Morozova K, Clement CC, Lawres L, Neal AJO, Mamoun CB, Mason KL, Hobbs BE, Scoles GA, Barry EM, Sonenshine DE, Pal U, Valenzuela JG, Sztein MB, Pasetti MF, Levin ML, Kotsyfakis M, Jay SM, Huntley JF, Miller LS, Santambrogio L, Pedra JHF. 2021. Tick extracellular vesicles enable arthropod feeding and promote distinct outcomes of bacterial infection. Nat Commun 12:3696.

[0158] 34. Lawres LA, Garg A, Kumar V, Bruzual I, Forquer IP, Renard I, Virji AZ, Boulard P, Rodriguez EX, Allen AJ, Pou S, Wegmann KW, Winter RW, Nilsen A, Mao J, Preston DA, Belperron AA, Bockenstedt LK, Hinrichs DJ, Riscoe MK, Doggett JS, Ben Mamoun C. 2016. Radical cure of experimental babesiosis in immunodeficient mice using a combination of an endochin-like quinolone and atovaquone. J Exp Med 213:1307-18.

[0159] 35. Moitra P, Zheng H, Anantharaman V, Banerjee R, Takeda K, Kozakai Y, Lepore T, Krause PJ, Aravind L, Kumar S. 2015. Expression, Purification, and Biological Characterization of Babesia microti Apical Membrane Antigen 1. Infect Immun 83:3890-901.

[0160] 36. Shikano S, Nakada K, Hashiguchi R, Shimada T, Ono K. 1995. A short term in vitro cultivation of Babesia rodhaini and Babesia microti. J Vet Med Sci 57:955-7.

[0161] 37. Choveaux DL, Przyborski JM, Goldring JP. 2012. A Plasmodium falciparum copper-binding membrane protein with copper transport motifs. Malar J 11:397.

[0162] 38. Salman AA, Goldring JPD. 2023. Expression and copper binding studies of a Plasmodium falciparum protein with Cox 19 copper binding motifs. Exp Parasitol 251 : 108572.

[0163] 39. Silva JC, Comillot E, McCracken C, Usmani-Brown S, Dwivedi A, Ifeonu 00, Crabtree J, Gotia HT, Virji AZ, Reynes C, Colinge J, Kumar V, LawresL, Pazzi JE, Pablo JV, Hung C, Brancato J, Kumari P, Orvis J, Tretina K, ChibucosM, Ott S, Sadzewicz L, Sengamalay N, Shetty AC, Su Q, Tallon L, Fraser CM, Frutos R, Molina DM, Krause PJ, Ben Mamoun C. 2016. Genome-wide diversity and gene expression profiling of Babesia microti isolates identify polymorphic genes that mediate host-pathogen interactions. Sci Rep 6:35284.

[0164] 40. Trager W, Jenson JB. 1978. Cultivation of malarial parasites. Nature 273:621-2.

[0165] 41. Herwaldt BL, Linden JV, Bosserman E, Young C, Olkowska D, Wilson M. 2011. Transfusion-associated babesiosis in the United States: a description of cases. Ann Intern Med 155:509-19.

[0166] 42. Drews SJ, Kjemtrup AM, Krause PJ, Lambert G, Leiby DA, Lewin A, O'Brien SF, Renaud C, Tonnetti L, Bloch EM. 2023. Transfusion-transmitted Babesia spp.: a changing landscape of epidemiology, regulation, and risk mitigation. J Clin Microbiol 61:e0126822.

[0167] 43. Thekkiniath J, Mootien S, Lawres L, Perrin BA, Gewirtz M, Krause PJ, Williams S, Doggett JS, Ledizet M, Ben Mamoun C. 2018. BmGPAC, an Antigen Capture Assay for Detection of Active Babesia microti Infection. J Clin Microbiol 56.

[0168] 44. Gagnon J, Timalsina S, Choi JY, Chand M, Singh P, Lamba P, Gaur G, Pal AC, Mootien S, Marcos LA, Ben Mamoun C, Ledizet M. 2022. Specific and Sensitive Diagnosis of Babesia microti Active Infection Using Monoclonal Antibodies to the Immunodominant Antigen BmGPI12. J Clin Microbiol 60:e0092522.

[0169] 45. Sim BK, Chitnis CE, Wasniowska K, Hadley TJ, Miller LH. 1994. Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum. Science 264:1941-4.

[0170] 46. Thekkiniath J, Kilian N, Lawres L, Gewirtz MA, Graham MM, Liu X, Ledizet M, Ben Mamoun C. 2019. Evidence for vesicle-mediated antigen export by the human pathogen Babesia microti. Life Sci Alliance 2.

[0171] 47. Baldwin M, Yamodo I, Ranjan R, Li X, Mines G, Marinkovic M, Hanada T, Oh SS, Chishti AH. 2014. Human erythrocyte band 3 functions as a receptor for the sialic acid-independent invasion of Plasmodium falciparum. Role of the RhopH3-MSPl complex. Biochim Biophys Acta 1843:2855-2870.

[0172] Example 2: Immunization of mice with a recombinant Babesia microti immunoreactive antigen

[0173] The efficacy of neutralizing antibodies raised by immunization of mice with a recombinant BMN1-20 (AA 134-346) segment was evaluated in an established preclinical model of babesiosis. The parasite reservoir was maintained in RAG-1 immunodeficient mice for the propagation of B. microti. While taking into consideration the self-limiting resolution of the B. microti infection in immunocompetent mouse models, female BALB / c mice were injected with the purified soluble recombinant BMN1-20 (AA 134-346) segment. The thioredoxin-HIS-BMNl- 20 (AA 134-346) fusion protein was purified using Ni-affinity chromatography and MonoQ AKTA-EPLC. Purified recombinant protein was emulsified in an equal volume of TiterMax Gold adjuvant, and 50 pg antigen was injected intraperitoneally at two-week intervals for a total of four doses in six female BALB / c mice (8-12 weeks old). The results are shown in EIG. 8.

[0174] Immunized mice generated a robust antibody response in contrast to the TRX injected control mice (EIG. 8). This observation is consistent with the detection of antibodies against BMN1-20 (AA 134-346) segment in all 60 patients tested by ELISA. See Table 2 below.

[0175] Example 3: Suppression of Babesia microti infection in vivo

[0176] The functional role of the BMN1-20 (AA 134-346) segment in protection against babesiosis was evaluated in vivo. Female BALB / c mice were inoculated with total of 107B. microti positive iRBCs and the parasitemia was measured by GIEMSA-stained blood smear slides. The results are shown in FIG. 9. Parasitemia increased to -5.5% after 10-13 days and then decreased consistently with the immunosuppression in naive BALB / c mice (FIG. 9, top curve). Six BALB / c micewere immunized with bacterially expressed and FPLC purified recombinant BMN1- 20 (AA 134-346) segment and evaluated for their susceptibility to infection by B. microti for 20 days (FIG. 9, bottom curve). As compared to TRX-injected mice, immunization with BMN1-20 (AA 134-346) segment showed -50% inhibition of parasitemia.

[0177] Example 4: Functional characterization of Babesia microti copper transporter.

[0178] Multiple phage clones were identified encoding a specific segment of BMN1-20 (AA 134-346) using plasma samples from patients with Babesiosis (1). Since the properties of this copper transporter appear to be unique, its biological function is determined to exploit potential vulnerabilities for therapeutic intervention. In general, there is little known about the function of copper importers in intracellular pathogens. The N-terminus of copper importer in the related malaria parasite binds to copper, may act as a chaperone, and its expression changes during intraerythrocytic development (20, 21). Genetic inactivation of malaria copper importer is lethal (22, 23). Similarly, the gene inactivation of mammalian copper importer CTR1 is lethal (2). A remarkable feature of B. microti BMN1-20, a putative copper importer, is its unique extracellular domain that does not share any sequence similarity with known copper importers (see FIGs. 2A and 2B and Tablel below). The function of its highly immunogenic segment (AA 134-346) remains enigmatic. While evaluating blood smears from mice immunized with BMN1-20 (AA 134-346) segment, impaired invasion of B. microti in host RBCs was frequently observed (see FIG. 10, right panel). This finding led to the hypothesis that B. microti BMN1-20 plays an essential role both in the regulation of copper homeostasis as well as parasite invasion during intraerythrocytic infection.

[0179] Subcellular localization ofBMNl-20 in infected erythrocytes.

[0180] There is considerable interest in the precise localization of B. microti antigens during intraerythrocytic development. We have considerable experience with similar studies in malaria parasite-infected erythrocytes (10, 11, 24). To determine BMN1-20 localization in iRBCs, we visualized BMN1-20 by immunofluorescence microscopy using polyclonal antibodies generated in mice (see FIG. 8). Surprisingly, the BMN1-20 immunoreactivity was detected at the parasite membrane with no signaldetected on the surface of iRBCs (see FIG. 11). This unexpected observation implies that BMN1-20 (AA 134-346) segment regulates copper import from erythrocyte cytosol into the parasite as well as plays a signaling role in parasite invasion and growth. There remains a possibility that polyclonal serum generated in mice might have missed the low abundance BMN1-20 on the surface of iRBCs. If so, then BMN1-20 may regulate copper import both at the host and parasite membranes.

[0181] Specific monoclonal antibodies against BMN1-20 will be obtained using established protocols (25). The precise localization of BMN1-20 is necessary to gain new insights into the stage- specific targeting of B. microti and its functional role in the proposed model of invasion by immune-gold electron microscopy. The monoclonal antibodies will be used to perform unbiased mass spectrometry of BMN1-20 immune-complexes to identify potential regulators of BMN1-20. Further, availability of specific monoclonals against BMN1-20 (AA 134-346) segment will be valuable in testing their inhibitory role in parasite invasion and development relevant to therapeutic applications.

[0182] Quantification ofBMNl-20 copper uptake activity.

[0183] To investigate the functional role of BMN1-20 (AA 134-346) segment, the copper uptake activity of full length BMN1-20 is compared with human and malaria copper transporters (see FIG. 2B). Briefly, pcDNA3.1 plasmids encoding full length copper transporters are transfected into human embryonic kidney (HEK293) cells with 10% fetal bovine serum. After transfection and incubation for 24 hours, cells are evaluated by an established copper uptake assay (28, 29) or similar transfection-based assays (3, 10, 30). The linear response of copper uptake activity is measured for 20 minutes and compared with a standard curve by including appropriate negative controls. Experiments are performed in triplicate to compare the copper transport activity. An anti-Myc monoclonal antibody is used to quantify the expression of protein expression by Western blotting. BMN1-20 can show a differential response of its copper transport activity as compared to human and malaria copper under these conditions. High copper import activity of BMN1-20 is studied by in vitro mutagenesis targeting critical residues in the BMN1-20 (AA 134- 346) segment. If the replacement of BMN1-20 (AA 134-346) segment restores copper import activity of BMN1-20 to the levels similar to human and malaria coppertransporters, it will strongly suggest a regulatory role of the unique extracellular domain of BMN1-20 in copper uptake.

[0184] Copper import-independent function(s) ofBMNl-20

[0185] A comparison of the structural organization of parasite copper transporters with human CTR1 showed a unique extension of Babesia microti N- terminal extracellular domain (427 amino acids) (see FIG. 2A). Further scanning of 213 amino acids in BMN1-20 (AA 134-346) segment indicated the absence of any conserved methionine residues that are known to be critical for copper binding in human CTR 133-35. These observations suggest that the highly immunoreactive 213 amino acid segment in BMN1-20 may also perform biological function(s) that are independent of its copper binding activity. These functions can include facilitating intracellular subcellular localization of BMN1-20 and its potential role in the regulation of extracellular interactions.

[0186] As mentioned above, B. microti upon egress from iRBCs and coated with antibodies targeting BMN1-20 cannot invade erythrocytes (See FIG. 10), the possibility exists that the BMN1-20 (AA 134-346) segment may directly bind to host RBCs. To test this model, recombinant BMN1-20 (AA 134-346) segment is examined for its direct interaction with RBCs, endothelial cells, and serum factors using microscopy, pull down, and mass spectrometry techniques using the monoclonal antibodies discussed above. If BMN1-20 N-terminal segment interacts with intact RBCs, cognate host receptor(s) are identified. It has been shown that invasion of RBCs by Babesia divergens is significantly reduced by treatment of RBCs with neuraminidase (36, 37). A similar approach has been used to identify multiple erythrocyte receptors by Plasmodium species for host cell invasion (38). Similar experiments are performed with BMN1-20 (AA 134-346) segment using neuraminidase, trypsin, and chymotrypsin treatment (1, 10, 11). A new phage display cDNA library from TNFa-induced human endothelial cells is used for screening BMN1-20 binding receptors on the endothelial cells, consistent with a recent report showing increased adhesion of B. microti infected erythrocytes to vascular wall in placenta (39). Together, these experiments may reveal a copper- independent regulatory function of BMN1-20 thus leading to the development of novel intervention strategies against babesiosis.

[0187] Physiological function ofBMNl-20 in Babesia microti infection. Copper is an essential catalytic cofactor required for regulating the activity of many enzymes and proteins including cytochrome c oxidase, superoxide dismutase, and ceruloplasmin involved in diverse physiological processes (14, 40, 41). In mammals, the major copper influx transporter SCL31A1 / CTR1 is required for survival. The CTR1 is essential for mitochondrial function and is expressed throughout erythropoiesis until the expulsion of nucleus and organelles during terminal erythropoiesis. Mass spectrometry analysis of highly purified RBC membranes did not detect CTR1 in both human and mouse erythrocytes (3). Since B. microti primarily invades mature erythrocytes, it is likely that the parasite differentially anchors BMN1-20 at specific sites during erythrocyte life cycle. Evidence suggest that BMN1-20 is localized at the parasite membrane (see FIG. 11). Nonetheless, lack of BMN1-20 detection on the iRBC surface could be explained either by its low abundance or the closed conformation of BMN1-20 mediated by the immunogenic segment (AA 134-346) segment. Alternatively, other host transporters or pathways may function in the import of residual copper that is concentrated by the BMN1-20 localized on the surface of intraerythrocytic parasite (see FIG. 10). Upon egress from the erythrocyte, the open conformation of B. microti merozoite enables rapid accumulation of copper at the expense of being exposed to host immune response (see FIG. 10). This model, consistent with the protection of mice immunized with the BMN1-20 (AA 134-346) segment (see FIG. 9), suggests that B. microti is unable to invade erythrocytes because antibodies bound to the BMN1-20 (AA 134-346) segment block parasite invasion in vivo.MATERIAES AND METHODS

[0188] Construction of Babesia microti phage display cDNA library

[0189] A random primed cDNA library was constructed for each pool of mRNA. The Babesia microti RM / NS strain(29) was used as the source of parasite mRNA. Immunodeficient ragl- / - mice on a C57BE / 6 genetic background (The Jackson Eab / JAX) were inoculated with a frozen stock of RM / NS parasites. Blood was collected when parasitemia reached -45%. The buffy coat, which contains leukocytes, was removed following centrifugation and total RNA was isolated using TRIzol Plus RNA purification kit (Ambion). mRNA was isolated from total RNA bytwo rounds of purifications using Dynabeads mRNA purification kit (Ambion). The phage display cDNA library was constructed using OrientExpress random prime cDNA system (Novagen) and T7Select 10-3b phage display system (Novagen). cDNA was synthesized from 3.8 pg of mRNA using 1.0 pg of Hindlll random primers. cDNA was end-modified with EcoRVHindlll linkers and digested with EcoRI and Hindlll. Small cDNA products (<300 bp) and excess linkers were removed by gel filtration. Lastly, cDNA was ligated with T7Select 10-3b EcoRI / Hindlll vector arms and packaged into phages using T7Select packaging extract. The total number of cDNA clones in the library was determined by plaque assay and was 1.0 x 106. The library was amplified once using E. coli BLT5403 by the plate method with a titer of 1 x IO10pfu / mL. The majority of phage clones contained an insert ranging from 300 to 1,000 bp in length.

[0190] Construction of human reticulocyte random primed phage display cDNA library

[0191] A human reticulocyte cDNA library was prepared using reticulocytes isolated from blood donated by patients with iron overload disorders (Tufts IRB protocol# 9750), including hereditary hemochromatosis. Total RNA was isolated from freshly collected blood after mature erythrocytes were lysed in NH4C1. The poly- A RNA was purified using the Dynabeads mRNA purification kit (Ambion). As with the B. microti Orient Express cDNA construction protocol, the T7 select 10-3b system (Novagen) was used to construct the phage cDNA library. The amplified cDNA library had a phage titer of 5 x 109pfu / mL.

[0192] Phage display screen to identify erythrocyte-binding proteins

[0193] The Babesia microti phage display cDNA library was screened with human RBCs as bait. Using an established biopanning protocol (16, 17, 27), human RBCs were washed three times with phosphate buffered saline (PBS) and resuspended in the RBC biopanning buffer (RPMI 1640 medium containing 10% fetal bovine serum and stabilized with 10 mM HEPES at pH 7.4). Human RBCs (20 pL) and IxlO8pfu of phage display library were mixed in 1.0 mL of RBC biopanning buffer and incubated at room temperature for one hour on a rotator. After incubation, RBCs were separated from unbound phages by centrifugation through 500 pL of silicon oil at 15,000 g for one minute. RBCs were washed three times with PBS andincubated in 1.5 M NaCl to elute bound phages. Eluted phages were amplified in the liquid culture of E. coli BLT5403 and used for another round of biopanning. After four rounds of biopanning, phages were plated with E. coli BLT5403, and DNA was extracted from each plaque in 10 mM EDTA, pH 8.0 at 65°C for 10 minutes. Inserts were amplified using T7UP 5’-GGAGCTGTCGTATTCCAGTC (SEQ ID NO: 1) and T7D0WN 5’-AACCCCTCAAGACCCGTTTA (SEQ ID NO: 2). PCR products were purified using the E.Z.N.A. Cycle Pure kit (Omega Bio-tek / VWR), and the DNA sequence of each plaque was determined by Sanger sequencing using the T7D0WN primer (Tufts University Core Facility).

[0194] Pre-treatment of human plasma samples with phage extract-saturated nitrocellulose membranes

[0195] To reduce non-specific reactivity, human plasma samples from asymptomatic blood donors who were seropositive for Babesia microti (Creative Testing Solutions, Tempe, AZ) were pretreated with the phage extract prepared from a human reticulocyte phage display cDNA library as described above. See FIG. 7. Phage extract was prepared from the human reticulocyte cDNA library propagated in E. coli BLT5403. The lysate was clarified by high-speed centrifugation (17,700 x g) and incubated with the nitrocellulose membranes for 30 minutes. Treated nitrocellulose membranes were incubated in blocking buffer (1% Gelatin and 3% BSA in PBS with 0.05% Tween-20, i.e., “PBST”) for one hour at room temperature. Human plasma samples were diluted 1:100 fold in PBST and incubated with pretreated nitrocellulose membranes for 20 minutes at room temperature. Preadsorbed plasma samples were added with 0.05% sodium azide and stored at 4°C until subsequent plaque-lift screens.

[0196] Phage display cDNA library screening using the plaque-lift method

[0197] The Babesia microti phage display cDNA library was plated on 6 cm dishes at a density of 15,000 plaques per plate. Plates were left at 4°C for one hour to minimize the adhesion of nitrocellulose membranes to agarose. Nitrocellulose membranes were incubated with the plated phage cDNA library for several minutes. The nitrocellulose membrane was lifted and blocked in PBST for 30 minutes. Preadsorbed plasma samples were diluted 1:500 fold in PBST and incubated with the nitrocellulose membranes for one hour at room temperature. Nitrocellulosemembranes were washed five times with PBST for one hour each time. Membranes were incubated with an HRP-conjugated anti-human IgG antibody diluted to 1:4,000 fold for one hour at room temperature. Nitrocellulose membranes were washed five times (PBST) before identification of positive plaques by chemiluminescence (SuperSignal West Pico Chemiluminescent substrate from ThermoFisher Scientific).

[0198] PCR amplification of identified plaques

[0199] PCR reactions were performed with 1.0 pL of DNA template in 50 pL containing 1.25 U Taq polymerase, 5 pM T7 Select UP and DOWN primers, 10 pM dNTP mix, and 10X NovaTaq buffer containing MgCh. Cycling conditions included an initial denaturation cycle of 94°C for five minutes, 35 amplification cycles (94°C for 30 seconds, 55°C for one minute, and 72°C for one minute) and a final extension step of 72°C for six minutes. PCR products were visualized by agarose gel electrophoresis and purified using the E.Z.N.A ® Cycle Pure kit (Omega Bio- tek / VWR). Sanger sequencing of the PCR products was performed by the Tufts University Core Facility.

[0200] Erythrocyte binding assay

[0201] The cDNA insert (e.g., amino acids 146-213 of BmSAl) was amplified by PCR using T7-UP and T7-D0WN primers, digested with EcoRI and Hindlll, and cloned into pET32b plasmid (Novagen). This plasmid provided thioredoxin (Trx) and 6x His tag found at the N-terminus of the recombinant protein. Host E. coli BL21 (DE3) cells were transformed with the plasmid and the recombinant protein was purified using high density nickel affinity beads (Gold Biotechnology) as described before(16, 27, 47). Purified recombinant proteins were dialyzed against PBS. RBC binding was performed in PBS supplemented with 3% BSA as previously described (27). Upon elution with 1.5 M NaCl, the eluted protein was analyzed by SDS-PAGE and Western blotting using an anti-Trx mouse monoclonal antibody (GenScript) and an HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Bio-Rad). The signal was detected using the SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific).

[0202] Enzymatic treatment of human erythrocytes

[0203] Human RBCs (20 pL) were resuspended in PBS and incubated with neuraminidase 100 mU / mL (Sigma), trypsin 1.0 mg / mL (Sigma) or chymotrypsin 1.0mg / mL (Sigma) at 3°C for one hour. After incubation, trypsin- or chymotrypsin- treated human RBCs were treated with soybean trypsin / chymotrypsin inhibitor at 1.0 mg / mL (Sigma) at 37°C for 15 minutes. Enzyme-treated human RBCs were washed three times with PBS prior to use in the RBC binding assay as described earlier (16, 28).

[0204] Purification of mouse reticulocytes

[0205] To induce reticulocytes in vivo, male C57BL / 6 (The Jackson Laboratory / J AX) mice were injected intraperitoneally with 60 mg / kg phenylhydrazine hydrochloride (Sigma) on day 1 and day 2. Blood was collected via cardiac puncture in EDTA-coated tubes (BD Biosciences) on day 5. Reticulocytes were isolated by sequential Percoll centrifugation. Sterile isotonic Percoll solutions were prepared in RPMI 1640 medium containing 1.5 mg / mL bovine serum albumin. For the first round of centrifugation, 2 mL of blood was layered over 5 mL of Percoll solution (density 1.058 g / ml) and centrifuged at 440 x g for 30 minutes at 4°C in a swing-out tube holder centrifuge (Beckman model J-6B). Mature RBCs and reticulocytes were pelleted at the bottom of the tube. For the second round of centrifugation, the pellet from the first centrifugation was washed two times, resuspended in RPMI 1640 medium, layered over 5 mL of Percoll solution (density 1.100 g / ml) and centrifuged at 440 x g for 30 minutes at 4°C. Reticulocytes were recovered on the top of the Percoll solution and assessed for purity by microscopy after staining with Methylene Blue.

[0206] Development of an ELISA for detection of antibodies against Babesia microti

[0207] Immulon 2HB 96 well plates (ThermoFisher) were saturated with 50 pL of a 500 ng / well target antigen overnight in PBS at 4°C. Plates were washed five times with PBST and exposed to PBST for one hour at room temperature. After removal of PBST, diluted plasma samples were added to wells. Plates were incubated for one hour at room temperature. Wells were washed five times with PBST for five minutes each. A 1:4,000 dilution of HRP-conjugated anti-human IgG antibody (Jackson Immunoresearch Laboratories Inc.) in PBST was incubated in the wells for one hour at room temperature. Wells were washed five times with PBST for five minutes each. TMB substrate was added to let the color develop. After 10 minutes,the reaction was quenched with 1.0 M HC1 and the absorbance at 450 nm measured using a VersaMax plate reader.

[0208] ELISA screen using plasma samples from subjects with babesiosis

[0209] Plasma samples from 31 asymptomatic blood donors from Creative Testing Solutions, as described above, were used to assess the efficacy of the BmSAl, BMN1-20, and BM4.12 segments in detecting antibodies. Two plasma samples from healthy individuals termed Hl and H2 were used as negative controls. Four plasma samples from patients infected with malaria were included (17). Wells of the ELISA plates were coated with the purified segments from B. microti antigens by adding 50 pL of a solution containing each segment at 500 ng / well in PBS. The rest of the procedure was as outlined in the above paragraph. Patient and control plasma samples were initially tested in triplicate for quality assurance and statistical analysis. Given the reproducibility of these measurements, each plasma sample was subsequently tested in duplicate. A reactivity cutoff was statistically determined (BIOREBA “ELISA Data Analysis”) as follows: (x+ 3s)*«l.l where *x and s are the mean optical density value and standard deviation for the control plasma samples.

[0210] Preparation of ELISA plates for long-term storage

[0211] The stability of ELISA plates coated with three segments of interest was assessed over an extended period. Ten ELISA plates coated with pooled segments overnight and blocked with blocking buffer (PBST) and washed three times with 200 pL of PBST. Two hundred pL of lx ELISA Microwell Coating Stabilizer solution (Rockland Immunochemicals) was added to each well and plates were incubated for one hour. Following the removal of the coating stabilizer solution, plates were incubated at 37°C until completely dry, sealed in an air-tight container with desiccant, and stored at -20°C for 12-24 months. Freshly prepared ELISA plates and stored plates were evaluated as described above.

[0212] Human Blood. Samples

[0213] Human donor plasma samples were obtained under the approved Tufts IRB protocol #9750. Human donor plasma samples from patients with babesiosis were obtained under Tufts IRB protocol #11771. Plasma samples were obtained from the National Institutes of Health (NIH) as part of a screen to search for potential biomarkers of malaria infection in the endemic areas as reported earlier (17). Plasmasamples from asymptomatic donors were obtained from the blood bank repository through Creative Testing Solutions.

[0214] REFERENCES FOR EXAMPLE 4 AND MATERIALS AND METHODS

[0215] 1. Hanada T, Empitu MA, Mines GI, Ma Q, Omorodion IL, Link A, Schwake CJ, Krueger RM, DaRosa NS, Levin AE, Vannier E, Chishti AH. Identification of Babesia microti immunoreactive antigens by phage display cDNA screen. Infect Immun. 2024;92(7):e0021524. Epub 20240617. doi: 10.1128 / iai.00215- 24. PubMed PMID: 38884473; PMCID: PMC11238553.

[0216] 2. Kuo YM, Zhou B, Cosco D, Gitschier J. The copper transporter CTR1 provides an essential function in mammalian embryonic development. Proc Natl Acad Sci U S A. 2001;98(12):6836-41. doi: 10.1073 / pnas.l 11057298. PubMed PMID: 11391004; PMCID: PMC34439.

[0217] 3. Khan AA, Hanada T, Mohseni M, Jeong JJ, Zeng L, Gaetani M, Li D, Reed BC, Speicher DW, Chishti AH. Dematin and adducin provide a novel link between the spectrin cytoskeleton and human erythrocyte membrane by directly interacting with glucose transporter- 1. J Biol Chem. 2008;283(21): 14600-9. Epub 2008 / 03 / 19. doi: M707818200 [pii]

[0218] 10.1074 / jbc.M707818200 [doi]. PubMed PMID: 18347014; PMCID: 2386908.

[0219] 4. Zimnicka AM, Ivy K, Kaplan JH. Acquisition of dietary copper: a role for anion transporters in intestinal apical copper uptake. Am J Physiol Cell Physiol. 2011;300(3):C588-99. Epub 20101229. doi:10.1152 / ajpcell.00054.2010. PubMed PMID: 21191107; PMCID: PMC3063964.

[0220] 5. luchi Y, Okada F, Onuma K, Onoda T, Asao H, Kobayashi M, Fujii J. Elevated oxidative stress in erythrocytes due to a SOD1 deficiency causes anaemia and triggers autoantibody production. Biochem J. 2007;402(2):219-27. doi: 10.1042 / BJ20061386. PubMed PMID: 17059387; PMCID: PMC1798435.

[0221] 6. McMillin GA, Travis JJ, Hunt JW. Direct measurement of free copper in serum or plasma ultrafiltrate. Am J Clin Pathol. 2009; 131(2): 160-5. doi: 10.1309 / AJCP7Z9KBFINVGYF. PubMed PMID: 19141375.

[0222] 7. Smith GP, Petrenko VA. Phage Display. Chem Rev. 1997;97(2):391-410. doi: 10.1021 / cr960065d. PubMed PMID: 11848876.

[0223] 8. Li X, Chen H, Khan AA, Lauterbach SB, Lanzillotti R, Rai PR, Kane RS, Coetzer TL, Chishti AH. Receptor-based identification of an inhibitory peptide against blood stage malaria. Biochem Biophys Res Commun. 2008;376(3):489-93. Epub 2008 / 09 / 17. doi: 10.1016 / j.bbrc.2008.09.011. PubMed PMID: 18793615; PMCID: 2574861.

[0224] 9. Li X, Marinkovic M, Russo C, McKnight CJ, Coetzer TL, Chishti AH. Identification of a specific region of Plasmodium falciparum EBL-1 that binds to host receptor glycophorin B and inhibits merozoite invasion in human red blood cells. Mol Biochem Parasitol. 2012; 183(l):23-31. Epub 2012 / 01 / 26. doi: 10.1016 / j.molbiopara.2012.01.002. PubMed PMID: 22273481; PMCID: 3307866.

[0225] 10. Almukadi H, Schwake C, Kaiser MM, Mayer DCG, Schiemer J, Baldwin MR, Hegde S, Lu Y, Hanada T, Chishti AH. Human erythrocyte band 3 is a host receptor for Plasmodium falciparum glutamic acid-rich protein. Blood. 2019;133(5):470-80. Epub 2018 / 12 / 14. doi: 10.1182 / blood-2018-07-865451. PubMed PMID: 30545833; PMCID: PMC6356982.

[0226] 11. Schwake CJ, Krueger RM, Hanada T, Chishti AH. Plasmodium falciparum glutamic acid-rich protein (PfGARP)-independent polyclonal antibodies inhibit malaria parasite growth in human erythrocytes. J Infect Dis. 2024. Epub 20240131. doi: 10.1093 / infdis / jiae050. PubMed PMID: 38298126.

[0227] 12. Crosnier C, Bustamante LY, Bartholdson SJ, Bei AK, Theron M, Uchikawa M, Mboup S, Ndir O, Kwiatkowski DP, Duraisingh MT, Rayner JC, Wright GJ. Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum. Nature. 2011;480(7378):534-7. Epub 2011 / 11 / 15. doi: 10.1038 / nature 10606. PubMed PMID: 22080952; PMCID: 3245779.

[0228] 13. Lee SK, Crosnier C, Valenzuela-Leon PC, Dizon BLP, Atkinson JP, Mu J, Wright GJ, Calvo E, Gunalan K, Miller LH. Complement receptor 1 is the human erythrocyte receptor for Plasmodium vivax erythrocyte binding protein. Proc Natl Acad Sci U S A. 2024; 121(5):e2316304121. Epub 20240123. doi: 10.1073 / pnas.2316304121. PubMed PMID: 38261617; PMCID: PMC10835065.

[0229] 14. Pope CR, Flores AG, Kaplan JH, Unger VM. Structure and function of copper uptake transporters. Curr Top Membr. 2012;69:97-112. doi: 10.1016 / B978-0- 12-394390-3.00004-5. PubMed PMID: 23046648.

[0230] 15. Jackson AP, Otto TD, Darby A, Ramaprasad A, Xia D, Echaide IE, Farber M, Gahlot S, Gamble J, Gupta D, Gupta Y, Jackson L, Malandrin L, Maias TB, Moussa E, Nair M, Reid AJ, Sanders M, Sharma J, Tracey A, Quail MA, Weir W, Wastling JM, Hall N, Willadsen P, Lingelbach K, Shiels B, Tait A, Berriman M, Allred DR, Pain A. The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction. Nucleic Acids Res. 2014;42(ll):7113-31. Epub 20140505. doi: 10.1093 / nar / gku322. PubMed PMID: 24799432; PMCID: PMC4066756.

[0231] 16. Guan G, Korhonen PK, Young ND, Koehler AV, Wang T, Li Y, Liu Z, Luo J, Yin H, Gasser RB. Genomic resources for a unique, low-virulence Babesia taxon from China. Parasit Vectors. 2016;9(l):564. Epub 20161027. doi: 10.1186 / S13071-016-1846-1. PubMed PMID: 27784333; PMCID: PMC5081931.

[0232] 17. Virji AZ, Thekkiniath J, Ma W, Lawres L, Knight J, Swei A, Roch KL, Mamoun CB. Insights into the evolution and drug susceptibility of Babesia duncani from the sequence of its mitochondrial and apicoplast genomes. Int J Parasitol. 2019;49(2):105-13. Epub 20180831. doi: 10.1016 / j.ijpara.2018.05.008. PubMed PMID: 30176236; PMCID: PMC6395566.

[0233] 18. Yamagishi J, Ceylan O, Xuan X, Sevinc F. Whole genome sequence and diversity in multigene families of Babesia ovis. Front Cell Infect Microbiol. 2023; 13: 1194608. Epub 20230801. doi: 10.3389 / fcimb.2023.1194608. PubMed PMID: 37662008; PMCID: PMC10471129.

[0234] 19. Yamagishi J, Asada M, Hakimi H, Tanaka TQ, Sugimoto C, Kawazu SI. Whole-genome assembly of Babesia ovata and comparative genomics between closely related pathogens. BMC genomics. 2017;18(l):832. Epub 20171027. doi: 10.1186 / S12864-017-4230-4. PubMed PMID: 29078748; PMCID: PMC5660447.

[0235] 20. Salman AA, Goldring JPD. Expression and copper binding studies of a Plasmodium falciparum protein with Cox 19 copper binding motifs. Exp Parasitol. 2023;251:108572. Epub 20230620. doi: 10.1016 / j.exppara.2023.108572. PubMed PMID: 37348640.

[0236] 21. Choveaux DL, Przyborski JM, Goldring JP. A Plasmodium falciparum copper-binding membrane protein with copper transport motifs. Malar J. 2012;l l:397. Epub 20121129. doi: 10.1186 / 1475-2875-11-397. PubMed PMID: 23190769; PMCID: PMC3528452.

[0237] 22. Maier AG, Rug M, O'Neill MT, Brown M, Chakravorty S, Szestak T, Chesson J, Wu Y, Hughes K, Coppel RL, Newbold C, Beeson JG, Craig A, Crabb BS, Cowman AF. Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes. Cell. 2008;134(l):48-61. Epub 2008 / 07 / 11. doi: 10.1016 / j.cell.2008.04.051. PubMed PMID: 18614010; PMCID: 2568870.

[0238] 23. Zhang M, Wang C, Otto TD, Oberstaller J, Liao X, Adapa SR, Udenze K, Bronner IF, Casandra D, Mayho M, Brown J, Li S, Swanson J, Rayner JC, Jiang RHY, Adams JH. Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis. Science. 2018;360(6388). Epub 2018 / 05 / 05. doi: 10.1126 / science.aap7847. PubMed PMID: 29724925.

[0239] 24. Li X, Chen H, Oh SS, Chishti AH. A Presenilin-like protease associated with Plasmodium falciparum micronemes is involved in erythrocyte invasion. Mol Biochem Parasitol. 2008;158(l):22-31. Epub 2007 / 12 / 28. doi: S0166- 6851(07)00313-1 [pii]

[0240] 10.1016 / j.molbiopara.2007.11.007. PubMed PMID: 18160114; PMCID: 2268099.

[0241] 25. Nakamura RM. Monoclonal antibodies: methods and clinical laboratory applications. Clin Physiol Biochem. 1983; 1(2-5): 160-72. Epub 1983 / 01 / 01. PubMed PMID: 6396016.

[0242] 26. Quinn BJ, Welch EJ, Kim AC, Lokuta MA, Huttenlocher A, Khan AA, Kuchay SM, Chishti AH. Erythrocyte scaffolding protein p55 / MPPl functions as an essential regulator of neutrophil polarity. Proc Natl Acad Sci U S A. 2009; 106(47): 19842-7. Epub 2009 / 11 / 10. doi: 10.1073 / pnas.0906761106. PubMed PMID: 19897731; PMCID: 2785254.

[0243] 27. Lu Y, Hanada T, Fujiwara Y, Nwankwo JO, Wieschhaus AJ, Hartwig J, Huang S, Han J, Chishti AH. Gene disruption of dematin causes precipitous loss of erythrocyte membrane stability and severe hemolytic anemia.Blood. 2016;128(l):93-103. doi: 10.1182 / blood-2016-01-692251. PubMed PMID: 27073223; PMCID: PMC4937361.

[0244] 28. Quarles CD, Jr., Macke M, Michalke B, Zischka H, Karst U, Sullivan P, Field MP. LC-ICP-MS method for the determination of "extractable copper" in serum. Metallomics. 2020; 12(9): 1348-55. doi: 10.1039 / d0mt00132e. PubMed PMID: 32789408.

[0245] 29. Schoeberl A, Gutmann M, Theiner S, Corte-Rodriguez M, Braun G, Vician P, Berger W, Koellensperger G. The copper transporter CTR1 and cisplatin accumulation at the single-cell level by LA-ICP-TOFMS. Front Mol Biosci. 2022;9: 1055356. Epub 20221128. doi: 10.3389 / fmolb.2022.1055356. PubMed PMID: 36518851; PMCID: PMC9742377.

[0246] 30. Horiguchi K, Hanada T, Fukui Y, Chishti AH. Transport of PIP3 by GAKIN, a kinesin-3 family protein, regulates neuronal cell polarity. J Cell Biol. 2006;174(3):425-36. PubMed PMID: 16864656.

[0247] 31. Songyang Z, Fanning AS, Fu C, Xu J, Marfatia SM, Chishti AH, Crompton A, Chan AC, Anderson JM, Cantley LC. Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science. 1997;275(5296):73-7.

[0248] 32. Morais Cabral JH, Petosa C, Sutcliffe MJ, Raza S, Byron O, Poy F, Marfatia SM, Chishti AH, Liddington RC. Crystal structure of a PDZ domain. Nature. 1996;382(6592):649-52.

[0249] 33. Puig S, Lee J, Lau M, Thiele DJ. Biochemical and genetic analyses of yeast and human high affinity copper transporters suggest a conserved mechanism for copper uptake. J Biol Chem. 2002;277(29):26021-30. Epub 20020430. doi: 10.1074 / jbc.M202547200. PubMed PMID: 11983704.

[0250] 34. Boulet A, Vest KE, Maynard MK, Gammon MG, Russell AC, Mathews AT, Cole SE, Zhu X, Phillips CB, Kwong JQ, Dodani SC, Leary SC, Cobine PA. The mammalian phosphate carrier SLC25A3 is a mitochondrial copper transporter required for cytochrome c oxidase biogenesis. J Biol Chem. 2018;293(6):1887-96. Epub 20171213. doi: 10.1074 / jbc.RAl 17.000265. PubMed PMID: 29237729; PMCID: PMC5808751.

[0251] 35. Zhu X, Boulet A, Buckley KM, Phillips CB, Gammon MG, Oldfather LE, Moore SA, Leary SC, Cobine PA. Mitochondrial copper and phosphatetransporter specificity was defined early in the evolution of eukaryotes. Elife. 2021;10. Epub 20210216. doi: 10.7554 / eLife.64690. PubMed PMID: 33591272; PMCID: PMC7924939.

[0252] 36. Lobo CA. Babesia divergens and Plasmodium falciparum use common receptors, glycophorins A and B, to invade the human red blood cell. Infect Immun. 2005;73(l):649-51. doi: 10.1128 / IAI.73.1.649-651.2005. PubMed PMID: 15618210; PMCID: PMC538995.

[0253] 37. Lobo CA, Rodriguez M, Cursino-Santos JR. Babesia and red cell invasion. Curr Opin Hematol. 2012; 19(3): 170-5. Epub 2012 / 04 / 11. doi: 10.1097 / MOH.0b013e328352245a. PubMed PMID: 22488304.

[0254] 38. Gaur D, Mayer DC, Miller LH. Parasite ligand-host receptor interactions during invasion of erythrocytes by Plasmodium merozoites. Int J Parasitol. 2004;34(13- 14): 1413-29. Epub 2004 / 12 / 08. doi: 10.1016 / j.ijpara.2004.10.010. PubMed PMID: 15582519.

[0255] 39. Jasik KP, Kleczka A, Franielczyk A. Histopathological Aspects of the Influence of Babesia microti on the Placentas of Infected Female Rats. Vet Sci. 2024; 11(1). Epub 20240103. doi: 10.3390 / vetscil 1010018. PubMed PMID: 38250924; PMCID: PMC10819886.

[0256] 40. Lutsenko S. Dynamic and cell-specific transport networks for intracellular copper ions. J Cell Sci. 2021;134(21). Epub 20211104. doi: 10.1242 / jcs.240523. PubMed PMID: 34734631; PMCID: PMC8627558.

[0257] 41. Maryon EB, Molloy SA, Ivy K, Yu H, Kaplan JH. Rate and regulation of copper transport by human copper transporter 1 (hCTRl). J Biol Chem. 2013;288(25): 18035-46. Epub 20130508. doi: 10.1074 / jbc.M 112.442426. PubMed PMID: 23658018; PMCID: PMC3689948.

[0258] 42. Borggraefe I, Yuan J, Telford SR, 3rd, Menon S, Hunter R, Shah S, Spielman A, Gelfand JA, Wortis HH, Vannier E. Babesia microti primarily invades mature erythrocytes in mice. Infect Immun. 2006;74(6):3204-12. Epub 2006 / 05 / 23. doi: 10.1128 / IAI.01560-05. PubMed PMID: 16714547; PMCID: PMC 1479280.

[0259] 43. Schwake C, Baldwin MR, Bachovchin W, Hegde S, Schiemer J, Okure C, Levin AE, Vannier E, Hanada T, Chishti AH. HIV protease inhibitorsblock parasite signal peptide peptidases and prevent growth of Babesia microti parasites in erythrocytes. Biochem Biophys Res Commun. 2019;517(1): 125-31. Epub 2019 / 07 / 18. doi: 10.1016 / j.bbrc.2019.07.031. PubMed PMID: 31311649; PMCID: PMC6707064.

[0260] 44. Schwake C, Hyon M, Chishti AH. Signal peptide peptidase: a potential therapeutic target for parasitic and viral infections. Expert Opin Ther Targets. 2022;26(3):261-73. Epub 20220307. doi: 10.1080 / 14728222.2022.2047932. PubMed PMID: 35235480; PMCID: PMC9210924.

[0261] 45. Baldwin MR, Li X, Hanada T, Liu SC, Chishti AH. Merozoite surface protein 1 recognition of host glycophorin A mediates malaria parasite invasion of red blood cells. Blood. 2015;125(17):2704-l l. Epub 2015 / 03 / 18. doi: 10.1182 / blood-2014-l 1-611707. PubMed PMID: 25778531; PMCID: PMC4408295.

[0262] 46. Fritz DI, Ding Y, Merrill-Skoloff G, Flaumenhaft R, Hanada T, Chishti AH. Dematin Regulates Calcium Mobilization, Thrombosis, and Early Akt Activation in Platelets. Mol Cell Biol. 2023;43(6):283-99. Epub 20230522. doi: 10.1080 / 10985549.2023.2210033. PubMed PMID: 37216480; PMCID: PMC10251785.

[0263] Table 1: Protein identification of positive phage clones bound to human erythrocytes. Intact erythrocytes were used as bait to capture bound phage clones from the Babesia microti cDNA library. Bound phages were eluted after several washes and amplified through four rounds of biopanning. Sequencing of phage clones predicted peptides / proteins that bound selectively to human erythrocytes. Clones with unknown function were not characterized further.

[0264] Table 2: Selected babesiosis patients with known clinical history.Selected patients with splenectomy and drug treatment for babesiosis were screened for the validation of ELISA by the detection of antibodies against Babesia microti antigens. Data from ELISA screens are shown in FIGS. 4A-4D and 5A-5C.Table 2 (ctd.)Table 2 (ctd.)OTHER EMBODIMENTS

[0265] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0266] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method for detecting Babesia microti in a biological sample, the method comprising: obtaining a biological sample containing antibodies; contacting the biological sample with a set of Babesia microti antigens; and determining that antibodies in the biological sample bind specifically to the set of Babesia microti antigens, thereby detecting Babesia microti, wherein the set of Babesia microti antigens includes: a polypeptide consisting of the amino acid sequence of BmSAl-PLl or an amino acid sequence having at least 85% amino acid identity to BmSAl-PLl and one or more polypeptides consisting of the amino acid sequences selected from BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2 or amino acid sequences having at least 85% amino acid identity to BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2.

2. The method of claim 1, wherein the set of Babesia microti antigens includes polypeptides consisting of the amino acid sequences of BmSAl-PLl, BMN1-20-PC1, and BM4.12-4PC2.

3. The method of claim 1 or 2, wherein the determining step is carried out by one or more of an enzyme-linked immunosorbent assay (ELISA), Western blot assay, and Lateral flow assay.

4. The method of any of claims 1 to 3, wherein the biological sample is blood plasma, blood serum, or whole blood.

5. A vaccine for ameliorating Babesia microti infection in a subject, the vaccine comprising one or more polypeptides consisting of the amino acid sequencesof BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2 or one or more vectors that express the one or more polypeptides.

6. The vaccine of claim 5, wherein the vaccine comprises the polypeptides consisting of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2.

7. The vaccine of claim 5, wherein the one or more vectors express the polypeptides consisting of the amino acid sequences of BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2.

8. A composition for detecting Babesia microti, the composition comprising polypeptides consisting of the amino acid sequence of BmSAl-PLl and one or more of BmSAl-RBD, BMN1-20-PC1 and BM4.12-4PC2 or polypeptides consisting of amino acid sequences having at least 85% amino acid identity to BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2.

9. The composition of claim 8, wherein the polypeptides consist of the amino acid sequences of BmSAl-PLl, BMN1-20-PC1, and BM4.12-4PC2.

10. A kit for detecting Babesia microti, the kit comprising: a solid support having immobilized thereon polypeptides consisting of the amino acid sequence of BmSAl-PLl and one or more of BmSAl-RBD, BMN1-20- PC1, and BM4.12-4PC2 or polypeptides consisting of amino acid sequences having at least 85% amino acid identity to BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2; and reagents for determining specific binding of antibodies to the immobilized polypeptides.

11. The kit of claim 10, wherein the polypeptides consist of the amino acid sequence of BmSAl-PLl, BMN1-20-PC1, and BM4.12-4PC2.

12. The kit of claims 10 or 11, wherein the reagents comprise an antihuman IgG antibody.

13. The kit of claim 12, wherein the anti-human IgG antibody is conjugated to a detection reagent.

14. A method for treating Babesia microti infection in a subject, the method comprising: obtaining a biological sample from the subject; contacting the biological sample with a set of Babesia microti antigens; determining that antibodies in the biological sample bind specifically to the set of Babesia microti antigens, thereby detecting Babesia microti; and treating the subject with an antimicrobial compound effective against Babesia microti, wherein the set of Babesia microti antigens includes polypeptides consisting of the amino acid sequence of BmSAl-PLl and one or more of BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2 or polypeptides consisting of amino acid sequences having at least 85% amino acid identity to BmSAl-PLl, BmSAl-RBD, BMN1-20-PC1, and BM4.12-4PC2.

15. The method of claim 14, wherein the polypeptides consist of the amino acid sequences of BmSAl-PLl, BMN1-20-PC1, and BM4.12-4PC2.

16. The method of claim 14 or 15, wherein the determining step is carried out by one or more of an enzyme-linked immunosorbent assay (ELISA), Western blot assay, and Lateral flow assay.

17. The method of any of claims 14 to 16, wherein the biological sample is blood plasma.

18. The method of any of claims 14 to 17, wherein the antimicrobial compound is selected from the group consisting of atovaquone, azithromycin, quinine, tafenoquine, and clindamycin.

Images