Pre-clinical evaluation of a whole-parasite vaccine to control human babesiosis.

Babesia spp. are tick-transmitted intra-erythrocytic protozoan parasites that infect humans and animals, causing a flu-like illness and hemolytic anemia. There is currently no human vaccine available. People most at risk of severe disease are the elderly, immunosuppressed, and asplenic individuals. B. microti and B. divergens are the predominant species affecting humans. Here, we present a whole-parasite Babesia vaccine. To establish proof-of-principle, we employed chemically attenuated B. microti parasitized red blood cells from infected mice. To aid clinical translation, we produced liposomes containing killed parasite material. Vaccination significantly reduces peak parasitemia following challenge. B cells and anti-parasite antibodies do not significantly contribute to vaccine efficacy. Protection is abrogated by the removal of CD4+ T cells or macrophages prior to challenge. Importantly, splenectomized mice are protected by vaccination. To further facilitate translation, we prepared a culture-based liposomal vaccine and demonstrate that this performs as a universal vaccine inducing immunity against different human Babesia species.

Molecular Characterization and Immunological Evaluation of Truncated Babesia microti Rhoptry Neck Protein 2 as a Vaccine Candidate.

Babesia microti is a protozoan that infects red blood cells. Babesiosis is becoming a new global threat impacting human health. Rhoptry neck proteins (RONs) are proteins located at the neck of the rhoptry and studies indicate that these proteins play an important role in the process of red blood cell invasion. In the present study, we report on the bioinformatic analysis, cloning, and recombinant gene expression of two truncated rhoptry neck proteins 2 (BmRON2), as well as their potential for incorporation in a candidate vaccine for babesiosis. Western blot and immunofluorescence antibody (IFA) assays were performed to detect the presence of specific antibodies against BmRON2 in infected mice and the localization of N-BmRON2 in B. microti parasites. In vitro experiments were carried out to investigate the role of BmRON2 proteins during the B. microti invasion process and in vivo experiments to investigate immunoprotection. Homologous sequence alignment and molecular phylogenetic analysis indicated that BmRON2 showed similarities with RON2 proteins of other Babesia species. We expressed the truncated N-terminal (33-336 aa, designated rN-BmRON2) and C-terminal (915-1171 aa, designated rC-BmRON2) fragments of the BmRON2 protein, with molecular weights of 70 and 29 kDa, respectively. Western blot assays showed that the native BmRON2 protein is approximately 170 kDa, and that rN-BmRON2 was recognized by serum of mice experimentally infected with B. microti. Immunofluorescence analysis indicated that the BmRON2 protein was located at the apical end of merozoites, at the opposite end of the nucleus. In vitro red blood cell invasion inhibition studies with B. microti rBmRON2 proteins showed that relative invasion rate of rN-BmRON2 and rC-BmRON2 group is 45 and 56%, respectively. Analysis of the host immune response after immunization and B. microti infection showed that both rN-BmRON2 and rC-BmRON2 enhanced the immune response, but that rN-BmRON2 conferred better protection than rC-BmRON2. In conclusion, our results indicate that truncated rhoptry neck protein 2, especially its N-terminal fragment (rN-BmRON2), plays an important role in the invasion of host red blood cells, confers immune protection, and shows good potential as a candidate vaccine against babesiosis.

Protein regulation strategies of the mouse spleen in response to Babesia microti infection.

BACKGROUND: Babesia is a protozoan parasite that infects red blood cells in some vertebrates. Some species of Babesia can induce zoonoses and cause considerable harm. As the largest immune organ in mammals, the spleen plays an important role in defending against Babesia infection. When infected with Babesia, the spleen is seriously injured but still actively initiates immunomodulatory responses. METHODS: To explore the molecular mechanisms underlying the immune regulation and self-repair of the spleen in response to infection, this study used data-independent acquisition (DIA) quantitative proteomics to analyse changes in expression levels of global proteins and in phosphorylation modification in spleen tissue after Babesia microti infection in mice. RESULTS: After mice were infected with B. microti, their spleens were seriously damaged. Using bioinformatics methods to analyse dynamic changes in a large number of proteins, we found that the spleen still initiated immune responses to combat the infection, with immune-related proteins playing an important role, including cathepsin D (CTSD), interferon-induced protein 44 (IFI44), interleukin-2 enhancer-binding factor 2 (ILF2), interleukin enhancer-binding factor 3 (ILF3) and signal transducer and activator of transcription 5A (STAT5A). In addition, some proteins related to iron metabolism were also involved in the repair of the spleen after B. microti infection, including serotransferrin, lactoferrin, transferrin receptor protein 1 (TfR1) and glutamate-cysteine ligase (GCL). At the same time, the expression and phosphorylation of proteins related to the growth and development of the spleen also changed, including protein kinase C-δ (PKC-δ), mitogen-activated protein kinase (MAPK) 3/1, growth factor receptor-bound protein 2 (Grb2) and P21-activated kinase 2 (PAK2). CONCLUSIONS: Immune-related proteins, iron metabolism-related proteins and growth and development-related proteins play an important role in the regulation of spleen injury and maintenance of homeostasis. This study provides an important basis for the diagnosis and treatment of babesiosis.

Exploration of Serum Marker Proteins in Mice Induced by Babesia microti Infection Using a Quantitative Proteomic Approach.

Babesia microti is a protozoan that mainly parasitizes rodent and human erythrocytes. B. microti infection can result in changes in the expression levels of various proteins in the host serum. To explore the mechanism underlying the regulation of serum proteins by the host during B. microti infection, this study used a data-independent acquisition (DIA) quantitative proteomic approach to perform comprehensive quantitative proteomic analysis on the serum of B. microti-infected mice. We identified and analysed 333 serum proteins during the infectious stage and recovery stage within 30 days of infection by B. microti in mice. Through quantitative analysis, we found 57 proteins differentially expressed in the infection stage and 69 proteins differentially expressed in the recovery stage. Bioinformatics analysis revealed that these differentially expressed proteins were mainly concentrated in organelles, cell parts, and extracellular regions that are mainly involved in immune system, metabolic, and cellular processes. Additionally, the differentially expressed proteins mainly had catalytic activity. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis showed that many of the differentially expressed proteins participate in the complement and coagulation cascade reaction, including complement C3, complement FP, and coagulation factor XII. The results of this study can provide more information for the selection of biomarkers for the early clinical monitoring of babesiosis and help in the treatment of babesiosis.

Antigen Discovery, Bioinformatics and Biological Characterization of Novel Immunodominant Babesia microti Antigens.

Babesia microti is an intraerythrocytic parasite and the primary causative agent of human babesiosis. It is transmitted by Ixodes ticks, transfusion of blood and blood products, organ donation, and perinatally. Despite its global public health impact, limited progress has been made to identify and characterize immunodominant B. microti antigens for diagnostic and vaccine use. Using genome-wide immunoscreening, we identified 56 B. microti antigens, including some previously uncharacterized antigens. Thirty of the most immunodominant B. microti antigens were expressed as recombinant proteins in E. coli. Among these, the combined use of two novel antigens and one previously described antigen provided 96% sensitivity and 100% specificity in identifying B. microti antibody containing sera in an ELISA. Using extensive computational sequence and bioinformatics analyses and cellular localization studies, we have clarified the domain architectures, potential biological functions, and evolutionary relationships of the most immunodominant B. microti antigens. Notably, we found that the BMN-family antigens are not monophyletic as currently annotated, but rather can be categorized into two evolutionary unrelated groups of BMN proteins respectively defined by two structurally distinct classes of extracellular domains. Our studies have enhanced the repertoire of immunodominant B. microti antigens, and assigned potential biological function to these antigens, which can be evaluated to develop novel assays and candidate vaccines.

Clofazimine, a Promising Drug for the Treatment of Babesia microti Infection in Severely Immunocompromised Hosts.

BACKGROUND: Persistent and relapsing babesiosis caused by Babesia microti often occurs in immunocompromised patients, and has been associated with resistance to antimicrobial agents such as atovaquone. Given the rising incidence of babesiosis in the United States, novel drugs are urgently needed. In the current study, we tested whether clofazimine (CFZ), an antibiotic used to treat leprosy and drug-resistant tuberculosis, is effective against B. microti. METHODS: Mice with severe combined immunodeficiency were infected with 107B. microti-infected erythrocytes. Parasites were detected by means of microscopic examination of Giemsa-stained blood smears or nested polymerase chain reaction. CFZ was administered orally. RESULTS: Uninterrupted monotherapy with CFZ curtailed the rise of parasitemia and achieved radical cure. B. microti parasites and B. microti DNA were cleared by days 10 and 50 of therapy, respectively. A 7-day administration of CFZ delayed the rise of parasitemia by 22 days. This rise was caused by B. microti isolates that did not carry mutations in the cytochrome b gene. Accordingly, a 14-day administration of CFZ was sufficient to resolve high-grade parasitemia caused by atovaquone-resistant B. microti parasites. CONCLUSIONS: Clofazimine is effective against B. microti infection in the immunocompromised host. Additional preclinical studies are required to identify the minimal dose and dosage of CFZ for babesiosis.

Inclusion of PD-L1 into a recombinant profilin antigen enhances immunity against Babesia microti in a murine model.

Pathogens and cancer cells employ the programmed cell death-Ligand 1 (PD-L1)/ programmed cell death-1 (PD-1) signaling pathway to inhibit the immune response. Hence, blockade of PD-L1/PD-1 recognition through monoclonal antibodies enhances the immune response. Antibodies that block PD-L1 and PD-1 binding have been used for the prevention and therapy of human pathogenic diseases, but have not yet been evaluated for the treatment of infectious diseases of livestock. In the present study, a recombinant vaccine named PROF-PDL1E, was designed comprising the Babesia microti-derived vaccine candidate profilin and the host PD-L1 protein, and its effect on immunization against murine B. microti infection was evaluated. PD-L1-specific antibodies generated after vaccination blocked PD-L1 and PD-1 binding as shown by in vitro assays. PROF-PDL1E reduced the burden of B. microti in a mouse model and decreased PD-1 expression in T cells. Furthermore, no tissue damage could be observed after PROF-PDL1E vaccination as verified by hematoxylin and eosin tissue staining of essential organs. In conclusion, vaccines targeting immune checkpoints seem to be a promising strategy for anti-Babesia vaccine development.

Evidence for vesicle-mediated antigen export by the human pathogen Babesia microti.

The apicomplexan parasite Babesia microti is the primary agent of human babesiosis, a malaria-like illness and potentially fatal tick-borne disease. Unlike its close relatives, the agents of human malaria, B. microti develops within human and mouse red blood cells in the absence of a parasitophorous vacuole, and its secreted antigens lack trafficking motifs found in malarial secreted antigens. Here, we show that after invasion of erythrocytes, B. microti undergoes a major morphogenic change during which it produces an interlacement of vesicles (IOV); the IOV system extends from the plasma membrane of the parasite into the cytoplasm of the host erythrocyte. We developed antibodies against two immunodominant antigens of the parasite and used them in cell fractionation studies and fluorescence and immunoelectron microscopy analyses to monitor the mode of secretion of B. microti antigens. These analyses demonstrate that the IOV system serves as a major export mechanism for important antigens of B. microti and represents a novel mechanism for delivery of parasite effectors into the host by this apicomplexan parasite.

Vaccination against babesiosis using recombinant GPI-anchored proteins.

The increase in human babesiosis is of major concern to health authorities. In the USA, most of these cases are due to infections with Babesia microti, whereas in Europe B. divergens is the major cause of clinical disease in humans. Here we review the immunological and biological literature of glycosylphosphatidylinositol (GPI)-anchored merozoite proteins of human Babesia parasites with emphasis on their role in immunity, and provide some new bioinformatical information on B. microti GPI-Anchored Proteins (GPI-AP). Cattle can be vaccinated with soluble parasite antigens (SPA) of Babesia divergens that are released by the parasite during proliferation. The major component in SPA preparations appeared to be a 37 kDa merozoite surface protein that is anchored in the merozoite membrane by a GPI anchor. Animals could be protected by vaccination with the recombinant 37 kDa protein expressed in Escherichia coli, provided the protein had a hydrophobic terminal sequence. Based on this knowledge, a recombinant vaccine was developed against Babesia canis infection in dogs, successfully. In order to identify similar GPI-AP in B. microti, the genome was analysed. Here it is shown that B. microti encodes all proteins necessary for GPI assembly and its subsequent protein transfer. In addition, in total 21 genes encoding for GPI-AP were detected, some of which reacted particularly strongly with sera from B. microti-infected human patients. Reactivity of antibodies with GPI-anchored merozoite proteins appears to be dependent on the structural conformation of the molecule. It is suggested that the three-dimensional structure of the protein that is anchored in the membrane is different from that of the protein that has been shed from the merozoite surface. The significance of this protein's dynamics in parasite biology and immune evasion is discussed. Finally, we discuss developments in tick and Babesia vaccine research, and the role such vaccines could play in the control of human babesiosis.

A library of recombinant Babesia microti cell surface and secreted proteins for diagnostics discovery and reverse vaccinology.

Human babesiosis is an emerging tick-borne parasitic disease and blood transfusion-transmitted infection primarily caused by the apicomplexan parasite, Babesia microti. There is no licensed vaccine for B. microti and the development of a reliable serological screening test would contribute to ensuring the safety of the donated blood supply. The recent sequencing of the B. microti genome has revealed many novel genes encoding proteins that can now be tested for their suitability as subunit vaccine candidates and diagnostic serological markers. Extracellular proteins are considered excellent vaccine candidates and serological markers because they are directly exposed to the host humoral immune system, but can be challenging to express as soluble recombinant proteins. We have recently developed an approach based on a mammalian expression system that can produce large panels of functional recombinant cell surface and secreted parasite proteins. Here, we use the B. microti genome sequence to identify 54 genes that are predicted to encode surface-displayed and secreted proteins expressed during the blood stages, and show that 41 (76%) are expressed using our method at detectable levels. We demonstrate that the proteins contain conformational, heat-labile, epitopes and use them to serologically profile the kinetics of the humoral immune responses to two strains of B. microti in a murine infection model. Using sera from validated human infections, we show a concordance in the host antibody responses to B. microti infections in mouse and human hosts. Finally, we show that BmSA1 expressed in mammalian cells can elicit high antibody titres in vaccinated mice using a human-compatible adjuvant but these antibodies did not affect the pathology of infection in vivo. Our library of recombinant B. microti cell surface and secreted antigens constitutes a valuable resource that could contribute to the development of a serological diagnostic test, vaccines, and elucidate the molecular basis of host-parasite interactions.

Robust adaptive immune response against Babesia microti infection marked by low parasitemia in a murine model of sickle cell disease.

The intraerythrocytic parasite Babesia microti is the number 1 cause of transfusion-transmitted infection and can induce serious, often life-threatening complications in immunocompromised individuals including transfusion-dependent patients with sickle cell disease (SCD). Despite the existence of strong long-lasting immunological protection against a second infection in mouse models, little is known about the cell types or the kinetics of protective adaptive immunity mounted following Babesia infection, especially in infection-prone SCD that are thought to have an impaired immune system. Here, we show, using a mouse B microti infection model, that infected wild-type (WT) mice mount a very strong adaptive immune response, characterized by (1) coordinated induction of a robust germinal center (GC) reaction; (2) development of follicular helper T (TFH) cells that comprise ∼30% of splenic CD4+ T cells at peak expansion by 10 days postinfection; and (3) high levels of effector T-cell cytokines, including interleukin 21 and interferon γ, with an increase in the secretion of antigen (Ag)-specific antibodies (Abs). Strikingly, the Townes SCD mouse model had significantly lower levels of parasitemia. Despite a highly disorganized splenic architecture before infection, these mice elicited a surprisingly robust adaptive immune response (including comparable levels of GC B cells, TFH cells, and effector cytokines as control and sickle trait mice), but higher immunoglobulin G responses against 2 Babesia-specific proteins, which may contain potential immunogenic epitopes. Together, these studies establish the robust emergence of adaptive immunity to Babesia even in immunologically compromised SCD mice. Identification of potentially immunogenic epitopes has implications to identify long-term carriers, and aid Ag-specific vaccine development.

Screening for biomarkers reflecting the progression of Babesia microti infection.

BACKGROUND: Babesiosis is caused by the invasion of erythrocytes by parasites of the Babesia spp. Babesia microti is one of the primary causative agents of human babesiosis. To better understand the status of the disease, discovering key biomarkers of the different infection stages is crucial. RESULTS: This study investigated B. microti infection in the mouse model from 0 to 270 days post-infection (dpi), using blood smears, PCR assays and ELISA. PCR assays showed a higher sensitivity when compared to microscopic examination. Specific IgG antibodies could be detected from 7 days to 270 dpi. Two-dimensional electrophoresis was combined with western blotting and mass spectrometric analysis to screen for specific reactive antigens during both the peak parasitaemia period (7 dpi) and IgG antibody response peak period (30 dpi) by the infected mice plasma. The 87 positive reactive proteins were identified and then expressed with the wheat germ cell-free system. Protein microarrays of all 87 targeted proteins were produced and hybridized with the serial plasma of infected mice model. Based on the antigen reaction profile during the infection procedure, 6 antigens were selected and expressed in Escherichia coli. Due to an early response to IgM, lower immunoreactivity levels of IgG after two months and higher immunoreactivity level IgG during nine months, four recombinant proteins were selected for further characterization, namely rBm2D97(CCF75281.1), rBm2D33(CCF74637.1), rBm2D41(CCF75408.1) and rBm7(CCF73510.1). The diagnostic efficacy of the four recombinant protein candidates was evaluated in a clinical setting using babesiosis patient plasma. The rBm2D33 showed the highest sensitivity with a positive rate of 62.5%. Additional characterization of the two candidate proteins using a mouse vaccination assay, demonstrated that rBm2D41 could reduce peak parasitaemia by 37.4%, indicating its efficacy in preventing severe babesiosis. CONCLUSIONS: The detection technologies of microscopic examination, PCR assays and antibody tests showed different sensitivities and accuracy during the different stages of B. microti infection. Antibody detection has a unique significance for B. microti infection in the asymptomatic stages. Using immunoreactivity profiles, biomarkers for disease progression were identified and represent useful information for future the diagnosis and vaccine development for this serious disease of public health significance.

Performance Evaluation of a Prototype Architect Antibody Assay for Babesia microti.

The tick-borne protozoan Babesia microti is responsible for more than 200 cases of transfusion-transmitted babesiosis (TTB) infection in the United States that have occurred over the last 30 years. Measures to mitigate the risk of TTB include nucleic acid testing (NAT) and B. microti antibody testing. A fully automated prototype B. microti antibody test was developed on the Architect instrument. The specificity was determined to be 99.98% in volunteer blood donors (n = 28,740) from areas considered to have low endemicity for B. microti The sensitivity of the prototype test was studied in experimentally infected macaques; a total of 128 samples were detected as positive whereas 125 were detected as positive with an indirect fluorescent antibody (IFA) test; additionally, 83 (89.2%) of the PCR-positive samples were detected in contrast to 81 (87.1%) using an IFA test. All PCR-positive samples that tested negative in the prototype antibody test were preseroconversion period samples. Following seroconversion, periods of intermittent parasitemia occurred; 17 PCR-negative samples drawn in between PCR-positive bleed dates tested positive both by the prototype test (robust reactivity) and IFA test (marginal reactivity) prior to the administration of therapeutic drugs, indicating that the PCR test failed to detect samples from persistently infected macaques. The prototype assay detected 56 of 58 (96.6%) human subjects diagnosed with clinical babesiosis by both PCR and IFA testing. Overall, the prototype anti-Babesia assay provides a highly sensitive and specific test for the diagnosis of B. microti infection. While PCR is preferred for detection of window-period parasitemia, antibody tests detect infected subjects during periods of low-level parasitemia.

Superior real-time polymerase chain reaction detection of Babesia microti parasites in whole blood utilizing high-copy BMN antigens as amplification targets.

BACKGROUND: Babesiosis is a zoonotic disease transmitted to humans by the bite of infected ticks and caused by apicomplexan parasites, most commonly Babesia microti. Additionally, blood and blood products collected from asymptomatically infected blood donors may cause transfusion-transmitted infections in recipients. Highly sensitive molecular assays that detect parasite nucleic acid are needed for laboratory diagnosis and to identify and defer clinically silent but parasitemic blood donors. STUDY DESIGN AND METHODS: Here we report the development and analytical and clinical characterization of a real-time polymerase chain reaction (RT-PCR)-based assay for the detection of B. microti genomic DNA in whole blood. We evaluate the detection of Babesia parasites using two separate targets, the traditional18S ribosomal subunit gene (Bm18S) and members of the abundant BMN family of seroreactive antigens (BmBMN). RESULTS: Analytical sensitivity determination using a probit analysis demonstrated an analytical sensitivity of 30.9 parasites/mL for 18S amplification and 10.0 parasites/mL for BMN amplification The BMN primer set also demonstrates superior sensitivity for serial dilution panels prepared from clinically diagnosed Babesia-infected blood samples, generally detecting 10-fold more dilute nucleic acid. CONCLUSIONS: Cumulatively, our data demonstrate that RT-PCR detection of the BMN family of seroreactive antigens reflects a sensitive and superior assay for the detection of B. microti in whole blood samples.

Evaluation of the protective effect of a prime-boost strategy with plasmid DNA followed by recombinant adenovirus expressing BmAMA1 as vaccines against Babesia microti infection in hamster.

In the present study, we have investigated the protective effect of a heterologous prime-boost strategy with priming plasmid DNA followed by recombinant adenovirus, both expressing BmAMA1, against Babesia microti infection. Four groups consisting of 3 hamsters per group were immunized with pBmAMA1/Ad5BmAMA1, pNull/Ad5BmAMA1, pBmAMA1/Ad5Null and pNull/Ad5Null, followed by challenge infection with B. microti. Our results showed that hamsters immunized with plasmid and adenovirus expressing BmAMA1 developed a robust IgG and IgG2a antibody response against BmAMA1, suggesting the DNA vaccine or viral vector vaccine tend to induce a Th1-biased response. Compared to the control hamsters, the hamsters vaccinated either with the prime-boost strategy or one of the two "vaccines" exhibited no significant protection against B. microti challenge. Although a slight difference in terms of parasitemia and hematocrit values at days 14-16 post challenge infection was observed, no other statistical difference was detected. Our results indicate that the prime-boost vaccination strategy of injection of plasmid and adenovirus expressing BmAMA1 is not efficient in protecting against B. microti infection.

Babesia microti and Malaria Infection in Africa: A Pilot Serosurvey in Kilosa District, Tanzania.

Babesia is a tick-borne intraerythrocytic parasite that is clinically and diagnostically similar to malaria parasite, conferring risk of misdiagnosis in areas where both parasites are endemic. Data on Babesia in humans in Africa are lacking, despite evidence that it is present in regional animal populations. Samples that were collected in November 2014 to July 2015 in Kilosa district, Tanzania, were evaluated for evidence of malaria and Babesia infection. Clinical data and laboratory samples (i.e., hemoglobin, rapid diagnostic testing [RDT] for malaria, peripheral blood smear, and dried blood spots) from a routine survey were available for analysis. Dried blood spots were tested using an investigational enzyme linked immunosorbent assay (ELISA) against Babesia microti. A total of 1,030 children aged 1 month to < 5 years were evaluated; 186 (18.1%) were malaria RDT positive, 180 (96.8%) of whom had peripheral smears reviewed; 70/180 (38.9%) were smear positive for parasites. The median (inter quartile range) and range of B. microti ELISA signal to cutoff (S/C) ratio was 0.10 (0.06-0.15) and 0.01-1.65, respectively; the S/C ratios were significantly higher in subjects ≥ 1 year as compared with those < 1 year old (P < 0.001). There was also a statistically significant association between a positive RDT for malaria and the Babesia S/C (median 0.09 versus 0.13 in RDT negative versus RDT positive, respectively; P < 0.001). The highest S/C ratios were disproportionately clustered in a few hamlets. The findings suggest that Babesia may be present in Kilosa district, Tanzania. However, serological cross-reactivity and false positivity, notably between Babesia and Plasmodium spp., cannot be definitively excluded and have implications for testing in other settings.

A pilot serosurvey of Babesia microti in Chinese blood donors.

BACKGROUND AND OBJECTIVES: Babesia spp. are tick-borne, intraerythrocytic protozoan parasites, several of which are transfusion-transmissible. Transfusion-transmitted babesiosis poses serious risk to a diverse patient population, including neonates, patients aged >50 years, the asplenic and the immunocompromised that are over-represented among transfusion recipients. Despite reports of B. microti and B. venatorum in People's Republic of China (PRC), no surveillance of blood donors for Babesia has previously been undertaken. We sought to determine the rates of B. microti seroreactivity in a sample of blood donors in the PRC. MATERIALS AND METHODS: A pilot serosurvey was conducted of community blood donors (n = 1000) who donated July-August 2016 at Mudanjiang Blood Center (Heilongjiang Province) using indirect fluorescent antibody testing for antibodies against B. microti. The slides were prepared using B. microti-infected hamster blood. Samples that were initially positive to a titre of 64 were subjected to repeat IFA testing. Final seroreactivity was based on repeat reactivity to ≥64. RESULTS: A total of 1000 individual donor samples were evaluated, comprising 888 whole blood and 112 platelet donations. Thirteen of 1000 (1·3%) donors were seroreactive for B. microti [8 (0·8%) and five (0·05%) at titres of 64 and 128, respectively]. CONCLUSION: Our preliminary findings support the need for expanded Babesia surveillance in Chinese blood donors, replete with molecular evaluation, to evaluate the risk to the blood supply.

Molecular characterization of Babesia microti seroreactive antigen 5-1-1 and development of rapid detection methods for anti-B. microti antibodies in serum.

Babesiosis has become a new global threat impacting human health, and most human babesiosis cases are caused by Babesia microti. Until now few antigens of B. microti have been described which can be used for the diagnosis of human babesiosis. In the present study, we report on the bioinformatic analysis, cloning and expression of the sequence encoding the B. microti seroreactive antigen 5-1-1 to investigate its potential incorporation in serologic diagnostic tools for babesiosis. Bioinformatic analysis and recombinant gene expression were performed to molecularly characterize seroreactive antigen 5-1-1. Enhanced chemiluminescence (ECL)-Western blot methods were used to detect specific antibodies in infected mice. Immunofluorescence antibody assays (IFA) were performed to detect the localization of BmSA5-1-1 in B. microti parasites. ELISA and immunochromatographic (ICT) tests were developed using recombinant BmSA5-1-1 to evaluate its potential use in rapid detection methods for B. microti antibodies and for the diagnosis of babesiosis. A recombinant expression plasmid was constructed by inserting the target gene fragment in the pET28a vector after double digestion with BamHI and XhoI restriction enzymes. The recombinant BmSA5-1-1 protein was expressed in Escherichia coli (rBmSA5-1-1) and purified by means of Ni-nitrilotriacetic acid (NTA) agarose columns. Polyclonal antibodies were generated against rBmSA5-1-1. Based on indirect immunofluorescence assay results, BmSA5-1-1 appeared to localize on the surface of B. microti. ELISA tests using the rBmSA5-1-1 antigen detected specific antibodies from infected mice as early as 4 days post-infection. Our results indicate that the two methods we developed can detect specific antibodies in mice at different stages of infection with sensitivities of 100% (rBmSA5-1-1 ELISA) and 90% (ICT). The specificity of the two methods was 100%. Sera of patients suffering from other closely related parasitic diseases, such as malaria and toxoplasmosis, produced negative results. In conclusion, seroreactive antigen 5-1-1, a member of the BMN1 protein family, is expressed on the outer surface of B. microti and is a promising candidate antigen for the early diagnosis of babesiosis. rBmSA5-1-1 ELISA and ICT methods show good potential for detecting specific antibodies in mice at different stages of infection.

Analysis of antigen-antibody cross-reactivity among lineages and sublineages of Babesia microti parasites using human babesiosis specimens.

BACKGROUND: Human babesiosis is caused mainly by Babesia microti and has recently become a public health concern due to an increase in transfusion-transmitted infection. Thus, the development of an antibody detection method with high specificity and sensitivity is a priority. Seroreactivity against B. microti has been reported to be highly specific not only to B. microti lineages but also to sublineages. This study aimed to elucidate the human antibody reactivity against various lineages, including US, Kobe, and Hobetsu, and sublineages (North America and East Asia) in the US lineage. STUDY DESIGN AND METHODS: Twenty samples obtained from individuals infected with B. microti in the United States were tested for the presence of anti-B. microti antibodies using indirect immunofluorescence assay (IFA) and Western blotting (WB) to indicate antigens of each (sub-)lineage. RESULTS: By IFA, 20 samples showed reactivity to the North America sublineage (titer range, 64-4096), 16 to the East Asia sublineage (64-512), 10 to the Kobe (64-128), and five to the Hobetsu (64). Antibody titers to the East Asia sublineage, Kobe, and Hobetsu were significantly lower than those to the North America sublineage (p < 0.01). By WB, in parallel with the IFA results, 18 samples showed strong reactions to the North America sublineage, weak reactions to the East Asia sublineage, and near-zero reactions to the Kobe and Hobetsu. CONCLUSION: Human antibodies induced by B. microti infection are highly specific against B. microti lineages and sublineages with low cross-reactivity. Developing a precise antibody detection method may require specific antigens based on B. microti lineages and sublineages.