Expression, Purification, and Biological Characterization of Babesia microti Apical Membrane Antigen 1.

The intraerythrocytic apicomplexan Babesia microti, the primary causative agent of human babesiosis, is a major public health concern in the United States and elsewhere. Apicomplexans utilize a multiprotein complex that includes a type I membrane protein called apical membrane antigen 1 (AMA1) to invade host cells. We have isolated the full-length B. microti AMA1 (BmAMA1) gene and determined its nucleotide sequence, as well as the amino acid sequence of the AMA1 protein. This protein contains an N-terminal signal sequence, an extracellular region, a transmembrane region, and a short conserved cytoplasmic tail. It shows the same domain organization as the AMA1 orthologs from piroplasm, coccidian, and haemosporidian apicomplexans but differs from all other currently known piroplasmida, including other Babesia and Theileria species, in lacking two conserved cysteines in highly variable domain III of the extracellular region. Minimal polymorphism was detected in BmAMA1 gene sequences of parasite isolates from six babesiosis patients from Nantucket. Immunofluorescence microscopy studies showed that BmAMA1 is localized on the cell surface and cytoplasm near the apical end of the parasite. Native BmAMA1 from parasite lysate and refolded recombinant BmAMA1 (rBmAMA1) expressed in Escherichia coli reacted with a mouse anti-BmAMA1 antibody using Western blotting. In vitro binding studies showed that both native BmAMA1 and rBmAMA1 bind to human red blood cells (RBCs). This binding is trypsin and chymotrypsin treatment sensitive but neuraminidase independent. Incubation of B. microti parasites in human RBCs with a mouse anti-BmAMA1 antibody inhibited parasite growth by 80% in a 24-h assay. Based on its antigenically conserved nature and potential role in RBC invasion, BmAMA1 should be evaluated as a vaccine candidate.

CD47 regulates the phagocytic clearance and replication of the Plasmodium yoelii malaria parasite.

Several Plasmodium species exhibit a strong age-based preference for the red blood cells (RBC) they infect, which in turn is a major determinant of disease severity and pathogenesis. The molecular basis underlying this age constraint on the use of RBC and its influence on parasite burden is poorly understood. CD47 is a marker of self on most cells, including RBC, which, in conjunction with signal regulatory protein alpha (expressed on macrophages), prevents the clearance of cells by the immune system. In this report, we have investigated the role of CD47 on the growth and survival of nonlethal Plasmodium yoelii 17XNL (PyNL) malaria in C57BL/6 mice. By using a quantitative biotin-labeling procedure and a GFP-expressing parasite, we demonstrate that PyNL parasites preferentially infect high levels of CD47 (CD47(hi))-expressing young RBC. Importantly, C57BL/6 CD47(-/-) mice were highly resistant to PyNL infection and developed a 9.3-fold lower peak parasitemia than their wild-type (WT) counterparts. The enhanced resistance to malaria observed in CD47(-/-) mice was associated with a higher percentage of splenic F4/80(+) cells, and these cells had a higher percentage of phagocytized parasitized RBC than infected WT mice during the acute phase of infection, when parasitemia was rapidly rising. Furthermore, injection of CD47-neutralizing antibody caused a significant reduction in parasite burden in WT C57BL/6 mice. Together, these results strongly suggest that CD47(hi) young RBC may provide a shield to the malaria parasite from clearance by the phagocytic cells, which may be an immune escape mechanism used by Plasmodium parasites that preferentially infect young RBC.

A slot blot immunoassay for quantitative detection of Plasmodium falciparum circumsporozoite protein in mosquito midgut oocyst.

There is still a need for sensitive and reproducible immunoassays for quantitative detection of malarial antigens in preclinical and clinical phases of vaccine development and in epidemiology and surveillance studies, particularly in the vector host. Here we report the results of sensitivity and reproducibility studies for a research-grade, quantitative enhanced chemiluminescent-based slot blot assay (ECL-SB) for detection of both recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP) and native PfCSP from Oocysts (Pf Oocyst) developing in the midguts of Anopheles stephensi mosquitoes. The ECL-SB detects as little as 1.25 pg of rPfCSP (linear range of quantitation 2.5-20 pg; R2 = 0.9505). We also find the earliest detectable expression of native PfCSP in Pf Oocyst by ECL-SB occurs on day 7 post feeding with infected blood meal. The ECL-SB was able to detect approximately as few as 0.5 day 8 Pf Oocysts (linear quantitation range 1-4, R2 = 0.9795) and determined that one Pf Oocyst expressed approximately 2.0 pg (0.5-3 pg) of native PfCSP, suggesting a similar range of detection for recombinant and native forms of Pf CSP. The ECL-SB is highly reproducible; the Coefficient of Variation (CV) for inter-assay variability for rPf CSP and native PfCSP were 1.74% and 1.32%, respectively. The CVs for intra-assay variability performed on three days for rPf CSP were 2.41%, 0.82% and 2% and for native Pf CSP 1.52%, 0.57%, and 1.86%, respectively. In addition, the ECL-SB was comparable to microscopy in determining the P. falciparum prevalence in mosquito populations that distinctly contained either high and low midgut Pf Oocyst burden. In whole mosquito samples, estimations of positivity for P. falciparum in the high and low burden groups were 83.3% and 23.3% by ECL-SB and 85.7% and 27.6% by microscopy. Based on its performance characteristics, ECL-SB could be valuable in vaccine development and to measure the parasite prevalence in mosquitoes and transmission-blocking interventions in endemic areas.

T-bet modulates the antibody response and immune protection during murine malaria.

CD4(+) T-cell subtypes govern the synthesis of different Ab isotypes and other immune functions. The influence of CD4(+) T-cell differentiation programs on isotype switching and other aspects of host immunological networks during malaria infection are currently poorly understood. Here, we used Tbx21(-/-) mice deficient for T-bet, a regulator of Th1 CD4(+) T-cell differentiation, to examine the effect of Th1 CD4(+) T cells on the immune protection to nonlethal murine malaria Plasmodium yoelii 17XNL. We found that Tbx21(-/-) mice exhibited significantly lower parasite burden that correlated with elevated levels of IgG1, indicating that T-bet-dependent Ab isotype switching may be responsible for lower parasite burden. Absence of T-bet was also associated with a transient but significant loss of T cells during the infection, suggesting that T-bet may suppress malaria-induced apoptosis or induce proliferation of T cells. However, Tbx21(-/-) mice produced greater numbers of Foxp3(+) CD25(+) regulatory CD4(+) T cells, which may contribute to the early contraction of T cells. Lastly, Tbx21(-/-) mice exhibited unimpaired production of IFN-γ by a diverse repertoire of immune cell subsets and a selective expansion of IFN-γ-producing T cells. These observations may have implications in malaria vaccine design.

Western blot assay for quantitative and qualitative antigen detection in vaccine development.

Immunological methods for quantitative measurement, antigenic characterization, and monitoring the stability of active immunogenic component(s) are a critical need in the vaccine development process. This unit describes an enhanced chemiluminescence-based western blot for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP), a major malaria candidate vaccine antigen. The most salient features of this assay are its high sensitivity and reproducibility; it can reliably detect ∼5 to 10 pg PfCSP expressed on native parasites or recombinantly expressed in Escherichia coli. Although described for a specific vaccine antigen, this assay should be applicable for any antigen-antibody combination for which relevant detection reagents are available. Detailed stepwise experimental procedures and methods for data acquisition and analysis are described.

The transcription factor T-bet regulates parasitemia and promotes pathogenesis during Plasmodium berghei ANKA murine malaria.

The pathogenesis of experimental cerebral malaria (ECM) is an immunologic process, mediated in part by Th1 CD4(+) T cells. However, the role of the Th1 CD4(+) T cell differentiation program on the ability to control parasitemia and susceptibility to ECM disease during blood stage malaria has never been assessed directly. Using the Plasmodium berghei ANKA murine model of ECM and mice deficient for the transcription factor T-bet (the master regulator of Th1 cells) on the susceptible C57BL/6 background, we demonstrate that although T-bet plays a role in the regulation of parasite burden, it also promotes the pathogenesis of ECM. T-bet-deficient (Tbx21(-/-)) mice had higher parasitemia than wild type controls did during the ECM phase of disease (17.7 ± 3.1% versus 10.9 ± 1.5%). In addition, although 100% (10/10) of wild type mice developed ECM by day 9 after infection, only 30% (3/10) of Tbx21(-/-) mice succumbed to disease during the cerebral phase of infection. Resistance to ECM in Tbx21(-/-) mice was associated with diminished numbers of IFN-γ-producing CD4(+) T cells in the spleen and a lower accumulation of CD4(+) and CD8(+) T cells in the brain. An augmented Th2 immune response characterized by enhanced production of activated GATA-3(+) CD4(+) T cells and elevated levels of the eotaxin, MCP-1, and G-CSF cytokines was observed in the absence of T-bet. Our results suggest that in virulent malarias, immune modulation or therapy resulting in an early shift toward a Th2 response may help to ameliorate the most severe consequences of malaria immunopathogenesis and the prospect of host survival.

A chemiluminescent-western blot assay for quantitative detection of Plasmodium falciparum circumsporozoite protein.

Highly sensitive and reliable assays based on the quantitation of immunologically relevant component(s) in recombinant or whole parasite-based vaccines would facilitate pre-clinical and clinical phases and the monitoring of malaria vaccine deployment. Here we report a laboratory-grade Western Blot assay for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP) in P. falciparum sporozoite (PfSPZ) and in recombinant (rPfCSP) product. This assay is based on the immuno-reactivity of an anti-P. falciparum CSP monoclonal antibody (mAb 2A10) with the NANP-repeat units on PfCSP. The antigen-antibody complex is detected by reaction with a commercially obtained chemiluminescence-linked Immunodetection system. The linear range for detecting the recombinant P. falciparum CSP (rPfCSP) in this assay is 3-12pg (R(2)=0.9399). The range for detecting the day 15 salivary-gland PfSPZ is between 0.0625 and 1 parasite (R(2)=0.9448) and approximately 10.0pg of PfCSP was detected on each sporozoite. The assay was highly reproducible in measuring the PfCSP on PfSPZ. The inter-assay Coefficient of Variation (CV%) was 10.31% while the intra-assay CV% on three different days was 6.05%, 2.03% and 1.42% respectively. These results suggest that this ECL-WB assay is highly sensitive and robust with a low degree of inter-assay and intra-assay variations. To our knowledge, this is the most sensitive immunoassay for the detection of a recombinant or native malarial protein and may have a wider range of applications including the quantification of immunological component(s) in a vaccine formulation, determination of the antigenic integrity in adjuvanted-vaccine and in stability studies. In addition, this assay can be applied to measure the mosquito infectivity in malaria transmission areas and to determine the effects of intervention measures on malaria transmission.

Leishmania donovani argininosuccinate synthase is an active enzyme associated with parasite pathogenesis.

BACKGROUND: Gene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver. CONCLUSION/SIGNIFICANCE: Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target.

Protection from experimental cerebral malaria with a single dose of radiation-attenuated, blood-stage Plasmodium berghei parasites.

BACKGROUND: Whole malaria parasites are highly effective in inducing immunity against malaria. Due to the limited success of subunit based vaccines in clinical studies, there has been a renewed interest in whole parasite-based malaria vaccines. Apart from attenuated sporozoites, there have also been efforts to use live asexual stage parasites as vaccine immunogens. METHODOLOGY AND RESULTS: We used radiation exposure to attenuate the highly virulent asexual blood stages of the murine malaria parasite P. berghei to a non-replicable, avirulent form. We tested the ability of the attenuated blood stage parasites to induce immunity to parasitemia and the symptoms of severe malaria disease. Depending on the mouse genetic background, a single high dose immunization without adjuvant protected mice from parasitemia and severe disease (CD1 mice) or from experimental cerebral malaria (ECM) (C57BL/6 mice). A low dose immunization did not protect against parasitemia or severe disease in either model after one or two immunizations. The protection from ECM was associated with a parasite specific antibody response and also with a lower level of splenic parasite-specific IFN-γ production, which is a mediator of ECM pathology in C57BL/6 mice. Surprisingly, there was no difference in the sequestration of CD8+ T cells and CD45+ CD11b+ macrophages in the brains of immunized, ECM-protected mice. CONCLUSIONS: This report further demonstrates the effectiveness of a whole parasite blood-stage vaccine in inducing immunity to malaria and explicitly demonstrates its effectiveness against ECM, the most pathogenic consequence of malaria infection. This experimental model will be important to explore the formulation of whole parasite blood-stage vaccines against malaria and to investigate the immune mechanisms that mediate protection against parasitemia and cerebral malaria.