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

Pathogenic Roles of CD14, Galectin-3, and OX40 during Experimental Cerebral Malaria in Mice

An in-depth knowledge of the host molecules and biological pathways that contribute towards the pathogenesis of cerebral malaria would help guide the development of novel prognostics and therapeutics. Genome-wide transcriptional profiling of the brain tissue during experimental cerebral malaria (ECM ) caused by Plasmodium berghei ANKA parasites in mice, a well established surrogate of human cerebral malaria, has been useful in predicting the functional classes of genes involved and pathways altered during the course of disease. To further understand the contribution of individual genes to the pathogenesis of ECM, we examined the biological relevance of three molecules – CD14, galectin-3, and OX40 that were previously shown to be overexpressed during ECM. We find that CD14 plays a predominant role in the induction of ECM and regulation of parasite density; deletion of the CD14 gene not only prevented the onset of disease in a majority of susceptible mice (only 21% of CD14-deficient compared to 80% of wildtype mice developed ECM, p<0.0004) but also had an ameliorating effect on parasitemia (a 2 fold reduction during the cerebral phase). Furthermore, deletion of the galectin-3 gene in susceptible C57BL/6 mice resulted in partial protection from ECM (47% of galectin-3-deficient versus 93% of wildtype mice developed ECM, p<0.0073). Subsequent adherence assays suggest that galectin-3 induced pathogenesis of ECM is not mediated by the recognition and binding of galectin-3 to P. berghei ANKA parasites. A previous study of ECM has demonstrated that brain infiltrating T cells are strongly activated and are CD44+CD62L− differentiated memory T cells [1]. We find that OX40, a marker of both T cell activation and memory, is selectively upregulated in the brain during ECM and its distribution among CD4+ and CD8+ T cells accumulated in the brain vasculature is approximately equal

Comparison of cellular and humoral responses to recombinant protein and synthetic peptides of exon2 region of Plasmodium falciparum erythrocyte membrane protein1 (PfEMP1) among malaria patients from an endemic region

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on the surface of parasitized red blood cells (PRBCs) mediate adhesion of PRBCs to host vascular endothelial receptors and is considered responsible for pathogenesis of severe P. falciparum malaria. The present study was undertaken to measure cellular immune responses and serum antibody responses against recombinant exon2 protein, the most conserved region of PfEMP1, and its synthetic peptides. T cell recognizing this domain could provide universal help to B cells in recognizing variant epitopes located in the extracellular region of PfEMP1. Human peripheral blood mononuclear cells from malaria-exposed immune adults (IA), malaria patients with varying severity, and malaria unexposed healthy donors were stimulated with recombinant exon2 protein and six synthetic peptides from its sequence to estimate the proliferative, IFN-γ, and IL-4 responses. Antibody responses against these synthetic peptides and exon2 protein were also studied. Positive proliferative, IFN-γ, and IL-4 responses in IA group each were 60% with recombinant exon2 protein and 27-47% with different synthetic peptides. Antibody recognition was observed in 67% with exon2 and between 40 and 53% with different peptides. In malaria patients, frequency and magnitude of proliferative response, IL-4 concentration, and antibody recognition were far less than immune adults but IFN-γ response was almost similar. Proportion of positive responders and the magnitude of response to synthetic peptides were low. Also, there was no consistency in response of different peptides towards proliferative, cytokine, and antibody responses in IA and malaria patient groups except for peptide 1. We presume peptide 1 is a potential vaccine candidate and different cocktails containing peptide 1 are being evaluated for their T cell immunogenicity

Host Biomarkers and Biological Pathways That Are Associated with the Expression of Experimental Cerebral Malaria in Mice▿ †

Cerebral malaria (CM) is a primary cause of malaria-associated deaths among young African children. Yet no diagnostic tools are available that could be used to predict which of the children infected with Plasmodium falciparum malaria will progress to CM. We used the Plasmodium berghei ANKA murine model of experimental cerebral malaria (ECM) and high-density oligonucleotide microarray analyses to identify host molecules that are strongly associated with the clinical symptoms of ECM. Comparative expression analyses were performed with C57BL/6 mice, which have an ECM-susceptible phenotype, and with mice that have ECM-resistant phenotypes: CD8 knockout and perforin knockout mice on the C57BL/6 background and BALB/c mice. These analyses allowed the identification of more than 200 host molecules (a majority of which had not been identified previously) with altered expression patterns in the brain that are strongly associated with the manifestation of ECM. Among these host molecules, brain samples from mice with ECM expressed significantly higher levels of p21, metallothionein, and hemoglobin α1 proteins by Western blot analysis than mice unaffected by ECM, suggesting the possible utility of these molecules as prognostic biomarkers of CM in humans. We suggest that the higher expression of hemoglobin α1 in the brain may be associated with ECM and could be a source of excess heme, a molecule that is considered to trigger the pathogenesis of CM. Our studies greatly enhance the repertoire of host molecules for use as diagnostics and novel therapeutics in CM

Identification, Cloning, Expression, and Characterization of the Gene for Plasmodium knowlesi Surface Protein Containing an Altered Thrombospondin Repeat Domain

Proteins present on the surface of malaria parasites that participate in the process of invasion and adhesion to host cells are considered attractive vaccine targets. Aided by the availability of the partially completed genome sequence of the simian malaria parasite Plasmodium knowlesi, we have identified a 786-bp DNA sequence that encodes a 262-amino-acid-long protein, containing an altered version of the thrombospondin type I repeat domain (SPATR). Thrombospondin type 1 repeat domains participate in biologically diverse functions, such as cell attachment, mobility, proliferation, and extracellular protease activities. The SPATR from P. knowlesi (PkSPATR) shares 61% and 58% sequence identity with its Plasmodium falciparum and Plasmodium yoelii orthologs, respectively. By immunofluorescence analysis, we determined that PkSPATR is a multistage antigen that is expressed on the surface of P. knowlesi sporozoite and erythrocytic stage parasites. Recombinant PkSPATR produced in Escherichia coli binds to a human hepatoma cell line, HepG2, suggesting that PkSPATR is a parasite ligand that could be involved in sporozoite invasion of liver cells. Furthermore, recombinant PkSPATR reacted with pooled sera from P. knowlesi-infected rhesus monkeys, indicating that native PkSPATR is immunogenic during infection. Further efficacy evaluation studies in the P. knowlesi-rhesus monkey sporozoite challenge model will help to decide whether the SPATR molecule should be developed as a vaccine against human malarias

Protection from parasitemia and severe disease in CD1 mice after immunization with irradiated blood-stage <i>Pb-A</i>.

<p><b>A</b>) Protection in CD1 mice after a single immunization with 10⁷ irradiation attenuated <i>Pb-A</i> blood-stage parasites (IrrPb). 10³ or 10⁷ IrrPb were injected iv into groups mice. On day 24 after injection, immunized and naïve mice were challenged with 10⁴ virulent <i>Pb-A</i> pRBC and parasitemia was monitored by blood smear (mean +/− SD) Daggers indicate mice that were euthanized or that died during the experiment. All naïve mice (4/4, filled circles) and 10³ IrrPb immunized mice (5/5, open triangles) displayed uncontrolled parasite growth and succumbed to acute blood-stage infection by day 12 after challenge. In contrast, mice immunized with 10⁷ IrrPb (4/5, open squares) controlled parasite growth after challenge and managed blood parasitemia down to low or undetectable levels. <b>B</b>) Protection in CD1 mice after a two immunizations with 10⁷ IrrPb. Groups of mice were immunized with 10³ or 10⁷ IrrPb, and then given a boost immunization 24 days later with an equal number of IrrPb (Boost). 24 days after the final immunization, immunized and naïve mice were challenged with 10⁴ virulent <i>Pb-A</i> pRBC. In mice immunized with 2 doses of 10³ IrrPb (n = 5 mice, open triangles), parasitemia levels continued to increase after challenge, similar to naïve mice (n = 5 mice, filled circles). In contrast, when mice were immunized with 2 doses of 10⁷ IrrPb (n = 5 mice, open squares), blood parasitemia declined over time to low or undetectable levels.</p

Determination of the radiation dose necessary to attenuate blood-stage <i>Pb-A</i> parasites.

<p>Virulent <i>Pb-A</i> parasitized RBC (pRBC) were exposed to increasing doses of gamma irradiation (10, 20, 40, 60, 80, 100 kilorads). 10⁷ irradiated pRBC or non-irradiated control pRBC (Ctrl) were injected intravenously (iv) into groups of naïve CD1 mice and parasitemia was monitored by blood smear. The graph shows the percentage of mice without patent parasitemia in each group over time beginning with day 2. Data are pooled from 3 experiments. Groups of mice infected with control pRBC or with pRBC exposed to 10, 20, 40, or 60 krad irradiation all developed patent parasitemia by day 7 (n = 4 mice each group). Accordingly, the curves describing these groups approach 0% by day 7. 70% (10/14) of mice infected with 80 krad irradiated pRBC remained free from patent blood-stage disease by day 14 (open circles, dashed line). 100% (10/10) of the mice injected with 100 krad irradiated pRBC remained free from blood stage infection under the period of observation (open squares, solid line). 100 krad irradiation was chosen as the attenuating dose in subsequent experiments.</p

Protection from experimental cerebral malaria (ECM) in C57BL/6 mice after immunization with IrrPb.

<p><b>A and B</b>) Protection from ECM after a single immunization of 10⁷ IrrPb. Data are pooled from two independent experiments. Groups of mice were immunized with 10³ or 10⁷ IrrPb and then challenged with 10⁴ virulent <i>Pb-A</i> pRBC on day 19 or 28 after immunization. To assess ECM, challenged mice were monitored for neurological symptoms, the ability to survive beyond day 10 (A), and blood parasitemia levels (B). <b>A</b>) 10% (1/10) of naïve mice (filled circles) and 12% (2/17) of mice immunized with 10³ IrrPb (open triangles) survived beyond day 10 after challenge. In contrast, 81% (17/21) of mice immunized with 10⁷ IrrPb (open squares) survived beyond day 10 (two-sided p = .0003, 10⁷ IrrPb group vs. naïve group, Fisher's exact test). <b>B</b>) In each group, mice that succumbed to ECM before day 10 had blood parasitemias less than 10%, while mice that survived beyond day 10 eventually developed hyperparasitemia. <b>C and D</b>) Protection from ECM after two immunizations with 10⁷ IrrPb. Mice were immunized with 10³ or 10⁷ IrrPb twice with a 28 day interval (Boost), challenged with 10⁴ virulent pRBC, and then monitored for survival (C) and blood parasitemia (D). <b>C</b>) 20% (1/5) of naïve mice (filled circles) and none (0/10) of the mice immunized twice with 10³ attenuated parasites (open triangles) survived beyond day 10 after challenge. In contrast, all (10/10) of the mice immunized with 10⁷ attenuated parasites (open squares) survived beyond day 10 (p = .0037, 10⁷ IrrPb group vs. naïve group, Fisher's exact test), and eventually developed hyperparasitemia (D).</p

Spleen weights of mice immunized with IrrPb increase during a virulent infection.

<p>C57BL/6 mice were immunized once with 10⁷ IrrPb, challenged with virulent parasites, and then spleens were harvested on day 6 after challenge. Spleens from immunized mice after challenge were an average 0.17 grams (0.09, 0.24) larger than the spleens from naïve mice after challenge (two-sided p<.001, Two Way ANOVA, Bonferroni post test). Results (n = 8 mice in each group) are pooled from two independent experiments.</p

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 (R2=0.9399). The range for detecting the day 15 salivary-gland PfSPZ is between 0.0625 and 1 parasite (R2=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. © 2013