Persistence of Protective Immunity to Malaria Induced by DNA Priming and Poxvirus Boosting: Characterization of Effector and Memory CD8+-T-Cell Populations

The persistence of immunity to malaria induced in mice by a heterologous DNA priming and poxvirus boosting regimen was characterized. Mice were immunized by priming with DNA vaccine plasmids encoding the Plasmodium yoelii circumsporozoite protein (PyCSP) and murine granulocyte-macrophage colony-stimulating factor and boosting with recombinant vaccinia encoding PyCSP. BALB/c mice immunized with either high-dose (100 µg of p PyCSP plus 30 µg of pGM-CSF) or low-dose (1 µg of p PyCSP plus 1 µg of pGM-CSF DNA) priming were protected against challenge with 50 P. yoelii sporozoites. Protection 2 weeks after immunization was 70 to 100%, persisted at this level for at least 20 weeks, and declined to 30 to 40% by 28 weeks. Eight of eight mice protected at 20 weeks were still protected when rechallenged at 40 weeks. The antigen (Ag)-specific effector CD8+-T-cell population present 2 weeks after boosting had ex vivo Ag-specific cytolytic activity, expressed both gamma interferon (IFN-{gamma}) and tumor necrosis factor alpha, and constituted 12 to 20% of splenic CD8+ T cells. In contrast, the memory CD8+-Ag-specific-cell population at 28 weeks lacked cytolytic activity and constituted only 6% of splenic CD8+ T cells, but at the single-cell level it produced significantly higher levels of IFN-{gamma} than the effectors. High levels of Ag- or parasite-specific antibodies present 2 weeks after boosting had declined three- to sevenfold by 28 weeks. Low-dose priming was similarly immunogenic and as protective as high-dose priming against a 50-, but not a 250-, sporozoite challenge. These results demonstrate that a heterologous priming and boosting vaccination can provide lasting protection against malaria in this model system

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

A Novel Chimeric Plasmodium vivax Circumsporozoite Protein Induces Biologically Functional Antibodies That Recognize both VK210 and VK247 Sporozoites

A successful vaccine against Plasmodium vivax malaria would significantly improve the health and quality of the lives of more than 1 billion people around the world. A subunit vaccine is the only option in the absence of long-term culture of P. vivax parasites. The circumsporozoite protein that covers the surface of Plasmodium sporozoites is one of the best-studied malarial antigens and the most promising vaccine in clinical trials. We report here the development of a novel “immunologically optimal” recombinant vaccine expressed in Escherichia coli that encodes a chimeric CS protein encompassing repeats from the two major alleles, VK210 and VK247. This molecule is widely recognized by sera from patients naturally exposed to P. vivax infection and induces a highly potent immune response in genetically disparate strains of mice. Antibodies from immunized animals recognize both VK210 and VK247 sporozoites. Furthermore, these antibodies appear to be protective in nature since they cause the agglutination of live sporozoites, an in vitro surrogate of sporozoite infectivity. These results strongly suggest that recombinant CS is biologically active and highly immunogenic across major histocompatibility complex strains and raises the prospect that in humans this vaccine may induce protective immune responses

Molecular Correlates of Experimental Cerebral Malaria Detectable in Whole Blood ▿ †

Cerebral malaria (CM) is a primary cause of deaths caused by Plasmodium falciparum in young children in sub-Saharan Africa. Laboratory tests based on early detection of host biomarkers in patient blood would help in the prognosis and differential diagnosis of CM. Using the Plasmodium berghei ANKA murine model of experimental cerebral malaria (ECM), we have identified over 300 putative diagnostic biomarkers of ECM in the circulation by comparing the whole-blood transcriptional profiles of resistant mice (BALB/c) to those of two susceptible strains (C57BL/6 and CBA/CaJ). Our results suggest that the transcriptional profile of whole blood captures the molecular and immunological events associated with the pathogenesis of disease. We find that during ECM, erythropoiesis is dysfunctional, thrombocytopenia is evident, and glycosylation of cell surface components may be modified. Furthermore, analysis of immunity-related genes suggests that slightly distinct mechanisms of immunopathogenesis may operate in susceptible C57BL/6 and CBA/CaJ mice. Furthermore, our data set has allowed us to create a molecular signature of ECM composed of a subset of circulatory markers. Complement component C1q, β-chain, nonspecific cytotoxic cell receptor protein 1, prostate stem cell antigen, DnaJC, member 15, glutathione S-transferase omega-1, and thymidine kinase 1 were overexpressed in blood during the symptomatic phase of ECM, as measured by quantitative real-time PCR analysis. These studies provide the first host transcriptome database that is uniquely altered during the pathogenesis of ECM in blood. A subset of these mediators of ECM warrant validation in P. falciparum-infected young African children as diagnostic markers of CM

Induction of galectin-3 during experimental cerebral malaria.

<p>Expression of galectin-3 was measured in brain tissue samples in individual moribund (n = 5), non-moribund (n = 5), CD8-KO ( = 3), and normal (non-infected, n = 3) C57BL/6 mice by ECL-based western blot analysis. Expression levels were determined based on the intensity of protein bands using Meta1 Morph 6.1 software and are represented as average integrated optical densities (IOD) units. The IOD units shown are values×1000. Details of antibodies and western blot reagents used can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006793#s4" target="_blank">Materials and Methods</a>.</p

OX40 expression on T cell subsets present in the brain of moribund mice.

<p>Total brain leukocytes (sequestered and nonsequestered) were stained with fluorescent-labeled antibodies against TCRβ, CD4, CD8, and OX40. The proportion of <i>A:</i> CD4⁺ and CD8⁺<i>B:</i> OX40⁺CD4⁺ and <i>C:</i> OX40⁺ CD8⁺ T cells was determined by flow cytometry. <i>D:</i> Although OX40 is preferentially expressed on CD4⁺ T cells, due to the comparatively higher number of CD8⁺ T cells in the brain, the absolute number of OX40⁺CD4⁺ and OX40⁺CD8⁺ T cells accumulated in the brain vasculature is approximately the same.</p

<i>Pb-A</i> parasites collected from whole blood at approximately 10% parasitemia were cultured overnight to obtain schizont stage parasites.

<p>Parasites were then incubated with <i>A:</i> no galectin-3 <i>B:</i> galectin-3 <i>C:</i> galectin-3 and sucrose and <i>D:</i> galectin-3 and lactose and subsequently stained with a goat antibody specific for galectin-3 and a fluorescent donkey antibody specific for goat IgG. Right panels show galectin-3 adherence to mouse lymphocytes. Images were collected on an epifluorescence microscope.</p

Expression of OX40 in the brain of mice with experimental cerebral malaria.

<p>Brain sections from <i>A:</i> moribund and <i>B:</i> non-moribund <i>Pb-A</i> infected mice were stained with a goat antibody specific for mouse OX40 and visualized at 40×magnification. Immunostaining demonstrated strong but highly specific staining of a subset of lymphoid cells within and adjacent to blood vessels in the brains of moribund mice (A) but much fewer positive cells were observed in non-moribund (B) mice.</p

Galectin-3 deficient mice are partially protected against experimental cerebral malaria and developed higher peripheral parasitemia.

<p><i>A:</i> Fourteen of 15 (93%) WT mice versus 8 of 17 (47%) galectin-3-KO mice succumbed to ECM by day 8 post-infection. Data shown is cumulated from two independent experiments. <i>B:</i> In galectin-3-KO (n = 5) and WT (n = 9) mice that developed ECM, galectin-3-KO mice had moderately higher parasitemia than WT mice (p<0.0137, two-way ANOVA). <i>C:</i> Among mice that did not develop ECM, although parasitemia did not differ markedly between days 4 and 12, parasitemia was higher on day 20 in the galectin-3-KO (53.3±8.29%, n = 4) versus WT (36%, n = 1) group.</p