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Protective CD8+ T lymphocytes in Primates Immunized with Malaria Sporozoites  [PDF]
Walter R. Weiss, Chengyong George Jiang
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0031247
Abstract: Live attenuated malaria vaccines are more potent than the recombinant protein, bacterial or viral platform vaccines that have been tested, and an attenuated sporozoite vaccine against falciparum malaria is being developed for humans. In mice, attenuated malaria sporozoite vaccines induce CD8+ T cells that kill parasites developing in the liver. We were curious to know if CD8+ T cells were also important in protecting primates against malaria. We immunized 9 rhesus monkeys with radiation attenuated Plasmodium knowlesi sporozoites, and found that 5 did not develop blood stage infections after challenge with live sporozoites. We then injected 4 of these protected monkeys with cM-T807, a monoclonal antibody to the CD8 molecule which depletes T cells. The fifth monkey received equivalent doses of normal IgG. In 3 of the 4 monkeys receiving cM-T807 circulating CD8+ T cells were profoundly depleted. When re-challenged with live sporozoites all 3 of these depleted animals developed blood stage malaria. The fourth monkey receiving cM-T807 retained many circulating CD8+ T cells. This monkey, and the vaccinated monkey receiving normal IgG, did not develop blood stage malaria at re-challenge with live sporozoites. Animals were treated with antimalarial drugs and rested for 4 months. During this interval CD8+ T cells re-appeared in the circulation of the depleted monkeys. When all vaccinated animals received a third challenge with live sporozoites, all 5 monkeys were once again protected and did not develop blood stage malaria infections. These data indicate that CD8+ T cells are important effector cells protecting monkeys against malaria sporozoite infection. We believe that malaria vaccines which induce effector CD8+ T cells in humans will have the best chance of protecting against malaria.
Intradermal immunization of mice with radiation-attenuated sporozoites of Plasmodium yoelii induces effective protective immunity
Tatiana Voza, Chahnaz Kebaier, Jerome P Vanderberg
Malaria Journal , 2010, DOI: 10.1186/1475-2875-9-362
Abstract: Mice were immunized with two injections of isolated, radiation-attenuated P. yoelii sporozoites, either by intravenous (IV) or intradermal (ID) inoculation. In an attempt to enhance protective immunogenicity of ID-injections, one group of experimental mice received topical application of an adjuvant, Imiquimod, while another group had their injections accompanied by local "tape-stripping" of the skin, a procedure known to disrupt the stratum corneum and activate local immunocytes. Challenge of immunized and non-immunized control mice was by bite of sporozoite-infected mosquitoes. Degree of protection among the various groups of mice was determined by microscopic examination of stained blood smears. Statistical significance of protection was determined by a one-way ANOVA followed by Tukey's post hoc test.Two intravenous immunizations produced 94% protection to mosquito bite challenge; intradermal immunization produced 78% protection, while intradermal immunization accompanied by "tape-stripping" produced 94% protection. There were no statistically significant differences in degree of protective immunity between immunizations done by intravenous versus intradermal injection.The use of a sub-microlitre syringe for intradermal injections yielded excellent protective immunity. ID-immunization with large numbers of radiation-attenuated P. yoelii sporozoites led to levels of protective immunity comparable to those achieved by IV-immunization. It remains to be determined whether an adjuvant treatment can be found to substantially reduce the numbers of attenuated sporozoites required to achieve a strong protective immunity with as few doses as possible for possible extension to immunization of humans.In spite of the huge global morbidity and mortality inflicted by malaria, an effective and practical vaccine against this disease has not yet been achieved. Early studies on immunization against sporozoite-induced rodent malaria resulted in close to 100% protection when mice wer
Sporozoite neutralizing antibodies elicited in mice and rhesus macaques immunized with a Plasmodium falciparum repeat peptide conjugated to meningococcal outer membrane protein complex  [PDF]
Craig Przysiecki,Daniel Carapau,Elizabeth Nardin
Frontiers in Cellular and Infection Microbiology , 2012, DOI: 10.3389/fcimb.2012.00146
Abstract: Antibodies that neutralize infectivity of malaria sporozoites target the central repeat region of the circumsporozoite (CS) protein, which in Plasmodium falciparum is comprised primarily of 30–40 tandem NANP tetramer repeats. We evaluated immunogenicity of an alum-adsorbed (NANP)6 peptide conjugated to an outer membrane protein complex (OMPC) derived from Neisseria meningitidis, a carrier protein used in a licensed Haemophilus influenzae pediatric vaccine. Mice immunized with (NANP)6-OMPC adsorbed to Merck's alum adjuvant (MAA), with or without Iscomatrix? as co-adjuvant, developed high levels of anti-repeat peptide antibody that inhibited in vitro invasion of human hepatoma cells by transgenic P. berghei sporozoites that express P. falciparum CS repeats (PfPb). Inhibition of sporozoite invasion in vitro correlated with in vivo resistance to challenge by the bites of PfPb-infected mosquitoes. Challenged mice had >90% reduction of hepatic stage parasites as measured by real-time PCR, and either sterile immunity, i.e., no detectable blood stage parasites, or delayed prepatent periods which indicate neutralization of a majority, but not all, sporozoites. Rhesus macaques immunized with two doses of (NANP)6-OMPC/MAA formulated with Iscomatrix? developed anti-repeat antibodies that persisted for ~2 years. A third dose of (NANP)6-OMPC/MAA+ Iscomatrix? at that time elicited strong anamnestic antibody responses. Rhesus macaque immune sera obtained post second and third dose of vaccine displayed high levels of sporozoite neutralizing activity in vitro that correlated with presence of high anti-repeat antibody titers. These preclinical studies in mice of different MHC haplotypes and a non-human primate support use of CS peptide-OMPC conjugates as a highly immunogenic platform to evaluate CS protective epitopes. Potential pre-erythrocytic vaccines can be combined with sexual blood stage vaccines as a multi-antigen malaria vaccine to block invasion and transmission of Plasmodium parasites.
In vivo CD8+ T Cell Dynamics in the Liver of Plasmodium yoelii Immunized and Infected Mice  [PDF]
Mynthia Cabrera, Lecia L. Pewe, John T. Harty, Ute Frevert
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0070842
Abstract: Plasmodium falciparum malaria remains one of the most serious health problems globally and a protective malaria vaccine is desperately needed. Vaccination with attenuated parasites elicits multiple cellular effector mechanisms that lead to Plasmodium liver stage elimination. While granule-mediated cytotoxicity requires contact between CD8+ effector T cells and infected hepatocytes, cytokine secretion should allow parasite killing over longer distances. To better understand the mechanism of parasite elimination in vivo, we monitored the dynamics of CD8+ T cells in the livers of na?ve, immunized and sporozoite-infected mice by intravital microscopy. We found that immunization of BALB/c mice with attenuated P. yoelii 17XNL sporozoites significantly increases the velocity of CD8+ T cells patrolling the hepatic microvasculature from 2.69±0.34 μm/min in na?ve mice to 5.74±0.66 μm/min, 9.26±0.92 μm/min, and 7.11±0.73 μm/min in mice immunized with irradiated, early genetically attenuated (Pyuis4-deficient), and late genetically attenuated (Pyfabb/f-deficient) parasites, respectively. Sporozoite infection of immunized mice revealed a 97% and 63% reduction in liver stage density and volume, respectively, compared to na?ve controls. To examine cellular mechanisms of immunity in situ, na?ve mice were passively immunized with hepatic or splenic CD8+ T cells. Unexpectedly, adoptive transfer rendered the motile CD8+ T cells from immunized mice immotile in the liver of P. yoelii infected mice. Similarly, when mice were simultaneously inoculated with viable sporozoites and CD8+ T cells, velocities 18 h later were also significantly reduced to 0.68±0.10 μm/min, 1.53±0.22 μm/min, and 1.06±0.26 μm/min for CD8+ T cells from mice immunized with irradiated wild type sporozoites, Pyfabb/f-deficient parasites, and P. yoelii CS280–288 peptide, respectively. Because immobilized CD8+ T cells are unable to make contact with infected hepatocytes, soluble mediators could potentially play a key role in parasite elimination under these experimental conditions.
Conserved Protective Mechanisms in Radiation and Genetically Attenuated uis3(-) and uis4(-) Plasmodium Sporozoites  [PDF]
Kota Arun Kumar, Peter Baxter, Alice S. Tarun, Stefan H. I. Kappe, Victor Nussenzweig
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0004480
Abstract: Immunization with radiation attenuated Plasmodium sporozoites (RAS) elicits sterile protective immunity against sporozoite challenge in murine models and in humans. Similarly to RAS, the genetically attenuated sporozoites (GAPs) named uis3(-), uis4(-) and P36p(-) have arrested growth during the liver stage development, and generate a powerful protective immune response in mice. We compared the protective mechanisms in P. yoelii RAS, uis3(-) and uis4(-) in BALB/c mice. In RAS and GAPs, sterile immunity is only achieved after one or more booster injections. There were no differences in the immune responses to the circumsporozoite protein (CSP) generated by RAS and GAPs. To evaluate the role of non-CSP T-cell antigens we immunized antibody deficient, CSP-transgenic BALB/c mice, that are T cell tolerant to CSP, with P. yoelii RAS or with uis3(-) or uis4(-) GAPs, and challenged them with wild type sporozoites. In every instance the parasite liver stage burden was approximately 3 logs higher in antibody deficient CSP transgenic mice as compared to antibody deficient mice alone. We conclude that CSP is a powerful protective antigen in both RAS and GAPs viz., uis3(-) and uis4(-) and that the protective mechanisms are similar independently of the method of sporozoite attenuation.
T cell responses to repeat and non-repeat regions of the circumsporozoite protein detected in volunteers immunized with Plasmodium falciparum sporozoites
Nardin, Elizabeth;Munesinghe, Yamuna Dona;Moreno, Alberto;Clavijo, Pedro;Calle, Mauricio Calvo;Edelman, Robert;Davis, Jonathan;Herrington, Deirdre;Nussenzweig, Ruth S.;
Memórias do Instituto Oswaldo Cruz , 1992, DOI: 10.1590/S0074-02761992000700037
Abstract: the design of malarial vaccine based on the circumsporozoite (cs) protein, a majuor surface antigen of the sporozoite stage of the malaria parasite, requires the identification of t and b cell epitopes for inclusion in recombinant or synthetic vaccine candidates. we have investigated the specificity and function of a series of t cell clones, derived from volunteers immunized with plasmodium falciparum sporozoites in an effort to identify relevant epitopes in the immune response to the pre-erythrocytic stages of the parasite. cd4+ t cell clones were obtained wich specifically recognized a repetitive epitope located in the 5'repeat region of the cs protein. this epitope, when conjugated to the 3'repeat region in a synthetic maps construct, induced high titers of antisporozoite antibodies in c57b1 mice. a second t cell epitope, which mapped to aa 326-345 of the carboxy terminal, was recognized by lytic, as well as non-lytic, cd4+ t cells derived from the sporozoite-immunized volunteers. the demonstration of cd4+ ctl in the volunteers, and the recent studies inthe rodent model (renia et al., 1991; tsuji et al., 1990), suggested that cs-specific cd4+ t cells, in addition to their indirect role as helper cells in the induction of antibody and cd8 + effector cells, may also play a direct role in protection against sporozoite challenge by targeting eef within the liver.
Long Term Protection after Immunization with P. berghei Sporozoites Correlates with Sustained IFNγ Responses of Hepatic CD8+ Memory T Cells  [PDF]
Krystelle Nganou-Makamdop, Geert-Jan van Gemert, Theo Arens, Cornelus C. Hermsen, Robert W. Sauerwein
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0036508
Abstract: Protection against P. berghei malaria can successfully be induced in mice by immunization with both radiation attenuated sporozoites (RAS) arresting early during liver stage development, or sporozoites combined with chloroquine chemoprophylaxis (CPS), resulting in complete intra-hepatic parasite development before killing of blood-stages by chloroquine takes place. We assessed the longevity of protective cellular immune responses by RAS and CPS P. berghei immunization of C57BL/6j mice. Strong effector and memory (TEM) CD8+ T cell responses were induced predominantly in the liver of both RAS and CPS immunized mice while CD4+ T cells with memory phenotype remained at base line levels. Compared to unprotected na?ve mice, we found high sporozoite-specific IFNγ ex vivo responses that associated with induced levels of in vivo CD8+ TEM cells in the liver but not spleen. Long term evaluation over a period of 9 months showed a decline of malaria-specific IFNγ responses in RAS and CPS mice that significantly correlated with loss of protection (r2 = 0.60, p<0.0001). The reducing IFNγ response by hepatic memory CD8+ T cells could be boosted by re-exposure to wild-type sporozoites. Our data show that sustainable protection against malaria associates with distinct intra-hepatic immune responses characterized by strong IFNγ producing CD8+ memory T cells.
Kinome-Wide RNAi Screen Implicates at Least 5 Host Hepatocyte Kinases in Plasmodium Sporozoite Infection  [PDF]
Miguel Prudêncio equal contributor,Cristina D. Rodrigues equal contributor,Michael Hannus,Cécilie Martin,Eliana Real,Lígia A. Gon?alves,Céline Carret,Robert Dorkin,Ingo R?hl,Kerstin Jahn-Hoffmann,Adrian J. F. Luty,Robert Sauerwein,Christophe J. Echeverri,Maria M. Mota
PLOS Pathogens , 2008, DOI: 10.1371/journal.ppat.1000201
Abstract: Plasmodium sporozoites, the causative agent of malaria, are injected into their vertebrate host through the bite of an infected Anopheles mosquito, homing to the liver where they invade hepatocytes to proliferate and develop into merozoites that, upon reaching the bloodstream, give rise to the clinical phase of infection. To investigate how host cell signal transduction pathways affect hepatocyte infection, we used RNAi to systematically test the entire kinome and associated genes in human Huh7 hepatoma cells for their potential roles during infection by P. berghei sporozoites. The three-phase screen covered 727 genes, which were tested with a total of 2,307 individual siRNAs using an automated microscopy assay to quantify infection rates and qRT-PCR to assess silencing levels. Five protein kinases thereby emerged as top hits, all of which caused significant reductions in infection when silenced by RNAi. Follow-up validation experiments on one of these hits, PKC? (PKCzeta), confirmed the physiological relevance of our findings by reproducing the inhibitory effect on P. berghei infection in adult mice treated systemically with liposome-formulated PKC?-targeting siRNAs. Additional cell-based analyses using a pseudo-substrate inhibitor of PKC? added further RNAi-independent support, indicating a role for host PKC? on the invasion of hepatocytes by sporozoites. This study represents the first comprehensive, functional genomics-driven identification of novel host factors involved in Plasmodium sporozoite infection.
Immunization with Radiation-Attenuated Plasmodium berghei Sporozoites Induces Liver cCD8α+DC that Activate CD8+T Cells against Liver-Stage Malaria  [PDF]
Ousman Jobe, Gina Donofrio, Guangping Sun, Dmitry Liepinsh, Robert Schwenk, Urszula Krzych
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0005075
Abstract: Immunization with radiation (γ)-attenuated Plasmodia sporozoites (γ-spz) confers sterile and long-lasting immunity against malaria liver-stage infection. In the P. berghei γ-spz model, protection is linked to liver CD8+ T cells that express an effector/memory (TEM) phenotype, (CD44hiCD45RBloCD62Llo ), and produce IFN-γ. However, neither the antigen presenting cells (APC) that activate these CD8+ TEM cells nor the site of their induction have been fully investigated. Because conventional (c)CD8α+ DC (a subset of CD11c+ DC) are considered the major inducers of CD8+ T cells, in this study we focused primarily on cCD8α+ DC from livers of mice immunized with Pb γ-spz and asked whether the cCD8α+ DC might be involved in the activation of CD8+ TEM cells. We demonstrate that multiple exposures of mice to Pb γ-spz lead to a progressive and nearly concurrent accumulation in the liver but not the spleen of both the CD11c+NK1.1? DC and CD8+ TEM cells. Upon adoptive transfer, liver CD11c+NK1.1? DC from Pb γ-spz-immunized mice induced protective immunity against sporozoite challenge. Moreover, in an in vitro system, liver cCD8α+ DC induced na?ve CD8+ T cells to express the CD8+ TEM phenotype and to secrete IFN-γ. The in vitro induction of functional CD8+ TEM cells by cCD8α+ DC was inhibited by anti-MHC class I and anti-IL-12 mAbs. These data suggest that liver cCD8α+ DC present liver-stage antigens to activate CD8+ TEM cells, the pre-eminent effectors against pre-erythrocytic malaria. These results provide important implications towards a design of anti-malaria vaccines.
An early burst of IFN-γ induced by the pre-erythrocytic stage favours Plasmodium yoelii parasitaemia in B6 mice
Valérie Soulard, Jacques Roland, Olivier Gorgette, Eliane Barbier, Pierre-André Cazenave, Sylviane Pied
Malaria Journal , 2009, DOI: 10.1186/1475-2875-8-128
Abstract: Mice were infected with Plasmodium yoelii 265BY sporozoites, the natural invasive form of the parasite, in order to complete its full life cycle. The concentrations of three proinflammatory cytokines in the sera of mice were determined by ELISA at different time points of infection. The contribution of the liver and the spleen to this cytokinic response was evaluated and the cytokine-producing lymphocytes were identified by flow cytometry. The physiological relevance of these results was tested by monitoring parasitaemia in genetically deficient C57BL/6 mice or wild-type mice treated with anti-cytokine neutralizing antibody. Finally, the cytokinic response in sera of mice infected with parasitized-RBCs was analysed.The early immune response of C57BL/6 mice to sporozoite-induced malaria is characterized by a peak of IFN-γ in the serum at day 5 of infection and splenic CD4 T lymphocytes are the major producer of this cytokine at this time point. Somewhat unexpected, the parasitaemia is significantly lower in P. yoelii-infected mice in the absence of IFN-γ. More precisely, at early time points of infection, IFN-γ favours parasitaemia, whereas helping to clear efficiently the blood-stage parasites at later time points. Interestingly, the early IFN-γ burst is induced by the pre-erythrocytic stage.These results challenge the current view regarding the role of IFN-γ on the control of parasite growth since they show that IFN-γ is not an essential mediator of protection in P. yoelii-infected C57BL/6 mice. Moreover, the mice parasitaemia is more efficiently controlled in the absence of an early IFN-γ production, suggesting that this cytokine promotes parasite's growth. Finally, this early burst of IFN-γ is induced by the pre-erythrocytic stage, showing the impact of this stage on the immune response taking place during the subsequent erythrocytic stage.Malaria is initiated when sporozoites are injected into the mammalian host during the blood meal of an infected mosquito. The
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