[Show abstract][Hide abstract] ABSTRACT: The Plasmodium-infected hepatocyte has been considered necessary to prime the immune responses leading to sterile protection after vaccination with attenuated sporozoites. However, it has recently been demonstrated that priming also occurs in the skin. We wished to establish if sterile protection could be obtained in the absence of priming by infected hepatocytes. To this end, we developed a subcutaneous (s.c.) immunization protocol where few, possibly none, of the immunizing irradiated Plasmodium yoelii sporozoites infect hepatocytes, and also used CD81-deficient mice non-permissive to productive hepatocyte infections. We then compared and contrasted the patterns of priming with those obtained by intradermal immunization, where priming occurs in the liver. Using sterile immunity as a primary read-out, we exploited an inhibitor of T-cell migration, transgenic mice with conditional depletion of dendritic cells and adoptive transfers of draining lymph node-derived T cells, to provide evidence that responses leading to sterile immunity can be primed in the skin-draining lymph nodes with little, if any, contribution from the infected hepatocyte.
EMBO Molecular Medicine 02/2013; 5(2). DOI:10.1002/emmm.201201677 · 8.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sterile immunity against malaria has been obtained in mammalian hosts exclusively through vaccination with whole parasite preparations. Induction of complete protection against challenge was obtained using sporozoites attenuated by irradiation or genetic manipulations. It has been demonstrated recently that immunization with normal sporozoites under chloroquine cover confers sterile protection in mice and humans, using substantially fewer parasites and injections than with irradiated sporozoite immunization. Subsequently, it was shown that other drugs can substitute for chloroquine. We describe the immunization protocol using live sporozoites under chloroquine cover, which confers sterile immunity in rodents.
[Show abstract][Hide abstract] ABSTRACT: Plasmodium vivax merozoites specifically invade reticulocytes. Until recently, two reticulocyte-binding proteins (Pvrbp1 and Pvrbp2) expressed at the apical pole of the P. vivax merozoite were considered to be involved in reticulocyte recognition. The genome sequence recently obtained for the Salvador I (Sal-I) strain of P. vivax revealed additional genes in this family, and in particular Pvrbp2a, Pvrbp2b (Pvrbp2 has been renamed as Pvrbp2c) and two pseudogenes Pvrbp2d and Pvrbp3. It had been previously found that Pvrbp2c is substantially more polymorphic than Pvrbp1. The primary goal of this study was to ascertain the level of polymorphism of these new genes.
The sequence of the Pvrbp2a, Pvrbp2b, Pvrbp2d and Pvrbp3 genes were obtained by amplification/cloning using DNA purified from four isolates collected from patients that acquired the infection in the four cardinal regions of Thailand (west, north, south and east). An additional seven isolates from western Thailand were analyzed for gene copy number variation. There were significant polymorphisms exhibited by these genes (compared to the reference Sal-I strain) with the ratio of mutations leading to a non-synonymous or synonymous amino acid change close to 3∶1 for Pvrbp2a and Pvrbp2b. Although the degree of polymorphism exhibited by these two genes was higher than that of Pvrbp1, it did not reach the exceptional diversity noted for Pvrbp2c. It was interesting to note that variations in the copy number of Pvrbp2a and Pvrbp2b occurred in some isolates.
The evolution of different members of the Pvrbp2 family and their relatively high degree of polymorphism suggests that the proteins encoded by these genes are important for parasite survival and are under immune selection. Our data also shows that there are highly conserved regions in rbp2a and rbp2b, which might provide suitable targets for future vaccine development against the blood stage of P. vivax.
PLoS ONE 06/2012; 7(3):e32105. DOI:10.1371/journal.pone.0032105 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The liver stages of malaria parasites are inhibited by cytokines such as interferon-γ or Interleukin (IL)-6. Binding of these cytokines to their receptors at the surface of the infected hepatocytes leads to the production of nitric oxide (NO) and radical oxygen intermediates (ROI), which kill hepatic parasites. However, conflicting results were obtained with TNF-α possibly because of differences in the models used. We have reassessed the role of TNF-α in the different cellular systems used to study the Plasmodium pre-erythrocytic stages.
Human or mouse TNF-α were tested against human and rodent malaria parasites grown in vitro in human or rodent primary hepatocytes, or in hepatoma cell lines. Our data demonstrated that TNF-α treatment prevents the development of malaria pre-erythrocytic stages. This inhibitory effect however varies with the infecting parasite species and with the nature and origin of the cytokine and hepatocytes. Inhibition was only observed for all parasite species tested when hepatocytes were pre-incubated 24 or 48 hrs before infection and activity was directed only against early hepatic parasite. We further showed that TNF-α inhibition was mediated by a soluble factor present in the supernatant of TNF-α stimulated hepatocytes but it was not related to NO or ROI. Treatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified.
Treatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified. However, the nature of the cytokine-host cell-parasite combination must be carefully considered for extrapolation to the human infection.
PLoS ONE 03/2011; 6(3):e17464. DOI:10.1371/journal.pone.0017464 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immunization with live Plasmodium sporozoites under chloroquine prophylaxis (Spz plus CQ) induces sterile immunity against sporozoite challenge in rodents and, more importantly, in humans. Full protection is obtained with substantially fewer parasites than with the classic immunization with radiation-attenuated sporozoites. The sterile protection observed comprised a massive reduction in the hepatic parasite load and an additional effect at the blood stage level. Differences in the immune responses induced by the two protocols occur but are as yet little characterized. We have previously demonstrated that in mice immunized with irradiated sporozoites, immune responses against the circumsporozoite protein (CSP), the major component of the sporozoite's surface and the leading malaria vaccine candidate, were not essential for sterile protection. Here, we have employed transgenic Plasmodium berghei parasites in which the endogenous CSP was replaced by that of Plasmodium yoelii, another rodent malaria species, to assess the role of CSP in the sterile protection induced by the Spz-plus-CQ protocol. The data demonstrated that this role was minor because sterile immunity was obtained irrespective of the origin of CSP expressed by the parasites in this model of protection. The immunity was obtained through a single transient exposure of the host to the immunizing parasites (preerythrocytic and erythrocytic), a dose much smaller than that required for immunization with radiation-attenuated sporozoites.
Infection and immunity 03/2010; 78(5):2182-8. DOI:10.1128/IAI.01415-09 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The origin of Plasmodium falciparum, the etiological agent of the most dangerous forms of human malaria, remains controversial. Although investigations of homologous parasites in African Apes are crucial to resolve this issue, studies have been restricted to a chimpanzee parasite related to P. falciparum, P. reichenowi, for which a single isolate was available until very recently. Using PCR amplification, we detected Plasmodium parasites in blood samples from 18 of 91 individuals of the genus Pan, including six chimpanzees (three Pan troglodytes troglodytes, three Pan t. schweinfurthii) and twelve bonobos (Pan paniscus). We obtained sequences of the parasites' mitochondrial genomes and/or from two nuclear genes from 14 samples. In addition to P. reichenowi, three other hitherto unknown lineages were found in the chimpanzees. One is related to P. vivax and two to P. falciparum that are likely to belong to distinct species. In the bonobos we found P. falciparum parasites whose mitochondrial genomes indicated that they were distinct from those present in humans, and another parasite lineage related to P. malariae. Phylogenetic analyses based on this diverse set of Plasmodium parasites in African Apes shed new light on the evolutionary history of P. falciparum. The data suggested that P. falciparum did not originate from P. reichenowi of chimpanzees (Pan troglodytes), but rather evolved in bonobos (Pan paniscus), from which it subsequently colonized humans by a host-switch. Finally, our data and that of others indicated that chimpanzees and bonobos maintain malaria parasites, to which humans are susceptible, a factor of some relevance to the renewed efforts to eradicate malaria.
[Show abstract][Hide abstract] ABSTRACT: Immunization with irradiated Plasmodium sporozoites induces sterile immunity in rodents, monkeys and humans. The major surface component of the sporozoite the circumsporozoite protein (CS) long considered as the antigen predominantly responsible for this immunity, thus remains the leading candidate antigen for vaccines targeting the parasite's pre-erythrocytic (PE) stages. However, this role for CS was questioned when we recently showed that immunization with irradiated sporozoites (IrrSpz) of a P. berghei line whose endogenous CS was replaced by that of P. falciparum still conferred sterile protection against challenge with wild type P. berghei sporozoites. In order to investigate the involvement of CS in the cross-species protection recently observed between the two rodent parasites P. berghei and P. yoelii, we adopted our gene replacement approach for the P. yoelii CS and exploited the ability to conduct reciprocal challenges. Overall, we found that immunization led to sterile immunity irrespective of the origin of the CS in the immunizing or challenge sporozoites. However, for some combinations, immune responses to CS contributed to the acquisition of protective immunity and were dependent on the immunizing IrrSpz dose. Nonetheless, when data from all the cross-species immunization/challenges were considered, the immune responses directed against non-CS parasite antigens shared by the two parasite species played a major role in the sterile protection induced by immunization with IrrSpz. This opens the perspective to develop a single vaccine formulation that could protect against multiple parasite species.
PLoS ONE 11/2009; 4(11):e7717. DOI:10.1371/journal.pone.0007717 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immunity to malaria has long been thought to be stage-specific. In this study we show that immunization of BALB/c mice with live erythrocytes infected with nonlethal strains of Plasmodium yoelii under curative chloroquine cover conferred protection not only against challenge by blood stage parasites but also against sporozoite challenge. This cross-stage protection was dose-dependent and long lasting. CD4(+) and CD8(+) T cells inhibited malaria liver but not blood stage. Their effect was mediated partially by IFN-gamma, and was completely dependent of NO. Abs against both pre-erythrocytic and blood parasites were elicited and were essential for protection against blood stage and liver stage parasites. Our results suggest that Ags shared by liver and blood stage parasites can be the foundation for a malaria vaccine that would provide effective protection against both pre-erythrocytic and erythrocytic asexual parasites found in the mammalian host.
The Journal of Immunology 01/2009; 181(12):8552-8. DOI:10.4049/jimmunol.181.12.8552 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown that IFN-gamma is essential for the pathogenesis of cerebral malaria (CM) induced by Plasmodium berghei ANKA (PbA) in mice. However, the exact role of IFN-gamma in the pathway (s) leading to CM has not yet been described. Here, we used 129P2Sv/ev mice which develop CM between 7 and 14 days post-infection with PbA. In this strain, both CD4(+) and CD8(+) T cells were involved in the effector phase of CM. When 129P2Sv/ev mice deficient in the IFN-gamma receptor alpha chain (IFN-gammaR1) were infected with PbA, CM did not occur. Migration of leucocytes to the brain at the time of CM was observed in wild type (WT) but not in deficient mice. However, in the latter, there was an accumulation of T cells in the lungs. Analysis of chemokines and their receptors in WT and in deficient mice revealed a complex, organ-specific pattern of expression. Up-regulation of RANTES/CCL5, IP-10/CCL3 and CCR2 was associated with leucocyte migration to the brain and increased expression of MCP-1/CCL2, IP-10/CCL3 and CCR5 with leucocyte migration to the lung. This shows that IFN-gamma controls trafficking of pathogenic T cells in the brain, thus providing an explanation for the organ-specific pathology induced by PbA infection.
[Show abstract][Hide abstract] ABSTRACT: Malaria parasites are obligate intracellular parasites whose invasive stages select and invade the unique host cell in which they can develop with exquisite specificity and efficacy. Most studies aimed at elucidating the molecules and the mechanisms implicated in the selection and invasion processes have been conducted on the merozoite, the stage that invades erythrocytes to perpetuate the pathological cycles of parasite multiplication in the blood. Bioinformatic analysis has helped identify the members of two parasite protein families, the reticulocyte-binding protein homologues (RBL) and erythrocyte binding like (EBL), in recently sequenced genomes of different Plasmodium species. In this article we review data from classical studies and gene disruption experiments that are helping to illuminate the role of these proteins in the selection-invasion processes. The manner in which subsets of proteins from each of the families act in concert suggests a model to explain the ability of the parasites to use alternate pathways of invasion. Future perspectives and implications are discussed.
[Show abstract][Hide abstract] ABSTRACT: Most C57BL/6 mice infected i.p. with Plasmodium berghei ANKA (PbA) die between 7 and 14 days with neurologic signs, and the remainder die later (>15 days) with severe anemia. Daily i.p. injections of a recombinant human IFN-alpha (active on mouse cells) prevented death by cerebral malaria (87% deaths in the control mice vs 6% in IFN-alpha-treated mice). The mechanisms of this IFN-alpha protective effect were multiple. IFN-alpha-treated, PbA-infected mice showed 1) a marked decrease in the number of PbA parasites in the blood mediated by IFN-gamma, 2) less sequestered parasites in cerebral vessels, 3) reduced up-regulation of ICAM-1 expression in brain endothelial cells, 4) milder rise of blood levels of TNF, 5) increased levels of IFN-gamma in the blood resulting from an increased production by splenic CD8+ T cells, and 6) fewer leukocytes (especially CD8+ T cells) sequestered in cerebral vessels. On the other hand, IFN-alpha treatment did not affect the marked anemia observed in PbA-infected mice. Survival time in IFN-alpha-treated mice was further increased by performing three blood transfusions over consecutive days.
The Journal of Immunology 06/2007; 178(10):6416-25. DOI:10.4049/jimmunol.178.10.6416 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Research aimed at developing vaccines against infectious diseases generally seeks to induce robust immune responses to immunodominant antigens. This approach has led to a number of efficient bacterial and viral vaccines, but it has yet to do so for parasitic pathogens. For malaria, a disease of global importance due to infection by Plasmodium protozoa, immunization with radiation-attenuated sporozoites uniquely leads to long lasting sterile immunity against infection. The circumsporozoite protein (CSP), an important component of the sporozoite's surface, remains the leading candidate antigen for vaccines targeting the parasite's pre-erythrocytic stages. Difficulties in developing CSP-based vaccines that reproduce the levels of protection afforded by radiation-attenuated sporozoites have led us to question the role of CSP in the acquisition of sterile immunity. We have used a parasite transgenic for the CSP because it allowed us to test whether a major immunodominant Plasmodium antigen is indeed needed for the induction of sterile protective immunity against infection.
We employed a P. berghei parasite line that expresses a heterologous CSP from P. falciparum in order to assess the role of the CSP in the protection conferred by vaccination with radiation-attenuated P. berghei parasites. Our data demonstrated that sterile immunity could be obtained despite the absence of immune responses specific to the CSP expressed by the parasite used for challenge.
We conclude that other pre-erythrocytic parasite antigens, possibly hitherto uncharacterised, can be targeted to induce sterile immunity against malaria. From a broader perspective, our results raise the question as to whether immunodominant parasite antigens should be the favoured targets for vaccine development.
PLoS ONE 02/2007; 2(12):e1371. DOI:10.1371/journal.pone.0001371 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite nearly 80 years of vaccine research and control efforts, malaria remains one of the most prevalent of all infectious diseases. The fact that people living in regions in which malaria is endemic eventually develop immunity to the parasite and the disease suggest that it might be possible to develop vaccines against malaria. Although few vaccination trials were conducted with whole parasites, the only protocol that leads to the induction of sterile immunity in humans relies on immunization with attenuated parasites. This observation has spurred the search for subunit vaccines that aim to reproduce this protection. As yet, none of the current candidate subunit vaccines have achieved complete protection reproducibly. This failure, coupled with the recent advent of the genetically modified Plasmodium parasites, has led to a renewed interest in the use of live parasites for vaccination. This article reviews past studies, summarizes recent developments in this field and discusses the challenges to be overcome before mass immunization with live parasites could be envisaged.
[Show abstract][Hide abstract] ABSTRACT: Malaria remains a major global health problem and cerebral malaria (CM) is one of the most serious complications of this disease. Recent years have seen important advances in our understanding of the pathogenesis of cerebral malaria. Parasite sequestration, a hallmark of this syndrome, is thought to be solely responsible for the pathological process. However, this phenomenon cannot explain all aspects of the pathogenesis of CM. The use of an animal model, Plasmodium berghei ANKA in mice, has allowed the identification of specific pathological components of CM. Although multiple pathways may lead to CM, an important role for CD8+ T cells has been clarified. Other cells, including platelets, and mediators such as cytokines also have an important role. In this review we have focused on the role of T cells, and discuss what remains to be studied to understand the pathways by which these cells mediate CM.
International Journal for Parasitology 06/2006; 36(5):547-54. DOI:10.1016/j.ijpara.2006.02.007 · 3.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The outcome of the Phase IIb trial of RTS,S (a vaccine comprising the polypeptides RTS and S) in young Mozambican children consolidated hopes that effective vaccination against malaria is a step nearer, and even elicited a generous promise of commitment from the Chancellor of the Exchequer of the UK. However, it seems that both optimism and generosity should be moderated by the failure of this vaccine to induce meaningful protection against infection by Plasmodium falciparum and that we should await confirmation of its efficacy in diminishing the incidence of severe malaria.
Trends in Parasitology 11/2005; 21(10):456-61. DOI:10.1016/j.pt.2005.08.002 · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protective cellular immune responses depend on MHC presentation of pathogen-derived Ag fragments. MHC diversity renders this process sensitive to point mutations coding for altered amino acid sequence of the short target Ag-derived peptides epitopes. Thus, in a given host, a pathogen with an altered epitope sequence will be more likely to escape detection and elimination by the immune system. At a population level, selection by immune pressure will increase the likelihood of polymorphism in important pathogen antigenic epitopes. This mechanism of immune evasion is found in viruses and other pathogens. The detection of polymorphic hot spots in an Ag is often taken as a strong indication of its role in protective immunity. We provide evidence that polymorphisms in the T cell epitopes of a malaria vaccine candidate are unlikely to have been selected by immune pressure in the human host.
The Journal of Immunology 10/2005; 175(6):3935-9. DOI:10.4049/jimmunol.175.6.3935 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During their complex life cycle, malaria parasites adopt morphologically, biochemically and immunologically distinct forms. The intra-hepatic form is the least known, yet of established value in the induction of sterile immunity and as a target for chemoprophylaxis. Using Plasmodium yoelii as a model we present here a novel approach to the elucidation of the transcriptome of this poorly studied stage. Sequences from Plasmodium were obtained in 388 of the 3533 inserts (11%) isolated from liver stages cDNA obtained from optimized cultures with high yields. These corresponded to a total of 88 putative P. yoelii genes. The majority of the transcribed genes identified, code for predicted proteins of as yet unknown function. The RT-PCR analysis carried out for 29 of these genes, confirmed expression at the hepatic stage and provided evidence for complex patterns of genes transcription in the distinct stages found in the mosquito and vertebrate host. The results demonstrate the efficacy of the approach that can now be applied to further detailed analysis of the hepatic stage transcriptome of Plasmodium.
[Show abstract][Hide abstract] ABSTRACT: Recent epidemiological observations suggest that clinical evolution of Plasmodium falciparum infections might be influenced by the concurrent presence of another Plasmodium species, and such mixed-species infections are now known to occur frequently in residents of most areas of endemicity. We used mice infected with P. berghei ANKA (PbA), a model for cerebral malaria (CM), to investigate the influence of experimental mixed-species infections on the expression of this pathology. Remarkably, the development of CM was completely inhibited by the simultaneous presence of P. yoelii yoelii but not that of P. vinckei or another line of P. berghei. In the protected coinfected mice, the accumulation of CD8(+) T cells in the brain vasculature, a pivotal step in CM pathogenesis, was found to be abolished. Protection from CM was further found to be associated with species-specific suppression of PbA multiplication. These observations establish the concept of mixed Plasmodium species infections as potential modulators of pathology and open novel avenues to investigate mechanisms implicated in the pathogenesis of malaria.
Infection and Immunity 09/2005; 73(8):4777-86. DOI:10.1128/IAI.73.8.4777-4786.2005 · 3.73 Impact Factor