A Role for Immune Responses against Non-CS Components in the Cross-Species Protection Induced by Immunization with Irradiated Malaria Sporozoites

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.     

Immunization with Pre-Erythrocytic Antigen CelTOS from Plasmodium falciparum Elicits Cross-Species Protection against Heterologous Challenge with Plasmodium berghei

Background

The Plasmodium protein Cell-traversal protein for ookinetes and sporozoites (CelTOS) plays an important role in cell traversal of host cells in both, mosquito and vertebrates, and is required for successful malaria infections. CelTOS is highly conserved among the Plasmodium species, suggesting an important functional role across all species. Therefore, targeting the immune response to this highly conserved protein and thus potentially interfering with its biological function may result in protection against infection even by heterologous species of Plasmodium.

Methodology/Principal Findings

To test this hypothesis, we developed a recombinant codon-harmonized P. falciparum CelTOS protein that can be produced to high yields in the E. coli expression system. Inbred Balb/c and outbred CD-1 mice were immunized with various doses of the recombinant protein adjuvanted with Montanide ISA 720 and characterized using in vitro and in vivo analyses.

Conclusions/Significance

Immunization with PfCelTOS resulted in potent humoral and cellular immune responses and most importantly induced sterile protection against a heterologous challenge with P. berghei sporozoites in a proportion of both inbred and outbred mice. The biological activity of CelTOS-specific antibodies against the malaria parasite is likely linked to the impairment of sporozoite motility and hepatocyte infectivity. The results underscore the potential of this antigen as a pre-erythrocytic vaccine candidate and demonstrate for the first time a malaria vaccine that is cross-protective between species.     

Wild Anopheles funestus Mosquito Genotypes Are Permissive for Infection with the Rodent Malaria Parasite,Plasmodium berghei

Background

Malaria parasites undergo complex developmental transitions within the mosquito vector. A commonly used laboratory model for studies of mosquito-malaria interaction is the rodent parasite, P. berghei. Anopheles funestus is a major malaria vector in sub-Saharan Africa but has received less attention than the sympatric species, Anopheles gambiae. The imminent completion of the A. funestus genome sequence will provide currently lacking molecular tools to describe malaria parasite interactions in this mosquito, but previous reports suggested that A. funestus is not permissive for P. berghei development.

Methods

An A. funestus population was generated in the laboratory by capturing female wild mosquitoes in Mali, allowing them to oviposit, and rearing the eggs to adults. These F1 progeny of wild mosquitoes were allowed to feed on mice infected with a fluorescent P. berghei strain. Fluorescence microscopy was used to track parasite development inside the mosquito, salivary gland sporozoites were tested for infectivity to mice, and parasite development in A. funestus was compared to A. gambiae.

Results

P. berghei oocysts were detectable on A. funestus midguts by 7 days post-infection. By 18–20 days post-infection, sporozoites had invaded the median and distal lateral lobes of the salivary glands, and hemocoel sporozoites were observed in the hemolymph. Mosquitoes were capable of infecting mice via bite, demonstrating that A. funestus supports the complete life cycle of P. berghei. In a random sample of wild mosquito genotypes, A. funestus prevalence of infection and the characteristics of parasite development were similar to that observed in A. gambiae-P. berghei infections.

Conclusions

The data presented in this study establish an experimental laboratory model for Plasmodium infection of A. funestus, an important vector of human malaria. Studying A. funestus-Plasmodium interactions is now feasible in a laboratory setting. This information lays the groundwork for exploitation of the awaited genome sequence of A. funestus.     

Plasmodium berghei circumvents immune responses induced by merozoite surface protein 1- and apical membrane antigen 1-based vaccines

Background

Two current leading malaria blood-stage vaccine candidate antigens for Plasmodium falciparum, the C-terminal region of merozoite surface protein 1 (MSP1₁₉) and apical membrane antigen 1 (AMA1), have been prioritized because of outstanding protective efficacies achieved in a rodent malaria Plasmodium yoelii model. However, P. falciparum vaccines based on these antigens have had disappointing outcomes in clinical trials. Discrepancies in the vaccine efficacies observed between the P. yoelii model and human clinical trials still remain problematic.

Methodology and Results

In this study, we assessed the protective efficacies of a series of MSP1₁₉- and AMA1-based vaccines using the P. berghei rodent malarial parasite and its transgenic models. Immunization of mice with a baculoviral-based vaccine (BBV) expressing P. falciparum MSP1₁₉ induced high titers of PfMSP1₁₉-specific antibodies that strongly reacted with P. falciparum blood-stage parasites. However, no protection was achieved following lethal challenge with transgenic P. berghei expressing PfMSP1₁₉ in place of native PbMSP1₁₉. Similarly, neither P. berghei MSP1₁₉- nor AMA1-BBV was effective against P. berghei. In contrast, immunization with P. yoelii MSP1₁₉- and AMA1-BBVs provided 100% and 40% protection, respectively, against P. yoelii lethal challenge. Mice that naturally acquired sterile immunity against P. berghei became cross-resistant to P. yoelii, but not vice versa.

Conclusion

This is the first study to address blood-stage vaccine efficacies using both P. berghei and P. yoelii models at the same time. P. berghei completely circumvents immune responses induced by MSP1₁₉- and AMA1-based vaccines, suggesting that P. berghei possesses additional molecules and/or mechanisms that circumvent the host''s immune responses to MSP1₁₉ and AMA1, which are lacking in P. yoelii. Although it is not known whether P. falciparum shares these escape mechanisms with P. berghei, P. berghei and its transgenic models may have potential as useful tools for identifying and evaluating new blood-stage vaccine candidate antigens for P. falciparum.     

Conserved Protective Mechanisms in Radiation and Genetically Attenuated uis3(-) and uis4(-) Plasmodium Sporozoites

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.     

Complex Minigene Library Vaccination for Discovery of Pre-Erythrocytic Plasmodium T Cell Antigens

Development of a subunit vaccine targeting liver-stage Plasmodium parasites requires the identification of antigens capable of inducing protective T cell responses. However, traditional methods of antigen identification are incapable of evaluating T cell responses against large numbers of proteins expressed by these parasites. This bottleneck has limited development of subunit vaccines against Plasmodium and other complex intracellular pathogens. To address this bottleneck, we are developing a synthetic minigene technology for multi-antigen DNA vaccines. In an initial test of this approach, pools of long (150 bp) antigen-encoding oligonucleotides were synthesized and recombined into vectors by ligation-independent cloning to produce two DNA minigene library vaccines. Each vaccine encoded peptides derived from 36 (vaccine 1) and 53 (vaccine 2) secreted or transmembrane pre-erythrocytic P. yoelii proteins. BALB/cj mice were vaccinated three times with a single vaccine by biolistic particle delivery (gene gun) and screened for interferon-γ-producing T cell responses by ELISPOT. Library vaccination induced responses against four novel antigens. Naïve mice exposed to radiation-attenuated sporozoites mounted a response against only one of the four novel targets (PyMDH, malate dehydrogenase). The response to PyMDH could not be recalled by additional homologous sporozoite immunizations but could be partially recalled by heterologous cross-species sporozoite exposure. Vaccination against the dominant PyMDH epitope by DNA priming and recombinant Listeria boosting did not protect against sporozoite challenge. Improvements in library design and delivery, combined with methods promoting an increase in screening sensitivity, may enable complex minigene screening to serve as a high-throughput system for discovery of novel T cell antigens.     

Immune response to pre-erythrocytic stages of malaria parasites

Immunization with radiation-attenuated Plasmodium spp. sporozoites induces sterile protective immunity against parasite challenge. This immunity is targeted primarily against the intrahepatic parasite and appears to be sustained long term even in the absence of sporozoite exposure. It is mediated by multifactorial mechanisms, including T cells directed against parasite antigens expressed in the liver stage of the parasite life cycle and antibodies directed against sporozoite surface proteins. In rodent models, CD8+ T cells have been implicated as the principal effector cells, and IFN-gamma as a critical effector molecule. IL-4 secreting CD4+ T cells are required for induction of the CD8+ T cell responses, and Th1 CD4+ T cells provide help for optimal CD8+ T cell effector activity. Components of the innate immune system, including gamma-delta T cells, natural killer cells and natural killer T cells, also play a role. The precise nature of pre-erythrocytic stage immunity in humans, including the contribution of these immune responses to the age-dependent immunity naturally acquired by residents of malaria endemic areas, is still poorly defined. The importance of immune effector targets at the pre-erythrocytic stage of the parasite life cycle is highlighted by the fact that infection-blocking immunity in humans rarely, if ever, occurs under natural conditions. Herein, we review our current understanding of the molecular and cellular aspects of pre-erythrocytic stage immunity.     

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.     

Identification of Pre-Erythrocytic Malaria Antigens That Target Hepatocytes for Killing In Vivo and Contribute to Protection Elicited by Whole-Parasite Vaccination

Pre-erythrocytic malaria vaccines, including those based on whole-parasite approaches, have shown protective efficacy in animal and human studies. However few pre-erythocytic antigens other than the immunodominant circumsporozoite protein (CSP) have been studied in depth with the goal of developing potent subunit malaria vaccines that are suited for use in endemic areas. Here we describe a novel technique to identify pre-erythrocytic malaria antigens that contribute to protection elicited by whole-parasite vaccination in the mouse model. Our approach combines immunization with genetically attenuated parasites and challenge with DNA plasmids encoding for potential protective pre-erythrocytic malaria antigens as luciferase fusions by hydrodynamic tail vein injection. After optimizing the technique, we first showed that immunization with Pyfabb/f⁻, a P. yoelii genetically attenuated parasite, induces killing of CSP-presenting hepatocytes. Depletion of CD8⁺ but not CD4⁺ T cells diminished the killing of CSP-expressing hepatocytes, indicating that killing is CD8⁺ T cell-dependent. Finally we showed that the use of heterologous prime/boost immunization strategies that use genetically attenuated parasites and DNA vaccines enabled the characterization of a novel pre-erythrocytic antigen, Tmp21, as a contributor to Pyfabb/f⁻ induced protection. This technique will be valuable for identification of potentially protective liver stage antigens and has the potential to contribute to the understanding of immunity elicited by whole parasite vaccination, as well as the development of effective subunit malaria vaccines.     

Imaging mosquito transmission of Plasmodium sporozoites into the mammalian host: Immunological implications

The malaria infection is initiated in mammals by injection of the sporozoite stage of the parasite through the bite of Plasmodium-infected, female Anopheles mosquitoes. Sporozoites are injected into extravascular portions of the skin while the mosquito is probing for a blood source. Sporozoite gliding motility allows them to locate and penetrate blood vessels of the dermis or subcutaneous tissues; once in the blood, they reach the liver, within which they continue their development. Some of the injected parasites invade dermal lymph vessels and travel to the proximal draining lymphatic node, where they interact with host immunocytes. The host responds to viable or attenuated sporozoites with antibodies directed against the immunodominant circumsporozoite protein (CSP), as well as against other sporozoite proteins. These CSP antibodies can inhibit the numbers of sporozoites injected by mosquitoes and the motility of those injected into the skin. This first phase of the immune response is followed by cell-mediated immunity involving CD8 T-cells directed against the developing liver stage of the parasite. This review discusses the early history of imaging studies, and focuses on the role that imaging has played in enabling a better understanding of both the induction and effector functions of the immune responses against sporozoites.     

Inhibition of Plasmodium sporozoites infection by targeting the host cell

There is a great need of new drugs against malaria because of the increasing spread of parasite resistance against the most commonly used drugs in the field. We found that monensin, a common veterinary antibiotic, has a strong inhibitory effect in Plasmodium berghei and Plasmodium yoelii sporozoites hepatocyte infection in vitro. Infection of host cells by another apicomplexan parasite with a similar mechanism of host cell invasion, Toxoplasma tachyzoites, was also inhibited. Treatment of mice with monensin abrogates liver infection with P. berghei sporozoites in vivo. We also found that at low concentrations monensin inhibits the infection of Plasmodium sporozoites by rendering host cells resistant to infection, rather than having a direct effect on sporozoites. Monensin effect is targeted to the initial stages of parasite invasion of the host cell with little or no effect on development, suggesting that this antibiotic affects an essential host cell component that is required for Plasmodium sporozoite invasion.     

Protective Antibody and CD8+ T-Cell Responses to the Plasmodium falciparum Circumsporozoite Protein Induced by a Nanoparticle Vaccine

Background

The worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8⁺ and CD4⁺ T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN) vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects.

Methodology/Principal Findings

To establish the basis for a SAPN-based vaccine, B- and CD8⁺ T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP) and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein (∼125 amino acids) that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP). We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year) protective antibody and poly-functional (IFNγ⁺, IL-2⁺) long-lived central memory CD8⁺ T-cells. Furthermore, we demonstrated that these Ab or CD8⁺ T–cells can independently provide sterile protection against a lethal challenge of the transgenic parasites.

Conclusion

The SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP) and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP.     

Sterile Immunity to Malaria after DNA Prime/Adenovirus Boost Immunization Is Associated with Effector Memory CD8+T Cells Targeting AMA1 Class I Epitopes

Background

Fifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) and boosted with human adenovirus-5 (Ad) expressing the same antigens (DNA/Ad). Four volunteers (27%) demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA) did not develop sterile protection.

Methodology/Principal Findings

We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans.

Conclusions/Significance

We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches that elicit responses to these epitopes will increase the potency of next generation gene-based vaccines.     

A new role for an old antimicrobial: lysozyme c-1 can function to protect malaria parasites in Anopheles mosquitoes

Background

Plasmodium requires an obligatory life stage in its mosquito host. The parasites encounter a number of insults while journeying through this host and have developed mechanisms to avoid host defenses. Lysozymes are a family of important antimicrobial immune effectors produced by mosquitoes in response to microbial challenge.

Methodology/Principal Findings

A mosquito lysozyme was identified as a protective agonist for Plasmodium. Immunohistochemical analyses demonstrated that Anopheles gambiae lysozyme c-1 binds to oocysts of Plasmodium berghei and Plasmodium falciparum at 2 and 5 days after infection. Similar results were observed with Anopheles stephensi and P. falciparum, suggesting wide occurrence of this phenomenon across parasite and vector species. Lysozyme c-1 did not bind to cultured ookinetes nor did recombinant lysozyme c-1 affect ookinete viability. dsRNA-mediated silencing of LYSC-1 in Anopheles gambiae significantly reduced the intensity and the prevalence of Plasmodium berghei infection. We conclude that this host antibacterial protein directly interacts with and facilitates development of Plasmodium oocysts within the mosquito.

Conclusions/Significance

This work identifies mosquito lysozyme c-1 as a positive mediator of Plasmodium development as its reduction reduces parasite load in the mosquito host. These findings improve our understanding of parasite development and provide a novel target to interrupt parasite transmission to human hosts.     

Highly Sensitive Quantitative Real-Time PCR for the Detection of Plasmodium Liver-Stage Parasite Burden following Low-Dose Sporozoite Challenge

The pre-erythrocytic stages of Plasmodium spp. are increasingly recognised as ideal targets for prophylactic vaccines and drug treatments. Intense research efforts in the last decade have been focused on in vitro culture and in vivo detection and quantification of liver stage parasites to assess the effects of candidate vaccines or drugs. Typically, the onset of blood stage parasitaemia is used as a surrogate endpoint to estimate the efficacy of vaccines and drugs targeting pre-erythrocytic parasite stages in animal models. However, this provides no information on the parasite burden in the liver after vaccination or treatment and therefore does not detect partial efficacy of any vaccine or drug candidates. Herein, we describe a quantitative RT-PCR method adapted to detect and quantitate Plasmodium yoelii liver stages in mice with increased sensitivity even after challenge with as few as 50 cryopreserved sporozoites (corresponding to approximately 5-10 freshly isolated sporozoites). We have validated our quantitative RT-PCR assay according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and established high reproducibility and accuracy. Our assay provides a rapid and reproducible assessment of liver stage parasite burden in rodent malaria models, thereby facilitating the evaluation of the efficacy of anti-malarial drugs or prophylactic vaccines with high precision and efficacy.     

The origin of malarial parasites in orangutans

Background

Recent findings of Plasmodium in African apes have changed our perspectives on the evolution of malarial parasites in hominids. However, phylogenetic analyses of primate malarias are still missing information from Southeast Asian apes. In this study, we report molecular data for a malaria parasite lineage found in orangutans.

Methodology/Principal Findings

We screened twenty-four blood samples from Pongo pygmaeus (Kalimantan, Indonesia) for Plasmodium parasites by PCR. For all the malaria positive orangutan samples, parasite mitochondrial genomes (mtDNA) and two antigens: merozoite surface protein 1 42 kDa (MSP-1₄₂) and circumsporozoite protein gene (CSP) were amplified, cloned, and sequenced. Fifteen orangutans tested positive and yielded 5 distinct mitochondrial haplotypes not previously found. The haplotypes detected exhibited low genetic divergence among them, indicating that they belong to one species. We report phylogenetic analyses using mitochondrial genomes, MSP-1₄₂ and CSP. We found that the orangutan malaria parasite lineage was part of a monophyletic group that includes all the known non-human primate malaria parasites found in Southeast Asia; specifically, it shares a recent common ancestor with P. inui (a macaque parasite) and P. hylobati (a gibbon parasite) suggesting that this lineage originated as a result of a host switch. The genetic diversity of MSP-1₄₂ in orangutans seems to be under negative selection. This result is similar to previous findings in non-human primate malarias closely related to P. vivax. As has been previously observed in the other Plasmodium species found in non-human primates, the CSP shows high polymorphism in the number of repeats. However, it has clearly distinctive motifs from those previously found in other malarial parasites.

Conclusion

The evidence available from Asian apes indicates that these parasites originated independently from those found in Africa, likely as the result of host switches from other non-human primates.     

Inhibitory effect of TNF-α on malaria pre-erythrocytic stage development: influence of host hepatocyte/parasite combinations

Background

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.

Methods and Findings

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.

Conclusions

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.