Jessica L. Miller

Interferon-Mediated Innate Immune Responses against Malaria Parasite Liver Stages

Mosquito-transmitted malaria parasites infect hepatocytes and asymptomatically replicate as liver stages. Using RNA sequencing, we show that a rodent malaria liver-stage infection stimulates a robust innate immune response including type I interferon (IFN) and IFNγ pathways. Liver-stage infection is suppressed by these infection-engendered innate responses. This suppression was abrogated in mice deficient in IFNγ, the type I IFN α/β receptor (IFNAR), and interferon regulatory factor 3. Natural killer and CD49b(+)CD3(+) natural killer T (NKT) cells increased in the liver after a primary infection, and CD1d-restricted NKT cells, which secrete IFNγ, were critical in reducing liver-stage burden of a secondary infection. Lack of IFNAR signaling abrogated the increase in NKT cell numbers in the liver, showing a link between type I IFN signaling, cell recruitment, and subsequent parasite elimination. Our findings demonstrate innate immune sensing of malaria parasite liver-stage infection and that the ensuing innate responses can eliminate the parasite.

Model for In Vivo Assessment of Humoral Protection against Malaria Sporozoite Challenge by Passive Transfer of Monoclonal Antibodies and Immune Serum

ABSTRACT Evidence from clinical trials of malaria vaccine candidates suggests that both cell-mediated and humoral immunity to pre-erythrocytic parasite stages can provide protection against infection. Novel pre-erythrocytic antibody (Ab) targets could be key to improving vaccine formulations, which are currently based on targeting antigens such as the circumsporozoite protein (CSP). However, methods to assess the effects of sporozoite-specific Abs on pre-erythrocytic infection in vivo remain underdeveloped. Here, we combined passive transfer of monoclonal Abs (MAbs) or immune serum with a luciferase-expressing Plasmodium yoelii sporozoite challenge to assess Ab-mediated inhibition of liver infection in mice. Passive transfer of a P. yoelii CSP MAb showed inhibition of liver infection when mice were challenged with sporozoites either intravenously or by infectious mosquito bite. However, inhibition was most potent for the mosquito bite challenge, leading to a more significant reduction of liver-stage burden and even a lack of progression to blood-stage parasitemia. This suggests that Abs provide effective protection against a natural infection. Inhibition of liver infection was also achieved by passive transfer of immune serum from whole-parasite-immunized mice. Furthermore, we demonstrated that passive transfer of a MAb against P. falciparum CSP inhibited liver-stage infection in a humanized mouse/P. falciparum challenge model. Together, these models constitute unique and sensitive in vivo methods to assess serum-transferable protection against Plasmodium sporozoite challenge.

Malaria Parasite Pre-erythrocytic Infection: Preparation Meets Opportunity

For those stricken with malaria, the classic clinical symptoms are caused by the parasites cyclic infection of red blood cells. However, this erythrocytic phase of the parasites life cycle initiates from an asymptomatic pre‐erythrocytic phase: the injection of sporozoites via the bite of a parasite‐carrying Anopheline mosquito, and the ensuing infection of the liver. With the increased capabilities of studying liver stages in mice, much progress has been made elucidating the cellular and molecular basis of the parasites progression through this bottleneck of its life cycle. Here we review relevant findings on how sporozoites prepare for infection of the liver and factors crucial to liver stage development as well as key host/parasite interactions.

Quantitative Bioluminescent Imaging of Pre-Erythrocytic Malaria Parasite Infection Using Luciferase-Expressing Plasmodium yoelii

The liver stages of Plasmodium parasites are important targets for the development of anti-malarial vaccine candidates and chemoprophylaxis approaches that aim to prevent clinical infection. Analyzing the impact of interventions on liver stages in the murine malaria model system Plasmodium yoelii has been cumbersome and requires terminal procedures. In vivo imaging of bioluminescent parasites has previously been shown to be an effective and non-invasive alternative to monitoring liver stage burden in the Plasmodium berghei model. Here we report the generation and characterization of a transgenic P. yoelii parasite expressing the reporter protein luciferase throughout the parasite life cycle. In vivo bioluminescent imaging of these parasites allows for quantitative analysis of P. yoelii liver stage burden and parasite development, which is comparable to quantitative RT-PCR analysis of liver infection. Using this system, we show that both BALB/cJ and C57BL/6 mice show comparable susceptibility to P. yoelii infection with sporozoites and that bioluminescent imaging can be used to monitor protective efficacy of attenuated parasite immunizations. Thus, this rapid, simple and noninvasive method for monitoring P. yoelii infection in the liver provides an efficient system to screen and evaluate the effects of anti-malarial interventions in vivo and in real-time.

A transgenic Plasmodium falciparum NF54 strain that expresses GFP–luciferase throughout the parasite life cycle

Plasmodium falciparum is the pathogenic agent of the most lethal of human malarias. Transgenic P. falciparum parasites expressing luciferase have been created to study drug interventions of both asexual and sexual blood stages but luciferase-expressing mosquito stage and liver stage parasites have not been created which has prevented the easy quantification of mosquito stage development (e.g. for transmission blocking interventions) and liver stage development (for interventions that prevent infection). To overcome this obstacle, we have created a transgenic P. falciparum NF54 parasite that expresses a GFP-luciferase transgene throughout the life cycle. Luciferase expression is robust and measurable at all life cycle stages, including midgut oocyst, salivary gland sporozoites and liver stages, where in vivo development is easily measurable using humanized mouse infections in conjunction with an in vivo imaging system. This parasite reporter strain will accelerate testing of interventions against pre-erythrocytic life cycle stages.

Plasmodium yoelii Macrophage Migration Inhibitory Factor Is Necessary for Efficient Liver-Stage Development

ABSTRACT Mammalian macrophage migration inhibitory factor (MIF) is a multifaceted cytokine involved in both extracellular and intracellular functions. Malaria parasites express a MIF homologue that might modulate host immune responses against blood-stage parasites, but the potential importance of MIF against other life cycle stages remains unstudied. In this study, we characterized the MIF homologue of Plasmodium yoelii throughout the life cycle, with emphasis on preerythrocytic stages. P. yoelii MIF (Py-MIF) was expressed in blood-stage parasites and detected at low levels in mosquito salivary gland sporozoites. MIF expression was strong throughout liver-stage development and localized to the cytoplasm of the parasite, with no evidence of release into the host hepatocyte. To examine the importance of Py-MIF for liver-stage development, we generated a Py-mif knockout parasite (P. yoelii Δmif). P. yoelii Δmif parasites grew normally as asexual erythrocytic-stage parasites and showed normal infection of mosquitoes. In contrast, the P. yoelii Δmif strain was attenuated during the liver stage. Mice infected with P. yoelii Δmif sporozoites either did not develop blood-stage parasitemia or exhibited a delay in the onset of blood-stage patency. Furthermore, P. yoelii Δmif parasites exhibited growth retardation in vivo. Combined, the data indicate that Plasmodium MIF is important for liver-stage development of P. yoelii, during which it is likely to play an intrinsic role in parasite development rather than modulating host immune responses to infection.

Extrahepatic exoerythrocytic forms of rodent malaria parasites at the site of inoculation: Clearance after immunization, susceptibility to primaquine, and contribution to blood-stage infection

ABSTRACT Plasmodium sporozoites are inoculated into the skin of the mammalian host as infected mosquitoes probe for blood. A proportion of the inoculum enters the bloodstream and goes to the liver, where the sporozoites invade hepatocytes and develop into the next life cycle stage, the exoerythrocytic, or liver, stage. Here, we show that a small fraction of the inoculum remains in the skin and begins to develop into exoerythrocytic forms that can persist for days. Skin exoerythrocytic forms were observed for both Plasmodium berghei and Plasmodium yoelii, two different rodent malaria parasites, suggesting that development in the skin of the mammalian host may be a common property of plasmodia. Our studies demonstrate that skin exoerythrocytic stages are susceptible to destruction in immunized mice, suggesting that their aberrant location does not protect them from the hosts adaptive immune response. However, in contrast to their hepatic counterparts, they are not susceptible to primaquine. We took advantage of their resistance to primaquine to test whether they could initiate a blood-stage infection directly from the inoculation site, and our data indicate that these stages are not able to initiate malaria infection.

Mechanisms of Stage-Transcending Protection Following Immunization of Mice with Late Liver Stage-Arresting Genetically Attenuated Malaria Parasites

Malaria, caused by Plasmodium parasite infection, continues to be one of the leading causes of worldwide morbidity and mortality. Development of an effective vaccine has been encumbered by the complex life cycle of the parasite that has distinct pre-erythrocytic and erythrocytic stages of infection in the mammalian host. Historically, malaria vaccine development efforts have targeted each stage in isolation. An ideal vaccine, however, would target multiple life cycle stages with multiple arms of the immune system and be capable of eliminating initial infection in the liver, the subsequent blood stage infection, and would prevent further parasite transmission. We have previously shown that immunization of mice with Plasmodium yoelii genetically attenuated parasites (GAP) that arrest late in liver stage development elicits stage-transcending protection against both a sporozoite challenge and a direct blood stage challenge. Here, we show that this immunization strategy engenders both T- and B-cell responses that are essential for stage-transcending protection, but the relative importance of each is determined by the host genetic background. Furthermore, potent anti-blood stage antibodies elicited after GAP immunization rely heavily on FC-mediated functions including complement fixation and FC receptor binding. These protective antibodies recognize the merozoite surface but do not appear to recognize the immunodominant merozoite surface protein-1. The antigen(s) targeted by stage-transcending immunity are present in both the late liver stages and blood stage parasites. The data clearly show that GAP-engendered protective immune responses can target shared antigens of pre-erythrocytic and erythrocytic parasite life cycle stages. As such, this model constitutes a powerful tool to identify novel, protective and stage-transcending T and B cell targets for incorporation into a multi-stage subunit vaccine.

Plasmodium yoelii inhibitor of cysteine proteases is exported to exomembrane structures and interacts with yoelipain‐2 during asexual blood‐stage development

Plasmodium falciparum (Pf) blood stages express falstatin, an inhibitor of cysteine proteases (ICP), which is implicated in regulating proteolysis during red blood cell infection. Recent data using the Plasmodium berghei rodent malaria model suggested an additional role for ICP in the infection of hepatocytes by sporozoites and during liver‐stage development. Here we further characterize the role of ICP in vivo during infection with Plasmodium yoelii (Py) and Pf. We found that Py‐ICP was refractory to targeted gene deletion indicating an essential function during asexual blood‐stage replication, but significant downregulation of ICP using a regulated system did not impact blood‐stage growth. Py‐ICP localized to vesicles within the asexual blood‐stage parasite cytoplasm, as well as the parasitophorous vacuole, and was exported to dynamic exomembrane structures in the infected RBC. In sporozoites, expression was observed in rhoptries, in addition to intracellular vesicles distinct from TRAP containing micronemes. During liver‐stage development, Py‐ICP was confined to the parasite compartment until the final phase of liver‐stage development when, after parasitophorous vacuolemembrane breakdown, it was released into the infected hepatocyte. Finally, we identified the cysteine protease yoelipain‐2 as a binding partner of Py‐ICP during blood‐stage infection. These data show that ICP may be important in regulating proteolytic processes during blood‐stage development, and is likely playing a role in liver stage‐hepatocyte interactions at the time of exoerythrocytic merozoite release.

Colocalization of a CD1d-Binding Glycolipid with a Radiation-Attenuated Sporozoite Vaccine in Lymph Node–Resident Dendritic Cells for a Robust Adjuvant Effect

A CD1d-binding glycolipid, α-Galactosylceramide (αGalCer), activates invariant NK T cells and acts as an adjuvant. We previously identified a fluorinated phenyl ring-modified αGalCer analog, 7DW8-5, displaying nearly 100-fold stronger CD1d binding affinity. In the current study, 7DW8-5 was found to exert a more potent adjuvant effect than αGalCer for a vaccine based on radiation-attenuated sporozoites of a rodent malaria parasite, Plasmodium yoelii, also referred to as irradiated P. yoelii sporozoites (IrPySpz). 7DW8-5 had a superb adjuvant effect only when the glycolipid and IrPySpz were conjointly administered i.m. Therefore, we evaluated the effect of distinctly different biodistribution patterns of αGalCer and 7DW8-5 on their respective adjuvant activities. Although both glycolipids induce a similar cytokine response in sera of mice injected i.v., after i.m. injection, αGalCer induces a systemic cytokine response, whereas 7DW8-5 is locally trapped by CD1d expressed by dendritic cells (DCs) in draining lymph nodes (dLNs). Moreover, the i.m. coadministration of 7DW8-5 with IrPySpz results in the recruitment of DCs to dLNs and the activation and maturation of DCs. These events cause the potent adjuvant effect of 7DW8-5, resulting in the enhancement of the CD8+ T cell response induced by IrPySpz and, ultimately, improved protection against malaria. Our study is the first to show that the colocalization of a CD1d-binding invariant NK T cell–stimulatory glycolipid and a vaccine, like radiation-attenuated sporozoites, in dLN-resident DCs upon i.m. conjoint administration governs the potency of the adjuvant effect of the glycolipid.