Daniel Y. Bargieri

On the Cytoadhesion of Plasmodium vivax–Infected Erythrocytes

BACKGROUND Plasmodium falciparum and Plasmodium vivax are responsible for most of the global burden of malaria. Although the accentuated pathogenicity of P. falciparum occurs because of sequestration of the mature erythrocytic forms in the microvasculature, this phenomenon has not yet been noted in P. vivax. The increasing number of severe manifestations of P. vivax infections, similar to those observed for severe falciparum malaria, suggests that key pathogenic mechanisms (eg, cytoadherence) might be shared by the 2 parasites. METHODS Mature P. vivax-infected erythrocytes (Pv-iEs) were isolated from blood samples collected from 34 infected patients. Pv-iEs enriched on Percoll gradients were used in cytoadhesion assays with human lung endothelial cells, Saimiri brain endothelial cells, and placental cryosections. RESULTS Pv-iEs were able to cytoadhere under static and flow conditions to cells expressing endothelial receptors known to mediate the cytoadhesion of P. falciparum. Although Pv-iE cytoadhesion levels were 10-fold lower than those observed for P. falciparum-infected erythrocytes, the strength of the interaction was similar. Cytoadhesion of Pv-iEs was in part mediated by VIR proteins, encoded by P. vivax variant genes (vir), given that specific antisera inhibited the Pv-iE-endothelial cell interaction. CONCLUSIONS These observations prompt a modification of the current paradigms of the pathogenesis of malaria and clear the way to investigate the pathophysiology of P. vivax infections.

Apical membrane antigen 1 mediates apicomplexan parasite attachment but is dispensable for host cell invasion

Apicomplexan parasites invade host cells by forming a ring-like junction with the cell surface and actively sliding through the junction inside an intracellular vacuole. Apical membrane antigen 1 is conserved in apicomplexans and a long-standing malaria vaccine candidate. It is considered to have multiple important roles during host cell penetration, primarily in structuring the junction by interacting with the rhoptry neck 2 protein and transducing the force generated by the parasite motor during internalization. Here, we generate Plasmodium sporozoites and merozoites and Toxoplasma tachyzoites lacking apical membrane antigen 1, and find that the latter two are impaired in host cell attachment but the three display normal host cell penetration through the junction. Therefore, apical membrane antigen 1, rather than an essential invasin, is a dispensable adhesin of apicomplexan zoites. These genetic data have implications on the use of apical membrane antigen 1 or the apical membrane antigen 1–rhoptry neck 2 interaction as targets of intervention strategies against malaria or other diseases caused by apicomplexans.

New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin

The present study evaluated the immunogenicity of new malaria vaccine formulations based on the 19kDa C-terminal fragment of Plasmodium vivax Merozoite Surface Protein-1 (MSP1(19)) and the Salmonella enterica serovar Typhimurium flagellin (FliC), a Toll-like receptor 5 (TLR5) agonist. FliC was used as an adjuvant either admixed or genetically linked to the P. vivax MSP1(19) and administered to C57BL/6 mice via parenteral (s.c.) or mucosal (i.n.) routes. The recombinant fusion protein preserved MSP1(19) epitopes recognized by sera collected from P. vivax infected humans and TLR5 agonist activity. Mice parenterally immunized with recombinant P. vivax MSP1(19) in the presence of FliC, either admixed or genetically linked, elicited strong and long-lasting MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass response. Incorporation of another TLR agonist, CpG ODN 1826, resulted in a more balanced response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response measured by interferon-gamma secretion. Finally, we show that MSP1(19)-specific antibodies recognized the native protein expressed on the surface of P. vivax parasites harvested from infected humans. The present report proposes a new class of malaria vaccine formulation based on the use of malarial antigens and the innate immunity agonist FliC. It contains intrinsic adjuvant properties and enhanced ability to induce specific humoral and cellular immune responses when administered alone or in combination with other adjuvants.

Independent Roles of Apical Membrane Antigen 1 and Rhoptry Neck Proteins during Host Cell Invasion by Apicomplexa

During invasion, apicomplexan parasites form an intimate circumferential contact with the host cell, the tight junction (TJ), through which they actively glide. The TJ, which links the parasite motor to the host cell cytoskeleton, is thought to be composed of interacting apical membrane antigen 1 (AMA1) and rhoptry neck (RON) proteins. Here we find that, in Plasmodium berghei, while both AMA1 and RON4 are important for merozoite invasion of erythrocytes, only RON4 is required for sporozoite invasion of hepatocytes, indicating that RON4 acts independently of AMA1 in the sporozoite. Further, in the Toxoplasma gondii tachyzoite, AMA1 is dispensable for normal RON4 ring and functional TJ assembly but enhances tachyzoite apposition to the cell and internalization frequency. We propose that while the RON proteins act at the TJ, AMA1 mainly functions on the zoite surface to permit correct attachment to the cell, which may facilitate invasion depending on the zoite-cell combination.

CD8+ T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2(d)-restricted CD8(+) T cell-specific epitope (CS(280-288)) derived from the Plasmodium yoelii circumsporozoite (CS) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS(280-288) peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS(280-288) peptide. The results showed that CS(280-288)-specific cytotoxic CD8(+) T cells were primed when BALB/c mice were orally inoculated with the expressing the CS(280-288) epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS(280-288) peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8(+) T cell responses without the need of a heterologous booster immunization. The CD8(+) T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c(+) dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations.

Host Cell Invasion by Apicomplexan Parasites: The Junction Conundrum

Apicomplexans form a large phylum of parasitic protists, some of which cause severe diseases in humans. Most notorious is Plasmodium, the agent of malaria, which kills around a million people each year, mostly young children in Africa. Most successful is Toxoplasma, which parasitizes nearly a third of the human population, making those people at risk of life-threatening complications, primarily encephalitis or pneumonia, in case of immunosuppression. Other apicomplexans of human importance include Cryptosporidium, Isospora, and Sarcocystis, which are opportunistic pathogens that cause severe diarrhea often associated with AIDS. Several apicomplexan parasites cause heavy losses in livestock, particularly Theileria and Babesia in cattle and Eimeria in poultry. Most apicomplexans are obligate intracellular parasites. Their extracellular stages, called zoites, display several conserved features: they are elongated and polarized cells, their shape is maintained by a set of microtubules running longitudinally, and their anterior pole contains secretory vesicles, called micronemes and rhoptries, which secrete their content at the anterior tip of the parasite. Most zoites also share two unique properties among eukaryotic cells. They move on substrate by a gliding type of motility, i.e., without overt deformation of the cell shape, at speeds of several microns per second. They also typically invade host cells by forming a ring-like junction with the host cell membrane. Zoites slide through the junction into an invagination of the host cell surface that becomes the parasitophorous vacuole (PV) after pinching off from the host cell plasma membrane, in a process that takes less than a minute. Once inside the PV niche, the zoite can multiply into multiple new zoites that eventually egress the infected cell to infect new host cells. Much work has been performed since the late 1970s to understand the cellular and molecular bases of host cell invasion by apicomplexans, using various zoites as models. The overall invasion process encompasses several steps, including loose followed by intimate attachment, reorientation relative to the host cell surface, and organelle discharge with junction formation. The ultimate step, sliding through the junction inside the PV and called here internalization, is commonly viewed as powered by the zoite submembrane actin-myosin motor. The junction is thought to act as a traction point for the motor, to bridge the cortical cytoskeletons in the two cells, and to be made of parasite proteins conserved in the apicomplexan phylum. In this review, we confront these established notions with genetic data recently obtained in Plasmodium and Toxoplasma parasites. The Junction: From “Moving” to Stationary The first observation of a junction between an apicomplexan zoite and its host cell was made using Plasmodium merozoites and their target cells, erythrocytes [1]. Electron microscopy showed that the merozoite, after initial random binding, reorients so that its apical tip faces the erythrocyte surface, and then induces a circumferential zone of close apposition of the zoite and erythrocyte membranes over ∼250 nm and the thickening of the inner leaflet of the erythrocyte membrane [1]. This junctional area was described as “actively moving down the body of the merozoite,” since the poorly motile merozoite was not thought to be capable of actively moving inside the cell, and was thus termed “moving junction” [1]. Studies in the 1980s focused on the highly motile Eimeria sporozoites. They showed that several activities at the zoite surface were dependent on parasite actin, including the posterior translocation (capping) of various surface ligands and beads [2]. Videomicroscopic studies revealed that host cell invasion by Eimeria sporozoites was continuous with extracellular gliding [3]. This led to the proposal that the zoite actin-based system would power both gliding motility and host cell invasion by capping substrate-binding ligands or the junction, respectively, which implied that the zoite actively moved inside the host cell [3], [4]. After myosins were identified in Toxoplasma [5] and in Plasmodium [6], it was assumed that an actin-myosin motor powered the zoite motile processes. The role of the host cell during zoite invasion has been studied mainly with Toxoplasma tachyzoites, which can be made to invade host cells at high frequency and synchronicity. The host cell was initially described as displaying no detectable actin reorganization and playing no active role during tachyzoite invasion [7], [8]. More recent work found that Toxoplasma tachyzoites induced, specifically at the junction, host actin polymerization and recruitment of the Arp2/3 complex, an actin-nucleating factor, which is important for tachyzoite entry [9]. Videomicroscopic studies showed a stationary ring of host F-actin at the parasite constriction, in agreement with the junction acting as an anchor for zoite traction inside the cell. In addition to de novo actin polymerization at the junction, tachyzoite invasion also requires disorganization of the host cortical actin meshwork. This activity is in part dependent on Toxofilin [10], a Toxoplasma protein that sequesters actin monomers in vitro [11] and promotes actin turnover at the leading edge of the cell [10]. Localized actin disassembly might thus release G-actin necessary to feed actin reassembly at the junction, regulated by recruited Arp2/3 complex, to anchor the junction to the host cortical cytoskeleton.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium.

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coli and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His(6)FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freunds adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP1(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity.

Plasmodium vivax apical membrane antigen-1: comparative recognition of different domains by antibodies induced during natural human infection

The Apical Membrane Antigen-1 (AMA-1) of Plasmodium sp. has been suggested as a vaccine candidate against malaria. This protein seems to be involved in merozoite invasion and its extra-cellular portion contains three distinct domains: DI, DII, and DIII. Previously, we described that Plasmodium vivax AMA-1 (PvAMA-1) ectodomain is highly immunogenic in natural human infections. Here, we expressed each domain, separately or in combination (DI-II or DII-III), as bacterial recombinant proteins to map immunodominant epitopes within the PvAMA-1 ectodomain. IgG recognition was assessed by ELISA using sera of P. vivax-infected individuals collected from endemic regions of Brazil or antibodies raised in immunized mice. The frequencies of responders to recombinant proteins containing the DII were higher than the others and similar to the ones observed against the PvAMA-1 ectodomain. Moreover, ELISA inhibition assays using the PvAMA-1 ectodomain as substrate revealed the presence of many common epitopes within DI-II that are recognized by human immune antibodies. Finally, immunization of mice with the PvAMA-1 ectodomain induced high levels of antibodies predominantly to DI-II. Together, our results indicate that DII is particularly immunogenic during natural human infections, thus indicating that this region could be used as part of an experimental sub-unit vaccine to prevent vivax malaria.

A recombinant vaccine based on domain II of Plasmodium vivax Apical Membrane Antigen 1 induces high antibody titres in mice

The Apical Membrane Antigen 1 (AMA-1) is considered a promising candidate for development of a malaria vaccine against asexual stages of Plasmodium. We recently identified domain II (DII) of Plasmodium vivax AMA-1 (PvAMA-1) as a highly immunogenic region recognised by IgG antibodies present in many individuals during patent infection with P. vivax. The present study was designed to evaluate the immunogenic properties of a bacterial recombinant protein containing PvAMA-1 DII. To accomplish this, the recombinant protein was administered to mice in the presence of each of the following six adjuvants: Complete/Incomplete Freunds Adjuvant (CFA/IFA), aluminium hydroxide (Alum), Quil A, QS21 saponin, CpG-ODN 1826 and TiterMax. We found that recombinant DII was highly immunogenic in BALB/c mice when administered in the presence of any of the tested adjuvants. Importantly, we show that DII-specific antibodies recognised the native AMA-1 protein expressed on the surface of P. vivax merozoites isolated from the blood of infected patients. These results demonstrate that a recombinant protein containing PvAMA-1 DII is immunogenic when administered in different adjuvant formulations, and indicate that this region of the AMA-1 protein should continue to be evaluated as part of a subunit vaccine against vivax malaria.

Calcium dynamics of Plasmodium berghei sporozoite motility.

Calcium is a key signalling molecule in apicomplexan parasites and plays an important role in diverse processes including gliding motility. Gliding is essential for the malaria parasite to migrate from the skin to the liver as well as to invade host tissues and cells. Here we investigated the dynamics of intracellular Ca2+ in the motility of Plasmodium berghei sporozoites by live imaging and flow cytometry. We found that cytosolic levels of Ca2+ increase when sporozoites are activated in suspension, which is sufficient to induce the secretion of integrin‐like adhesins that are essential for gliding motility. By increasing intracellular Ca2+ levels artificially with an ionophore, these adhesins are secreted onto the sporozoite surface, however, the parasite is not capable of gliding. A second level of Ca2+ modulation was observed during attachment to and detachment from a solid substrate, leading to a further increase or a decrease in the cytoplasmic levels of Ca2+ respectively. We also observed oscillations in the intracellular Ca2+ level during gliding. Finally, an intracellular Ca2+ chelator, an inhibitor of phosphoinositide‐specific phospholipase C (PI‐PLC), and an inhibitor of the inositol triphosphate (IP3) receptor blocked the rise in intracellular Ca2+, adhesin secretion, and motility of activated sporozoites, indicating that intracellular stores supply Ca2+ during sporozoite gliding. Our study indicates that a rise in intracellular Ca2+ is necessary but not sufficient to activate gliding, that Ca2+ levels are modulated in several ways during motility, and that a PI‐PLC/IP3 pathway regulates Ca2+ release during the process of sporozoite locomotion.