Application of B cell immortalization for the isolation of antibodies and B cell clones from vaccine and infection settings.

The isolation and characterization of neutralizing antibodies from infection and vaccine settings informs future vaccine design, and methodologies that streamline the isolation of antibodies and the generation of B cell clones are of great interest. Retroviral transduction to express Bcl-6 and Bcl-xL and transform primary B cells has been shown to promote long-term B cell survival and antibody secretion in vitro, and can be used to isolate antibodies from memory B cells. However, application of this methodology to B cell subsets from different tissues and B cells from chronically infected individuals has not been well characterized. Here, we characterize Bcl-6/Bcl-xL B cell immortalization across multiple tissue types and B cell subsets in healthy and HIV-1 infected individuals, as well as individuals recovering from malaria. In healthy individuals, naïve and memory B cell subsets from PBMCs and tonsil tissue transformed with similar efficiencies, and displayed similar characteristics with respect to their longevity and immunoglobulin secretion. In HIV-1-viremic individuals or in individuals with recent malaria infections, the exhausted CD27-CD21- memory B cells transformed with lower efficiency, but the transformed B cells expanded and secreted IgG with similar efficiency. Importantly, we show that this methodology can be used to isolate broadly neutralizing antibodies from HIV-infected individuals. Overall, we demonstrate that Bcl-6/Bcl-xL B cell immortalization can be used to isolate antibodies and generate B cell clones from different B cell populations, albeit with varying efficiencies.

Neutralizing and interfering human antibodies define the structural and mechanistic basis for antigenic diversion.

Defining mechanisms of pathogen immune evasion and neutralization are critical to develop potent vaccines and therapies. Merozoite Surface Protein 1 (MSP-1) is a malaria vaccine antigen and antibodies to MSP-1 are associated with protection from disease. However, MSP-1-based vaccines performed poorly in clinical trials in part due to a limited understanding of the protective antibody response to MSP-1 and of immune evasion by antigenic diversion. Antigenic diversion was identified as a mechanism wherein parasite neutralization by a MSP-1-specific rodent antibody was disrupted by MSP-1-specific non-inhibitory blocking/interfering antibodies. Here, we investigated a panel of MSP-1-specific naturally acquired human monoclonal antibodies (hmAbs). Structures of multiple hmAbs with diverse neutralizing potential in complex with MSP-1 revealed the epitope of a potent strain-transcending hmAb. This neutralizing epitope overlaps with the epitopes of high-affinity non-neutralizing hmAbs. Strikingly, the non-neutralizing hmAbs outcompete the neutralizing hmAb enabling parasite survival. These findings demonstrate the structural and mechanistic basis for a generalizable pathogen immune evasion mechanism through neutralizing and interfering human antibodies elicited by antigenic diversion, and provides insights required to develop potent and durable malaria interventions.

Atypical B cells up-regulate costimulatory molecules during malaria and secrete antibodies with T follicular helper cell support.

Several infectious and autoimmune diseases are associated with an expansion of CD21-CD27- atypical B cells (atBCs) that up-regulate inhibitory receptors and exhibit altered B cell receptor (BCR) signaling. The function of atBCs remains unclear, and few studies have investigated the biology of pathogen-specific atBCs during acute infection. Here, we performed longitudinal flow cytometry analyses and RNA sequencing of Plasmodium falciparum (Pf)-specific B cells isolated from study participants before and shortly after febrile malaria, with simultaneous analysis of influenza hemagglutinin (HA)-specific B cells as a comparator. At the healthy baseline before the malaria season, individuals had similar frequencies of Pf- and HA-specific atBCs that did not differ proportionally from atBCs within the total B cell population. BCR sequencing identified clonal relationships between Pf-specific atBCs, activated B cells (actBCs), and classical memory B cells (MBCs) and revealed comparable degrees of somatic hypermutation. At the healthy baseline, Pf-specific atBCs were transcriptionally distinct from Pf-specific actBCs and classical MBCs. In response to acute febrile malaria, Pf-specific atBCs and actBCs up-regulated similar intracellular signaling cascades. Pf-specific atBCs showed activation of pathways involved in differentiation into antibody-secreting cells and up-regulation of molecules that mediate B-T cell interactions, suggesting that atBCs respond to T follicular helper (TFH) cells. In the presence of TFH cells and staphylococcal enterotoxin B, atBCs of malaria-exposed individuals differentiated into CD38+ antibody-secreting cells in vitro, suggesting that atBCs may actively contribute to humoral immunity to infectious pathogens.

Comparative intravital imaging of human and rodent malaria sporozoites reveals the skin is not a species-specific barrier.

Malaria infection starts with the injection of Plasmodium sporozoites into the host's skin. Sporozoites are motile and move in the skin to find and enter blood vessels to be carried to the liver. Here, we present the first characterization of P. falciparum sporozoites in vivo, analyzing their motility in mouse skin and human skin xenografts and comparing their motility to two rodent malaria species. These data suggest that in contrast to the liver and blood stages, the skin is not a species-specific barrier for Plasmodium. Indeed, P. falciparum sporozoites enter blood vessels in mouse skin at similar rates to the rodent malaria parasites. Furthermore, we demonstrate that antibodies targeting sporozoites significantly impact the motility of P. falciparum sporozoites in mouse skin. Though the sporozoite stage is a validated vaccine target, vaccine trials have been hampered by the lack of good animal models for human malaria parasites. Pre-clinical screening of next-generation vaccines would be significantly aided by the in vivo platform we describe here, expediting down-selection of candidates prior to human vaccine trials.

Plasmodium falciparum-specific IgM B cells dominate in children, expand with malaria, and produce functional IgM.

IgG antibodies play a role in malaria immunity, but whether and how IgM protects from malaria and the biology of Plasmodium falciparum (Pf)-specific IgM B cells is unclear. In a Mali cohort spanning infants to adults, we conducted longitudinal analyses of Pf- and influenza-specific B cells. We found that Pf-specific memory B cells (MBCs) are disproportionally IgM+ and only gradually shift to IgG+ with age, in contrast to influenza-specific MBCs that are predominantly IgG+ from infancy to adulthood. B cell receptor analysis showed Pf-specific IgM MBCs are somatically hypermutated at levels comparable to influenza-specific IgG B cells. During acute malaria, Pf-specific IgM B cells expand and upregulate activation/costimulatory markers. Finally, plasma IgM was comparable to IgG in inhibiting Pf growth and enhancing phagocytosis of Pf by monocytes in vitro. Thus, somatically hypermutated Pf-specific IgM MBCs dominate in children, expand and activate during malaria, and produce IgM that inhibits Pf through neutralization and opsonic phagocytosis.

Increased circulation time of Plasmodium falciparum underlies persistent asymptomatic infection in the dry season.

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquito vectors to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the human host or compromising host survival, is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of individuals with febrile malaria in the transmission season, coinciding with longer circulation within each replicative cycle of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication but rather to increased splenic clearance of longer-circulating infected erythrocytes, which likely maintain parasitemias below clinical and immunological radar. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence.

Generation of Transmission-Competent Human Malaria Parasites with Chromosomally-Integrated Fluorescent Reporters.

Malaria parasites have a complex life cycle that includes specialized stages for transmission between their mosquito and human hosts. These stages are an understudied part of the lifecycle yet targeting them is an essential component of the effort to shrink the malaria map. The human parasite Plasmodium falciparum is responsible for the majority of deaths due to malaria. Our goal was to generate transgenic P. falciparum lines that could complete the lifecycle and produce fluorescent transmission stages for more in-depth and high-throughput studies. Using zinc-finger nuclease technology to engineer an integration site, we generated three transgenic P. falciparum lines in which tdtomato or gfp were stably integrated into the genome. Expression was driven by either stage-specific peg4 and csp promoters or the constitutive ef1a promoter. Phenotypic characterization of these lines demonstrates that they complete the life cycle with high infection rates and give rise to fluorescent mosquito stages. The transmission stages are sufficiently bright for intra-vital imaging, flow cytometry and scalable screening of chemical inhibitors and inhibitory antibodies.

Antibody-Mediated Protection against Plasmodium Sporozoites Begins at the Dermal Inoculation Site.

Plasmodium sporozoites are injected into the skin as mosquitoes probe for blood. From here, they migrate through the dermis to find blood vessels which they enter in order to be rapidly carried to the liver, where they invade hepatocytes and develop into the next life cycle stage, the exoerythrocytic stage. Once sporozoites enter the blood circulation, they are found in hepatocytes within minutes. In contrast, sporozoite exit from the inoculation site resembles a slow trickle and occurs over several hours. Thus, sporozoites spend the majority of their extracellular time at the inoculation site, raising the hypothesis that this is when the malarial parasite is most vulnerable to antibody-mediated destruction. Here, we investigate this hypothesis and demonstrate that the neutralizing capacity of circulating antibodies is greater at the inoculation site than in the blood circulation. Furthermore, these antibodies are working, at least in part, by impacting sporozoite motility at the inoculation site. Using actively and passively immunized mice, we found that most parasites are either immobilized at the site of injection or display reduced motility, particularly in their net displacement. We also found that antibodies severely impair the entry of sporozoites into the bloodstream. Overall, our data suggest that antibodies targeting the migratory sporozoite exert a large proportion of their protective effect at the inoculation site.IMPORTANCE Studies in experimental animal models and humans have shown that antibodies against Plasmodium sporozoites abolish parasite infectivity and provide sterile immunity. While it is well documented that these antibodies can be induced after immunization with attenuated parasites or subunit vaccines, the mechanisms by and location in which they neutralize parasites have not been fully elucidated. Here, we report studies indicating that these antibodies display a significant portion of their protective effect in the skin after injection of sporozoites and that one mechanism by which they work is by impairing sporozoite motility, thus diminishing their ability to reach blood vessels. These results suggest that immune protection against malaria begins at the earliest stages of parasite infection and emphasize the need of performing parasite challenge in the skin for the evaluation of protective immunity.

Alpha-v-containing integrins are host receptors for the Plasmodium falciparum sporozoite surface protein, TRAP.

Malaria-causing Plasmodium sporozoites are deposited in the dermis by the bite of an infected mosquito and move by gliding motility to the liver where they invade and develop within host hepatocytes. Although extracellular interactions between Plasmodium sporozoite ligands and host receptors provide important guidance cues for productive infection and are good vaccine targets, these interactions remain largely uncharacterized. Thrombospondin-related anonymous protein (TRAP) is a parasite cell surface ligand that is essential for both gliding motility and invasion because it couples the extracellular binding of host receptors to the parasite cytoplasmic actinomyosin motor; however, the molecular nature of the host TRAP receptors is poorly defined. Here, we use a systematic extracellular protein interaction screening approach to identify the integrin αvß3 as a directly interacting host receptor for Plasmodium falciparum TRAP. Biochemical characterization of the interaction suggests a two-site binding model, requiring contributions from both the von Willebrand factor A domain and the RGD motif of TRAP for integrin binding. We show that TRAP binding to cells is promoted in the presence of integrin-activating proadhesive Mn2+ ions, and that cells genetically targeted so that they lack cell surface expression of the integrin αv-subunit are no longer able to bind TRAP. P. falciparum sporozoites moved with greater speed in the dermis of Itgb3-deficient mice, suggesting that the interaction has a role in sporozoite migration. The identification of the integrin αvß3 as the host receptor for TRAP provides an important demonstration of a sporozoite surface ligand that directly interacts with host receptors.

Discovery of Plasmodium (M)TRAP-Aldolase Interaction Stabilizers Interfering with Sporozoite Motility and Invasion.

As obligate, intracellular parasites, Plasmodium spp. rely on invasion of host cells in order to replicate and continue their life cycle. The parasite needs to traverse the dermis and endothelium of blood vessels, invade hepatocytes and red blood cells, traverse the mosquito midgut, and enter the salivary glands to continue the cycle of infection and transmission. To traverse and invade cells, the parasite employs an actomyosin motor at the core of a larger invasion machinery complex known as the glideosome. The complex is comprised of multiple protein-protein interactions linking the motor to the internal cytoskeletal network of the parasite and to the extracellular adhesins, which directly contact the host tissue or cell surface. One key interaction is between the cytoplasmic tails of the thrombospondin related anonymous protein (TRAP) and aldolase, a bridging protein to the motor. Here, we present results from screening the Medicines for Malaria Venture (MMV) library of 400 compounds against this key protein-protein interaction. Using a surface plasmon resonance screen, we have identified several compounds that modulate the dynamics of the interaction between TRAP and aldolase. These compounds have been validated in vitro by studying their effects on sporozoite gliding motility and hepatocyte invasion. One of the MMV compounds identified reduced invasion levels by 89% at the lowest concentration tested (16 µM) and severely inhibited gliding at even lower concentrations (5 µM). By targeting protein-protein interactions, we investigated an under-explored area of parasite biology and general drug development, to identify potential antimalarial lead compounds.

Deletion of the rodent malaria ortholog for falcipain-1 highlights differences between hepatic and blood stage merozoites.

Proteases have been implicated in a variety of developmental processes during the malaria parasite lifecycle. In particular, invasion and egress of the parasite from the infected hepatocyte and erythrocyte, critically depend on protease activity. Although falcipain-1 was the first cysteine protease to be characterized in P. falciparum, its role in the lifecycle of the parasite has been the subject of some controversy. While an inhibitor of falcipain-1 blocked erythrocyte invasion by merozoites, two independent studies showed that falcipain-1 disruption did not affect growth of blood stage parasites. To shed light on the role of this protease over the entire Plasmodium lifecycle, we disrupted berghepain-1, its ortholog in the rodent parasite P. berghei. We found that this mutant parasite displays a pronounced delay in blood stage infection after inoculation of sporozoites. Experiments designed to pinpoint the defect of berghepain-1 knockout parasites found that it was not due to alterations in gliding motility, hepatocyte invasion or liver stage development and that injection of berghepain-1 knockout merosomes replicated the phenotype of delayed blood stage growth after sporozoite inoculation. We identified an additional role for berghepain-1 in preparing blood stage merozoites for infection of erythrocytes and observed that berghepain-1 knockout parasites exhibit a reticulocyte restriction, suggesting that berghepain-1 activity broadens the erythrocyte repertoire of the parasite. The lack of berghepain-1 expression resulted in a greater reduction in erythrocyte infectivity in hepatocyte-derived merozoites than it did in erythrocyte-derived merozoites. These observations indicate a role for berghepain-1 in processing ligands important for merozoite infectivity and provide evidence supporting the notion that hepatic and erythrocytic merozoites, though structurally similar, are not identical.

Interrogating the Plasmodium Sporozoite Surface: Identification of Surface-Exposed Proteins and Demonstration of Glycosylation on CSP and TRAP by Mass Spectrometry-Based Proteomics.

Malaria parasite infection is initiated by the mosquito-transmitted sporozoite stage, a highly motile invasive cell that targets hepatocytes in the liver for infection. A promising approach to developing a malaria vaccine is the use of proteins located on the sporozoite surface as antigens to elicit humoral immune responses that prevent the establishment of infection. Very little of the P. falciparum genome has been considered as potential vaccine targets, and candidate vaccines have been almost exclusively based on single antigens, generating the need for novel target identification. The most advanced malaria vaccine to date, RTS,S, a subunit vaccine consisting of a portion of the major surface protein circumsporozoite protein (CSP), conferred limited protection in Phase III trials, falling short of community-established vaccine efficacy goals. In striking contrast to the limited protection seen in current vaccine trials, sterilizing immunity can be achieved by immunization with radiation-attenuated sporozoites, suggesting that more potent protection may be achievable with a multivalent protein vaccine. Here, we provide the most comprehensive analysis to date of proteins located on the surface of or secreted by Plasmodium falciparum salivary gland sporozoites. We used chemical labeling to isolate surface-exposed proteins on sporozoites and identified these proteins by mass spectrometry. We validated several of these targets and also provide evidence that components of the inner membrane complex are in fact surface-exposed and accessible to antibodies in live sporozoites. Finally, our mass spectrometry data provide the first direct evidence that the Plasmodium surface proteins CSP and TRAP are glycosylated in sporozoites, a finding that could impact the selection of vaccine antigens.

Palmitoyl transferases have critical roles in the development of mosquito and liver stages of Plasmodium.

As the Plasmodium parasite transitions between mammalian and mosquito host, it has to adjust quickly to new environments. Palmitoylation, a reversible and dynamic lipid post-translational modification, plays a central role in regulating this process and has been implicated with functions for parasite morphology, motility and host cell invasion. While proteins associated with the gliding motility machinery have been described to be palmitoylated, no palmitoyl transferase responsible for regulating gliding motility has previously been identified. Here, we characterize two palmityol transferases with gene tagging and gene deletion approaches. We identify DHHC3, a palmitoyl transferase, as a mediator of ookinete development, with a crucial role for gliding motility in ookinetes and sporozoites, and we co-localize the protein with a marker for the inner membrane complex in the ookinete stage. Ookinetes and sporozoites lacking DHHC3 are impaired in gliding motility and exhibit a strong phenotype in vivo; with ookinetes being significantly less infectious to their mosquito host and sporozoites being non-infectious to mice. Importantly, genetic complementation of the DHHC3-ko parasite completely restored virulence. We generated parasites lacking both DHHC3, as well as the palmitoyl transferase DHHC9, and found an enhanced phenotype for these double knockout parasites, allowing insights into the functional overlap and compensational nature of the large family of PbDHHCs. These findings contribute to our understanding of the organization and mechanism of the gliding motility machinery, which as is becoming increasingly clear, is mediated by palmitoylation.

Longitudinal analysis of Plasmodium sporozoite motility in the dermis reveals component of blood vessel recognition.

Malaria infection starts with injection of Plasmodium sporozoites by an Anopheles mosquito into the skin of the mammalian host. How sporozoites locate and enter a blood vessel is a critical, but poorly understood process. In this study, we examine sporozoite motility and their interaction with dermal blood vessels, using intravital microscopy in mice. Our data suggest that sporozoites exhibit two types of motility: in regions far from blood vessels, they exhibit 'avascular motility', defined by high speed and less confinement, while in the vicinity of blood vessels their motility is more constrained. We find that curvature of sporozoite tracks engaging with vasculature optimizes contact with dermal capillaries. Imaging of sporozoites with mutations in key adhesive proteins highlight the importance of the sporozoite's gliding speed and its ability to modulate adhesive properties for successful exit from the inoculation site.

Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion.

Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary. Here we address this issue for the essential cGMP-dependent protein kinase, PfPKG. By employing chemical and genetic tools in combination with quantitative global phosphoproteomics, we identify the phosphorylation sites on 69 proteins that are direct or indirect cellular targets for PfPKG. These PfPKG targets include proteins involved in cell signalling, proteolysis, gene regulation, protein export and ion and protein transport, indicating that cGMP/PfPKG acts as a signalling hub that plays a central role in a number of core parasite processes. We also show that PfPKG activity is required for parasite invasion. This correlates with the finding that the calcium-dependent protein kinase, PfCDPK1, is phosphorylated by PfPKG, as are components of the actomyosin complex, providing mechanistic insight into the essential role of PfPKG in parasite egress and invasion.

The innate and adaptive response to mosquito saliva and Plasmodium sporozoites in the skin.

A malaria infection begins when an infected mosquito takes a blood meal and inoculates parasites into the skin of its mammalian host. The parasite then has to exit the skin and escape the immune cells that protect the body from infection and alert the system to intruding pathogens. It has become apparent that this earliest stage of infection is amenable to vaccine interventions. Here, we discuss how the innate and adaptive host response to both mosquito saliva and the parasite may interfere with the infection, as well as possible mechanisms the parasite might use to circumvent the host defense.

The repeat region of the circumsporozoite protein is critical for sporozoite formation and maturation in Plasmodium.

The circumsporozoite protein (CSP) is the major surface protein of the sporozoite stage of malaria parasites and has multiple functions as the parasite develops and then migrates from the mosquito midgut to the mammalian liver. The overall structure of CSP is conserved among Plasmodium species, consisting of a species-specific central tandem repeat region flanked by two conserved domains: the NH2-terminus and the thrombospondin repeat (TSR) at the COOH-terminus. Although the central repeat region is an immunodominant B-cell epitope and the basis of the only candidate malaria vaccine in Phase III clinical trials, little is known about its functional role(s). We used the rodent malaria model Plasmodium berghei to investigate the role of the CSP tandem repeat region during sporozoite development. Here we describe two mutant parasite lines, one lacking the tandem repeat region (ΔRep) and the other lacking the NH2-terminus as well as the repeat region (ΔNΔRep). We show that in both mutant lines oocyst formation is unaffected but sporozoite development is defective.

Targeting Plasmodium falciparum protein kinases with adenosine analogue-oligoarginine conjugates.

During the last decade, a vast number of inhibitors, ligands and fluorescent probes have evolved for mammalian protein kinases; however, the suitability of these compounds for studies of evolutionarily divergent eukaryotes has mostly been left beyond the scope of research. Here, we examined whether adenosine analogue-oligoarginine conjugates that had been extensively characterized as efficient inhibitors of the human protein kinases are applicable for targeting Plasmodium protein kinases. We demonstrated that ARCs were not only able to bind to and inhibit a representative member of Plasmodium falciparum kinome (cGMP-dependent protein kinase) in biochemical assay, but also affected the general phosphorylation levels in parasites released from the infected red blood cells upon saponin treatment. These findings urge advantaging of already existing biochemical tools, whose initially generic, but intrinsically "tunable" selectivity profiles could be used for dissection of signaling pathways outside the initially defined group of biological targets.

Spatiotemporal and functional characterisation of the Plasmodium falciparum cGMP-dependent protein kinase.

Signalling by 3'-5'-cyclic guanosine monophosphate (cGMP) exists in virtually all eukaryotes. In the apicomplexan parasite Plasmodium, the cGMP-dependent protein kinase (PKG) has previously been reported to play a critical role in four key stages of the life cycle. The Plasmodium falciparum isoform (PfPKG) is essential for the initiation of gametogenesis and for blood stage schizont rupture and work on the orthologue from the rodent malaria parasite P. berghei (PbPKG) has shown additional roles in ookinete differentiation and motility as well as liver stage schizont development. In the present study, PfPKG expression and subcellular location in asexual blood stages was investigated using transgenic epitope-tagged PfPKG-expressing P. falciparum parasites. In Western blotting experiments and immunofluorescence analysis (IFA), maximal PfPKG expression was detected at the late schizont stage. While IFA suggested a cytosolic location, a degree of overlap with markers of the endoplasmic reticulum (ER) was found and subcellular fractionation showed some association with the peripheral membrane fraction. This broad localisation is consistent with the notion that PfPKG, as with the mammalian orthologue, has numerous cellular substrates. This idea is further supported by the global protein phosphorylation pattern of schizonts which was substantially changed following PfPKG inhibition, suggesting a complex role for PfPKG during schizogony.