Towards a comprehensive Plasmodium falciparum merozoite cell surface and secreted recombinant protein library.

BACKGROUND: Plasmodium falciparum is the aetiological agent for malaria, a deadly infectious disease for which no vaccine has yet been licensed. The proteins displayed on the merozoite cell surface have long been considered attractive vaccine targets because of their direct exposure to host antibodies; however, progress in understanding the functional role of these targets has been hindered by technical challenges associated with expressing these proteins in a functionally active recombinant form. To address this, a method that enables the systematic expression of functional extracellular Plasmodium proteins was previously developed, and used to create a library of 42 merozoite proteins. METHODS: To compile a more comprehensive library of recombinant proteins representing the repertoire of P. falciparum merozoite extracellular proteins for systematic vaccine and functional studies, genome-wide expression profiling was used to identify additional candidates. Candidate proteins were recombinantly produced and their integrity and expression levels were tested by Western blotting and ELISA. RESULTS: Twenty-five additional genes that were upregulated during late schizogony, and predicted to encode secreted and cell surface proteins, were identified and expressed as soluble recombinant proteins. A band consistent with the entire ectodomain was observed by immunoblotting for the majority of the proteins and their expression levels were quantified. By using sera from malaria-exposed immune adults, the immunoreactivity of 20 recombinant proteins was assessed, and most of the merozoite ligands were found to carry heat-labile epitopes. To facilitate systematic comparative studies across the entire library, multiple Plasmodium proteins were simultaneously purified using a custom-made platform. CONCLUSIONS: A library of recombinant P. falciparum secreted and cell surface proteins was expanded by 20 additional proteins, which were shown to express at usable levels and contain conformational epitopes. This resource of extracellular P. falciparum merozoite proteins, which now contains 62 full-length ectodomains, will be a valuable tool in elucidating the function of these proteins during the blood stages of infection, and facilitate the comparative assessment of blood stage vaccine candidates.

Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1.

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expressing four transcription factors: Oct4, Sox2, Klf4, and c-Myc. Here we report that enhancing RA signaling by expressing RA receptors (RARs) or by RA agonists profoundly promoted reprogramming, but inhibiting it using a RAR-α dominant-negative form completely blocked it. Coexpressing Rarg (RAR-γ) and Lrh-1 (liver receptor homologue 1; Nr5a2) with the four factors greatly accelerated reprogramming so that reprogramming of mouse embryonic fibroblast cells to ground-state iPSCs requires only 4 d induction of these six factors. The six-factor combination readily reprogrammed primary human neonatal and adult fibroblast cells to exogenous factor-independent iPSCs, which resembled ground-state mouse ES cells in growth properties, gene expression, and signaling dependency. Our findings demonstrate that signaling through RARs has critical roles in molecular reprogramming and that the synergistic interaction between Rarg and Lrh1 directs reprogramming toward ground-state pluripotency. The human iPSCs described here should facilitate functional analysis of the human genome.

APEX2-based proximity proteomic analysis identifies candidate interactors for Plasmodium falciparum knob-associated histidine-rich protein in infected erythrocytes.

The interaction of Plasmodium falciparum-infected red blood cells (iRBCs) with the vascular endothelium plays a crucial role in malaria pathology and disease. KAHRP is an exported P. falciparum protein involved in iRBC remodelling, which is essential for the formation of protrusions or "knobs" on the iRBC surface. These knobs and the proteins that are concentrated within them allow the parasites to escape the immune response and host spleen clearance by mediating cytoadherence of the iRBC to the endothelial wall, but this also slows down blood circulation, leading in some cases to severe cerebral and placental complications. In this work, we have applied genetic and biochemical tools to identify proteins that interact with P. falciparum KAHRP using enhanced ascorbate peroxidase 2 (APEX2) proximity-dependent biotinylation and label-free shotgun proteomics. A total of 30 potential KAHRP-interacting candidates were identified, based on the assigned fragmented biotinylated ions. Several identified proteins have been previously reported to be part of the Maurer's clefts and knobs, where KAHRP resides. This study may contribute to a broader understanding of P. falciparum protein trafficking and knob architecture and shows for the first time the feasibility of using APEX2-proximity labelling in iRBCs.

Barcoding Genetically Distinct Plasmodium falciparum Strains for Comparative Assessment of Fitness and Antimalarial Drug Resistance.

The repeated emergence of antimalarial drug resistance in Plasmodium falciparum, including to the current frontline antimalarial artemisinin, is a perennial problem for malaria control. Next-generation sequencing has greatly accelerated the identification of polymorphisms in resistance-associated genes but has also highlighted the need for more sensitive and accurate laboratory tools to profile current and future antimalarials and to quantify the impact of drug resistance acquisition on parasite fitness. The interplay of fitness and drug response is of fundamental importance in understanding why particular genetic backgrounds are better at driving the evolution of drug resistance in natural populations, but the impact of parasite fitness landscapes on the epidemiology of drug resistance has typically been laborious to accurately quantify in the lab, with assays being limited in accuracy and throughput. Here we present a scalable method to profile fitness and drug response of genetically distinct P. falciparum strains with well-described sensitivities to several antimalarials. We leverage CRISPR/Cas9 genome-editing and barcode sequencing to track unique barcodes integrated into a nonessential gene (pfrh3). We validate this approach in multiplex competitive growth assays of three strains with distinct geographical origins. Furthermore, we demonstrate that this method can be a powerful approach for tracking artemisinin response as it can identify an artemisinin resistant strain within a mix of multiple parasite lines, suggesting an approach for scaling the laborious ring-stage survival assay across libraries of barcoded parasite lines. Overall, we present a novel high-throughput method for multiplexed competitive growth assays to evaluate parasite fitness and drug response. IMPORTANCE The complex interplay between antimalarial resistance and parasite fitness has important implications for understanding the development and spread of drug resistance alleles and the impact of genetic background on transmission. One limitation with current methodologies to measure parasite fitness is the ability to scale this beyond simple head-to-head competition experiments between a wildtype control line and test line, with a need for a scalable approach that allows tracking of parasite growth in complex mixtures. In our study, we have used CRISPR editing to insert unique DNA barcodes into a safe-harbor genomic locus to tag multiple parasite strains and use next-generation sequencing to read out strain dynamics. We observe inherent fitness differences between the strains, as well as sensitive modulation of responses to challenge with clinically relevant antimalarials, including artemisinin.

Immunomodulation of T- and NK-cell Responses by a Bispecific Antibody Targeting CD28 Homolog and PD-L1.

Checkpoint blockade therapies targeting PD-1/PD-L1 and CTLA-4 are clinically successful but also evoke adverse events due to systemic T-cell activation. We engineered a bispecific, mAb targeting CD28 homolog (CD28H), a newly identified B7 family receptor that is constitutively expressed on T and natural killer (NK) cells, with a PD-L1 antibody to potentiate tumor-specific immune responses. The bispecific antibody led to T-cell costimulation, induced NK-cell cytotoxicity of PD-L1-expressing tumor cells, and activated tissue-resident memory CD8+ T cells. Mechanistically, the CD28H agonistic arm of the bispecific antibody reduced PD-L1/PD-1-induced SHP2 phosphorylation while simultaneously augmenting T-cell receptor signaling by activating the MAPK and AKT pathways. This bispecific approach could be used to target multiple immune cells, including CD8+ T cells, tissue-resident memory T cells, and NK cells, in a tumor-specific manner that may lead to induction of durable, therapeutic antitumor responses.

Adaptation of Plasmodium falciparum to humans involved the loss of an ape-specific erythrocyte invasion ligand.

Plasmodium species are frequently host-specific, but little is currently known about the molecular factors restricting host switching. This is particularly relevant for P. falciparum, the only known human-infective species of the Laverania sub-genus, all other members of which infect African apes. Here we show that all tested P. falciparum isolates contain an inactivating mutation in an erythrocyte invasion associated gene, PfEBA165, the homologues of which are intact in all ape-infective Laverania species. Recombinant EBA165 proteins only bind ape, not human, erythrocytes, and this specificity is due to differences in erythrocyte surface sialic acids. Correction of PfEBA165 inactivating mutations by genome editing yields viable parasites, but is associated with down regulation of both PfEBA165 and an adjacent invasion ligand, which suggests that PfEBA165 expression is incompatible with parasite growth in human erythrocytes. Pseudogenization of PfEBA165 may represent a key step in the emergence and evolution of P. falciparum.

A defined mechanistic correlate of protection against Plasmodium falciparum malaria in non-human primates.

Malaria vaccine design and prioritization has been hindered by the lack of a mechanistic correlate of protection. We previously demonstrated a strong association between protection and merozoite-neutralizing antibody responses following vaccination of non-human primates against Plasmodium falciparum reticulocyte binding protein homolog 5 (PfRH5). Here, we test the mechanism of protection. Using mutant human IgG1 Fc regions engineered not to engage complement or FcR-dependent effector mechanisms, we produce merozoite-neutralizing and non-neutralizing anti-PfRH5 chimeric monoclonal antibodies (mAbs) and perform a passive transfer-P. falciparum challenge study in Aotus nancymaae monkeys. At the highest dose tested, 6/6 animals given the neutralizing PfRH5-binding mAb c2AC7 survive the challenge without treatment, compared to 0/6 animals given non-neutralizing PfRH5-binding mAb c4BA7 and 0/6 animals given an isotype control mAb. Our results address the controversy regarding whether merozoite-neutralizing antibody can cause protection against P. falciparum blood-stage infections, and highlight the quantitative challenge of achieving such protection.

Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites.

In malaria parasites, evolution of parasitism has been linked to functional optimisation. Despite this optimisation, most members of a calcium-dependent protein kinase (CDPK) family show genetic redundancy during erythrocytic proliferation. To identify relationships between phospho-signalling pathways, we here screen 294 genetic interactions among protein kinases in Plasmodium berghei. This reveals a synthetic negative interaction between a hypomorphic allele of the protein kinase G (PKG) and CDPK4 to control erythrocyte invasion which is conserved in P. falciparum. CDPK4 becomes critical when PKG-dependent calcium signals are attenuated to phosphorylate proteins important for the stability of the inner membrane complex, which serves as an anchor for the acto-myosin motor required for motility and invasion. Finally, we show that multiple kinases functionally complement CDPK4 during erythrocytic proliferation and transmission to the mosquito. This study reveals how CDPKs are wired within a stage-transcending signalling network to control motility and host cell invasion in malaria parasites.

Malaria Vaccine Development: Focusing Field Erythrocyte Invasion Studies on Phenotypic Diversity: The West African Merozoite Invasion Network (WAMIN).

Erythrocyte invasion by Plasmodium falciparum merozoites is an essential step for parasite survival and proliferation. Invasion is mediated by multiple ligands, which could be promising vaccine targets. The usage and sequence of these ligands differs between parasites, yet most studies of them have been carried out in only a few laboratory-adapted lines. To understand the true extent of natural variation in invasion phenotypes and prioritize vaccine candidates on a relevant evidence base, we need to develop and apply standardized assays to large numbers of field isolates. The West African Merozoite Invasion Network (WAMIN) has been formed to meet these goals, expand training in Plasmodium phenotyping, and perform large-scale field phenotyping studies in order to prioritize blood stage vaccine candidates.

Basigin is a druggable target for host-oriented antimalarial interventions.

Plasmodium falciparum is the parasite responsible for the most lethal form of malaria, an infectious disease that causes a large proportion of childhood deaths and poses a significant barrier to socioeconomic development in many countries. Although antimalarial drugs exist, the repeated emergence and spread of drug-resistant parasites limit their useful lifespan. An alternative strategy that could limit the evolution of drug-resistant parasites is to target host factors that are essential and universally required for parasite growth. Host-targeted therapeutics have been successfully applied in other infectious diseases but have never been attempted for malaria. Here, we report the development of a recombinant chimeric antibody (Ab-1) against basigin, an erythrocyte receptor necessary for parasite invasion as a putative antimalarial therapeutic. Ab-1 inhibited the PfRH5-basigin interaction and potently blocked erythrocyte invasion by all parasite strains tested. Importantly, Ab-1 rapidly cleared an established P. falciparum blood-stage infection with no overt toxicity in an in vivo infection model. Collectively, our data demonstrate that antibodies or other therapeutics targeting host basigin could be an effective treatment for patients infected with multi-drug resistant P. falciparum.