In Plasmodium falciparum–endemic areas, pregnancy-associated malaria (PAM) is an important health problem. The condition is precipitated by accumulation of parasite-infected erythrocytes (IEs) in the placenta, and this process is mediated by parasite-encoded variant surface antigens (VSA) binding to chondroitin sulfate A (CSA). Parasites causing PAM express unique VSA types, VSA_PAM, which can be serologically classified as sex specific and parity dependent. It is sex specific because men from malaria-endemic areas do not develop VSA_PAM antibodies; it is parity dependent because women acquire anti-VSA_PAM immunoglobulin (Ig) G as a function of parity. Previously, it was shown that transcription of var2csa is up-regulated in placental parasites and parasites selected for CSA binding. Here, we show the following: (a) that VAR2CSA is expressed on the surface of CSA-selected IEs; (b) that VAR2CSA is recognized by endemic plasma in a sex-specific and parity-dependent manner; (c) that high anti-VAR2CSA IgG levels can be found in pregnant women from both West and East Africa; and (d) that women with high plasma levels of anti-VAR2CSA IgG give birth to markedly heavier babies and have a much lower risk of delivering low birth weight children than women with low levels.

The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of P. falciparum–infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target. To survive within human red blood cells, malaria parasites must export a catalog of proteins that remodel the host cell and its surface. This enables parasites to acquire nutrients from outside the cell and to modify the cell surface in order to evade host defenses. Protein export involves proteolytic cleavage of the Plasmodium Export Element (PEXEL) by the aspartyl protease Plasmepsin V. We report here the development of a small molecule inhibitor that closely mimics the natural PEXEL substrate and blocks the activity of Plasmepsin V from the malarial parasites Plasmodium falciparum and Plasmodium vivax. The inhibitor impairs export and cellular remodeling and kills P. falciparum at the ring-trophozoite transition, providing direct evidence that Plasmepsin V activity is essential for export of PEXEL proteins and parasite survival within the host. These findings validate Plasmepsin V as a highly conserved antimalarial drug target.

The development of a highly effective vaccine remains a key strategic goal to aid the control and eventual eradication of Plasmodium falciparum malaria. In recent years, the reticulocyte-binding protein homolog 5 (RH5) has emerged as the most promising blood-stage P. falciparum candidate antigen to date, capable of conferring protection against stringent challenge in Aotus monkeys. We report on the first clinical trial to our knowledge to assess the RH5 antigen — a dose-escalation phase Ia study in 24 healthy, malaria-naive adult volunteers. We utilized established viral vectors, the replication-deficient chimpanzee adenovirus serotype 63 (ChAd63), and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA), encoding RH5 from the 3D7 clone of P. falciparum. Vaccines were administered i.m. in a heterologous prime-boost regimen using an 8-week interval and were well tolerated. Vaccine-induced anti-RH5 serum antibodies exhibited cross-strain functional growth inhibition activity (GIA) in vitro, targeted linear and conformational epitopes within RH5, and inhibited key interactions within the RH5 invasion complex. This is the first time to our knowledge that substantial RH5-specific responses have been induced by immunization in humans, with levels greatly exceeding the serum antibody responses observed in African adults following years of natural malaria exposure. These data support the progression of RH5-based vaccines to human efficacy testing.

Summary: Pregnancy‐associated malaria (PAM) is caused by Plasmodium falciparum‐infected erythrocytes (IEs) that bind to chondroitin sulphate A (CSA) in the placenta by PAM‐associated clonally variant surface antigens (VSA). Pregnancy‐specific VSA (VSA_PAM), which include the PfEMP1 variant VAR2CSA, are targets of IgG‐mediated protective immunity to PAM. Here, we report an investigation of the specificity of naturally acquired immunity to PAM, using eight human monoclonal IgG1 antibodies that react exclusively with intact CSA‐adhering IEs expressing VSA_PAM. Four reacted in Western blotting with high‐molecular‐weight (> 200 kDa) proteins, while seven reacted with either the DBL3‐X or the DBL5‐ε domains of VAR2CSA expressed either as Baculovirus constructs or on the surface of transfected Jurkat cells. We used a panel of recombinant antigens representing DBL3‐X domains from P. falciparum field isolates to evaluate B‐cell epitope diversity among parasite isolates, and identified the binding site of one monoclonal antibody using a chimeric DBL3‐X construct. Our findings show that there is a high‐frequency memory response to VSA_PAM, indicating that VAR2CSA is a primary target of naturally acquired PAM‐specific protective immunity, and demonstrate the value of human monoclonal antibodies and conformationally intact recombinant antigens in VSA characterization.

Significance: Malaria is one of the world’s most devastating infectious diseases, affecting hundreds of millions of people and resulting in nearly half a million deaths each year. The parasites that cause malaria must invade the red blood cells of an infected patient, while blocking this process prevents the disease. The PfRH5 protein is an exciting vaccine candidate required for red blood cell invasion by Plasmodium falciparum , the most deadly malaria parasite. Here, we describe our use of a streamlined computational methodology to design variants of PfRH5 that can be produced more simply and cheaply and that show greater thermal stability. This method has broad potential to help the design of vaccines that target many of the world’s most deadly diseases.

Plasmodium falciparum malaria is a major cause of mortality and severe morbidity. Its virulence is related to the parasite's ability to evade host immunity through clonal antigenic variation and tissue-specific adhesion of infected erythrocytes (IEs). The P. falciparum erythrocyte membrane protein 1 (PfEMP1) family is central to both. Here, we present evidence of a P. falciparum evasion mechanism not previously documented: the masking of PfEMP1-specific IgG epitopes by nonspecific IgM. Nonspecific IgM binding to erythrocytes infected by parasites expressing the PfEMP1 protein VAR2CSA (involved in placental malaria pathogenesis and protective immunity) blocked subsequent specific binding of human monoclonal IgG to the Duffy binding-like (DBL) domains DBL3X and DBL5ε of this PfEMP1 variant. Strikingly, a VAR2CSA-specific monoclonal antibody that binds outside these domains and can inhibit IE adhesion to the specific VAR2CSA receptor chondroitin sulfate A was unaffected. Nonspecific IgM binding protected the parasites from FcγR-dependent phagocytosis of VAR2CSA ⁺ IEs, but it did not affect IE adhesion to chondroitin sulfate A or lead to C1q deposition on IEs. Taken together, our results indicate that the VAR2CSA affinity for nonspecific IgM has evolved to allow placenta-sequestering P. falciparum to evade acquired protective immunity without compromising VAR2CSA function or increasing IE susceptibility to complement-mediated lysis. Furthermore, functionally important PfEMP1 epitopes not prone to IgM masking are likely to be particularly important targets of acquired protective immunity to P. falciparum malaria.