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Concerted efforts to fight malaria have caused significant reductions in global malaria cases and mortality. Sustaining this will be critical to avoid rebound and outbreaks of seasonal malaria. Identifying predictive attributes that define clinical malaria will be key to guide development of second-generation tools to fight malaria. Broadly reactive antibodies against variable surface antigens that are expressed on the surface of infected erythrocytes and merozoites stage antigens are targets of naturally acquired immunity and prime candidates for anti-malaria therapeutics and vaccines. However, predicting the relationship between the antigen-specific antibodies and protection from clinical malaria remains unresolved. Here, we used new datasets and multiple approaches combined with re-analysis of our previous data to assess the multi-dimensional and complex relationship between antibody responses and clinical malaria outcomes. We observed 22 antigens (17 PfEMP1 domains, 3 RIFIN family members, merozoite surface protein 3 (PF3D7_1035400), and merozoites-associated armadillo repeats protein (PF3D7_1035900) that were selected across three different clinical malaria definitions (1,000/2,500/5,000 parasites/µl plus fever). In addition, Principal Components Analysis (PCA) indicated that the first three components (Dim1, Dim2 and Dim3 with eigenvalues of 306, 48, and 29, respectively) accounted for 66.1% of the total variations seen. Specifically, the Dim1, Dim2 and Dim3 explained 52.8%, 8.2% and 5% of variability, respectively. We further observed a significant relationship between the first component scores and age with antibodies to PfEMP1 domains being the key contributing variables. This is consistent with a recent proposal suggesting that there is an ordered acquisition of antibodies targeting PfEMP1 proteins. Thus, although limited, and further work on the significance of the selected antigens will be required, these approaches may provide insights for identification of drivers of naturally acquired protective immunity as well as guide development of additional tools for malaria elimination and eradication.

Abstract Serine repeat antigen (SERA) is conserved among species of the genus Plasmodium. Sera genes form a multigene family and are generally tandemly clustered on a single chromosome. Although all Plasmodium species encode multiple sera genes, the number varies between species. Among species, the members share similar sequences and gene organization. SERA possess a central papain-like cysteine protease domain, however, in some members, the active site cysteine residue is substituted with a serine. Recent studies implicate this gene family in a number of aspects in parasite biology and induction of protective immune response. This review summarizes the current understanding on this important gene family in several Plasmodium species. The Plasmodium falciparum (Pf)-sera family, for example, consists of nine gene members. Unlike other multigene families in Plasmodium species, Pf-sera genes do not exhibit antigenic variation. Pf-sera5 nucleotide diversity is also low. Moreover, although Pf-sera5 is highly transcribed during the blood stage of malaria infection, and a large amount is released into the host blood following schizont rupture, in malaria endemic countries the sero-positive rates for Pf-SERA5 are low, likely due to Pf-SERA5 binding of host proteins to avoid immune recognition. As an antigen, the N-terminal 47 kDa domain of Pf-SERA5 is a promising vaccine candidate currently undergoing clinical trials. Pf-SERA5 and Pf-SERA6, as well as P. berghei (Pb)-SERA3, and Pb-SERA5, have been investigated for their roles in parasite egress. Two P. yoelii SERA, which have a serine residue at the protease active center, are implicated in parasite virulence. Overall, these studies provide insight that during the evolution of the Plasmodium parasite, the sera gene family members have increased by gene duplication, and acquired various functions that enable the parasite to survive and successfully maintain infection in the host.

Abstract Background Usage of chloroquine was discontinued from the treatment of Plasmodium falciparum infection in almost all endemic regions because of global spread of resistant parasites. Since the first report in Malawi, numerous epidemiological studies have demonstrated that the discontinuance led to re-emergence of chloroquine-susceptible P. falciparum, suggesting a possible role in future malaria control. However, most studies were cross-sectional, with few studies looking at the persistence of chloroquine recovery in long term. This study fills the gap by providing, for a period of at least 6 years, proof of persistent re-emergence/stable recovery of susceptible parasite populations using both molecular and phenotypic methods. Methods Ex vivo drug-susceptibility assays to chloroquine (n = 319) and lumefantrine (n = 335) were performed from 2013 to 2018 in Gulu, Northern Uganda, where chloroquine had been removed from the official malaria treatment regimen since 2006. Genotyping of pfcrt and pfmdr1 was also performed. Results Chloroquine resistance (≥ 100 nM) was observed in only 3 (1.3%) samples. Average IC50 values for chloroquine were persistently low throughout the study period (17.4–24.9 nM). Parasites harbouring pfcrt K76 alleles showed significantly lower IC50s to chloroquine than the parasites harbouring K76T alleles (21.4 nM vs. 43.1 nM, p-value = 3.9 × 10−8). Prevalence of K76 alleles gradually increased from 71% in 2013 to 100% in 2018. Conclusion This study found evidence of stable persistence of chloroquine susceptibility with the fixation of pfcrt K76 in Northern Uganda after discontinuation of chloroquine in the region. Accumulation of similar evidence in other endemic areas in Uganda could open channels for possible future re-use of chloroquine as an option for malaria treatment or prevention.

Clinical immunity to malaria develops after repeated exposure to Plasmodium falciparum parasites. Broadly reactive antibodies against parasite antigens expressed on the surface of infected erythrocytes (variable surface antigens; VSAs) are candidates for anti-malaria therapeutics and vaccines. Among the VSAs, several RIFIN, STEVOR, and SURFIN family members have been demonstrated to be targets of naturally acquired immunity against malaria. For example, RIFIN family members are important ligands for opsonization of P. falciparum infected erythrocytes with specific immunoglobulins (IgG) acquiring broad protective reactivity. However, the global repertoire of human anti-VSAs IgG, its variation in children, and the key protective targets remain poorly understood. Here, we report wheat germ cell-free system-based production and serological profiling of a comprehensive library of A-RIFINs, B-RIFINs, STEVORs, and SURFINs derived from the P. falciparum 3D7 parasite strain. We observed that >98% of assayed proteins (n = 265) were immunogenic in malaria-exposed individuals in Uganda. The overall breadth of immune responses was significantly correlated with age but not with clinical malaria outcome among the study volunteers. However, children with high levels of antibodies to four RIFINs (PF3D7_0201000, PF3D7_1254500, PF3D7_1040600, PF3D7_1041100), STEVOR (PF3D7_0732000), and SURFIN 1.2 (PF3D7_0113600) had prospectively reduced the risk of developing febrile malaria, suggesting that the 5 antigens are important targets of protective immunity. Further studies on the significance of repeated exposure to malaria infection and maintenance of such high-level antibodies would contribute to a better understanding of susceptibility and naturally acquired immunity to malaria.

Abstract Humans and dogs are the two major hosts of Strongyloides stercoralis, an intestinal parasitic nematode. To better understand the phylogenetic relationships among S. stercoralis isolates infecting humans and dogs and to assess the zoonotic potential of this parasite, we analyzed mitochondrial Cox1, nuclear 18S rDNA, 28S rDNA, and a major sperm protein domain-containing protein genes. Overall, our analyses indicated the presence of two distinct lineages of S. stercoralis (referred to as type A and type B). While type A parasites were isolated both from humans and dogs in different countries, type B parasites were found exclusively in dogs, indicating that the type B has not adapted to infect humans. These epidemiological data, together with the close phylogenetic relationship of S. stercoralis with S. procyonis, a Strongyloides parasite of raccoons, possibly indicates that S. stercoralis originally evolved as a canid parasite, and later spread into humans. The inability to infect humans might be an ancestral character of this species and the type B might be surmised to be an origin population from which human-infecting strains are derived.

Clinical manifestation of malaria is mainly due to intra-erythrocytic development of Plasmodium parasites. Plasmodium falciparum merozoites, the invasive form of the blood-stage parasite, invade human erythrocytes in a complex but rapid process. This multi-step progression involves interactions between parasite and human host proteins. Here we show that antibodies against a vaccine antigen, PfGAMA, co-immunoprecipitate with PfMSP10. This interaction was validated as direct by surface plasmon resonance analysis. We then demonstrate that antibodies against PfMSP10 have growth inhibitory activity against cultured parasites, with the region PfMSP10 R1 that is critical for its interaction with PfGAMA being the key target. We also observe that the PfMSP10 R1 region is highly conserved among African field isolates. Lastly, we show that high levels of antibodies against PfMSP10 R1 associate with reduced risk to clinical malaria in children resident in a malaria endemic region in northern Uganda. Put together, these findings provide for the first time the functional context of the important role of PfGAMA/PfMSP10 interaction in erythrocyte invasion and unveil a novel asexual blood-stage malaria vaccine target for attenuating P. falciparum merozoite invasion.

The malaria vaccine candidate antigen, SE36, is based on the N-terminal 47 kDa domain of Plasmodium falciparum serine repeat antigen 5 (SERA5). In epidemiological studies, we have previously shown the inhibitory effects of SE36 specific antibodies on in vitro parasite growth and the negative correlation between antibody level and malaria symptoms. A phase 1 b trial of the BK-SE36 vaccine in Uganda elicited 72% protective efficacy against symptomatic malaria in children aged 6-20 years during the follow-up period 130-365 days post-second vaccination. Here, we performed epitope mapping with synthetic peptides covering the whole sequence of SE36 to identify and map dominant epitopes in Ugandan adult serum presumed to have clinical immunity to P. falciparum malaria. High titer sera from the Ugandan adults predominantly reacted with peptides corresponding to two successive N-terminal regions of SERA5 containing octamer repeats and serine rich sequences, regions of SERA5 that were previously reported to have limited polymorphism. Affinity purified antibodies specifically recognizing the octamer repeats and serine rich sequences exhibited a high antibody-dependent cellular inhibition (ADCI) activity that inhibited parasite growth. Furthermore, protein structure predictions and structural analysis of SE36 using spectroscopic methods indicated that N-terminal regions possessing inhibitory epitopes are intrinsically unstructured. Collectively, these results suggest that strict tertiary structure of SE36 epitopes is not required to elicit protective antibodies in naturally immune Ugandan adults.

Malaria remains as one of the most devastating infectious disease, and continues to exact an enormous toll in medical cost and days of labor lost especially in the tropics. Effective malaria control and eventual eradication remain a huge challenge, with efficacious antimalarials as important intervention/management tool. Clearly new alternative drugs that are more affordable and with fewer side effects are desirable. After preliminary in vitro assays with plant growth regulators and inhibitors, here, we focus on biosynthetic inhibitors of gibberellin, a plant hormone with many important roles in plant growth, and show their inhibitory effect on the growth of both apicomplexa, Plasmodium falciparum and Toxoplasma gondii. Treatment of P. falciparum cultures with the gibberellin biosynthetic inhibitors resulted in marked morphological changes that can be reversed to a certain degree under hyperosmotic environment. These unique observations suggest that changes in the parasite membrane permeability may explain the pleiotropic effects observed within the intracellular parasites.

SERA gene sequences were newly determined from 11 primate Plasmodium species including two human parasites, P. ovale and P. malariae, and the evolutionary history of SERA genes was analyzed together with 7 known species. All have one each of Group I to III cysteine-type SERA genes and varying number of Group IV serine-type SERA genes in tandem cluster. Notably, Group IV SERA genes were ascertained in all mammalian parasite lineages; and in two primate parasite lineages gene events such as duplication, truncation, fragmentation and gene loss occurred at high frequency in a manner that mimics the birth-and-death evolution model. Transcription profile of individual SERA genes varied greatly among rodent and monkey parasites. Results support the lineage-specific evolution of the Plasmodium SERA gene family. These findings provide further impetus for studies that could clarify/provide proof-of-concept that duplications of SERA genes were associated with the parasites' expansion of host range and the evolutionary conundrums of multigene families in Plasmodium.