Meta-Analysis of Human Antibodies Against Plasmodium falciparum Variable Surface and Merozoite Stage Antigens

Abstract

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.

High-Throughput Antibody Profiling Identifies Targets of Protective Immunity against P. falciparum Malaria in Thailand

“…The protein library used in this study included a total of 691 proteins, which represented various protein families of P. falciparum , such as blood stage proteins (BSP; n = 157), selected from our previous study based on their significant immunoreactivities [ 13 ], cysteine-rich inter-domain regions of P. falciparum erythrocyte membrane protein 1 (PfEMP1) (CIDR; n = 108), Duffy binding-like domains of PfEMP1 (DBL; n = 163), repetitive interspersed family (RIFIN) proteins (n = 178), sub-telomeric variable open reading frame (STEVOR) proteins (n = 53), and surface-associated interspersed gene family (SURFIN) proteins (n = 32). The target genes were cloned into the pEU plasmid vector (CellFree Sciences, Matsuyama, Japan) as previously described [ 10 , 17 , 18 ].…”

“…Mono-biotinylated recombinant proteins were synthesized in vitro with WGCFS using a semi-automated GenDecoder 1000 robotic protein synthesizer (CellFree Sciences). All protein sequences were derived from the P. falciparum 3D7 reference strain [ 13 ].…”

“…Leveraging the availability of a well-annotated 3D7 P. falciparum genome, recent investigations have focused on identifying parasite proteins that are key targets of antibodies-mediated blood-stage immunity. This includes variable surface antigens (VSA) that are localized to the surface of infected red blood cells, such as P. falciparum erythrocyte membrane protein 1 (PfEMP1), repetitive interspersed family (RIFIN) proteins, sub-telomeric variable open reading frame (STEVOR) proteins, and surface-associated interspersed gene family (SURFIN) proteins, as well as other asexual-stage parasite proteins (here referred to as BSPs) [ 13 ]. In one such study, the evaluation of these proteins were conducted using serum samples collected from individuals living in a malaria-endemic region of Uganda, characterized by a high entomological inoculation rate (EIR) of more than 100 infective mosquito bites per year [ 13 ].…”

“…The analysis revealed that over 95% of the proteins examined exhibited immunoreactivity. Of the proteins tested, 22 induced antibody responses significantly associated with a reduced risk of malaria episodes, as determined by three different definitions of clinical malaria based on parasite density (1000/2500/5000 parasites/µL blood) along with the presence of fever [ 13 ]. Importantly, 20 of the 22 selected proteins belonged to VSAs.…”

“…In that study, we observed that antibody responses against MSP3, merozoite surface protein Duffy binding-like (MSPDBL) 1, reticulocyte binding protein 2 homolog b (RH2b), and MSP7 were significantly higher in asymptomatic than in symptomatic individuals [ 16 ]. However, the antigens used for the study did not include VSAs, which could be important seromarkers of reduced malaria episodes, as demonstrated elsewhere [ 13 , 17 ].…”