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As the intensity of malaria transmission has declined, Plasmodium falciparum parasite populations have displayed decreased clonal diversity resulting from the emergence of many parasites with common genetic signatures (CGS). We have monitored such CGS parasite clusters from 2006 to 2013 in Thiès, Senegal, using the molecular barcode. The first, and one of the largest observed clusters of CGS parasites, was present in 24% of clinical isolates in 2008, declined to 3.4% of clinical isolates in 2009, and then disappeared. To begin to explore the relationship between the immune responses of the population and the emergence and decline of specific parasite genotypes, we have determined whether antibodies to CGS parasites correlate with their prevalence. We measured (i) antibodies capable of inhibiting parasite growth in culture and (ii) antibodies recognizing the surfaces of infected erythrocytes (RBCs). IgG obtained from volunteers in 2009 showed increased reactivity to the surfaces of CGS-parasitized erythrocytes over IgG from 2008. Since P. falciparum EMP-1 (PfEMP-1) is a major variant surface antigen, we used var Ups quantitative reverse transcription-PCR (qRT-PCR) and sequencing with degenerate DBL1α domain primers to characterize the var genes expressed by CGS parasites after short-term in vitro culture. CGS parasites show upregulation of UpsA var genes and 2-cysteine-containing PfEMP-1 molecules and express the same dominant var transcript. Our work indicates that the CGS parasites in this cluster express similar var genes, more than would be expected by chance in the population, and that there is year-to-year variation in immune recognition of surface antigens on CGS parasite-infected erythrocytes. This study lays the groundwork for detailed investigations of the mechanisms driving the expansion or contraction of specific parasite clones in the population.

Plasmodium falciparum, the parasite that causes the deadliest form of malaria, has evolved multiple proteins known as invasion ligands that bind to specific erythrocyte receptors to facilitate invasion of human erythrocytes. The EBA-175/glycophorin A (GPA) and Rh5/basigin ligand-receptor interactions, referred to as invasion pathways, have been the subject of intense study. In this study, we focused on the less-characterized sialic acid-containing receptors glycophorin B (GPB) and glycophorin C (GPC). Through bioinformatic analysis, we identified extensive variation in glycophorin B (GYPB) transcript levels in individuals from Benin, suggesting selection from malaria pressure. To elucidate the importance of the GPB and GPC receptors relative to the well-described EBA-175/GPA invasion pathway, we used an ex vivo erythrocyte culture system to decrease expression of GPA, GPB, or GPC via lentiviral short hairpin RNA transduction of erythroid progenitor cells, with global surface proteomic profiling. We assessed the efficiency of parasite invasion into knockdown cells using a panel of wild-type P. falciparum laboratory strains and invasion ligand knockout lines, as well as P. falciparum Senegalese clinical isolates and a short-term-culture-adapted strain. For this, we optimized an invasion assay suitable for use with small numbers of erythrocytes. We found that all laboratory strains and the majority of field strains tested were dependent on GPB expression level for invasion. The collective data suggest that the GPA and GPB receptors are of greater importance than the GPC receptor, supporting a hierarchy of erythrocyte receptor usage in P. falciparum.

This study shows markedly different isotype distributions of antibodies to asexual blood stages of Plasmodium falciparum and to merozoite surface protein 1 in clinically immune Senegalese adults depending on the study site. The relationships between immunoglobulin M (IgM) and IgG and between IgG3 and IgG1 antibodies differed in settings where transmission is perennial compared to settings where it is seasonal. This suggests a role for antibody class and/or subclass production and utilization in the regulation of protective immunity to such antigens.

Forty-one African patients suffering from clinically defined severe malaria were studied in the intensive medical care unit of the main hospital in Dakar, Senegal, West Africa. All of these individuals lived in Greater Dakar, an area of low and seasonal Plasmodium falciparum endemicity. Twenty-seven patients (mean age +/- 1 standard deviation, 19.2 +/- 12.7 years) survived this life-threatening episode, but 14 (30.8 +/- 16.2 years old) died despite initiation of adequate treatment. On the day of admission (day 0) and 3 days later, one to two blood samples (i.e., approximately 10 to 15 ml) were obtained from each subject, and different biological parameters were evaluated in the two groups. Plasma samples were tested for their content in tumor necrosis factor alpha (TNF-alpha), soluble receptors I and II for TNF-alpha (TNF-alpha sRI and TNF-alpha sRII), interleukin-6 (IL-6), IL-6 sR, IL-10, and IL-2 sR. The concentrations of all these cytokines and/or their receptors was significantly elevated in patient plasma samples on day 0, and it rapidly decreased in the group of individuals who survived. By comparison, the mean concentration of the same parameters decreased slowly in the group of patients who died (except for IL-10, which dramatically fell in all patient plasma samples soon after initiation of antimalarial treatment). The TNF-alpha sRI level remained significantly elevated among the patients who died, and the highest levels of soluble TNF-alpha sRI receptor were found among the older patients. Parasite-specific immunoglobulin M (IgM), total IgG, IgG1, IgG2, IgG3, and IgG4 were evaluated by enzyme-linked immunosorbent assay using a crude extract of a local P. falciparum isolate as antigen and human class- and subclass-specific monoclonal antibodies. Parasite-specific IgM, total IgG, and IgG1 were detectable in the plasma samples of most of these African patients, whereas IgG2 and IgG4 mean values were low. The mean level of parasite-specific IgG3 was different (P = 0.024) at day 0, i.e., before initiation of intensive medical care, between the group of the 27 surviving subjects and the group of 14 patients dying of severe malaria. As a consequence, most of the African patients who died had only trace amounts or almost no detectable level of parasite-specific IgG3 at the time of admission. In contrast, the presence of even limited IgG3 activity at day 0 was found to be associated with a significantly increased probability of recovering from severe malaria. Therefore, in our study, both an elevated level of TNF-alpha sRI and absence of IgG3 activity were of bleak prognostic significance, whereas a favorable outcome was usually observed when parasite-specific IgG3 activity was detectable. This finding was strongly suggestive of a prime role for these parasite-specific immunoglobulins in the capacity to help recovery from severe malaria.