Effect of CD4+ cell count and HIV status on post-infection immune memory increase of anti-P. falciparum IgG.

<p>To evaluate the effect of CD4⁺ cell count and HIV status on post-infection increase of <i>P</i>. <i>falciparum</i> specific antibody, the CD4⁺ cell counts of PLWHAs (n = 24) and HIV negative participants (n = 91) were first determined. There was no significant difference found in CD4⁺ cell counts of HIV negative and PLWHAs (p: 0.81, Mann Whitney test). Medians of the respective ratios for summative ELISA OD (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124297#pone.0124297.g004" target="_blank">Fig 4</a>) of day 42 and day 0 sera from PLWHAs and HIV negative participants were likewise not significantly different (Mann Whitney test, p: 0.56).</p

Binding sites for MSP2 monoclonal antibodies.

<p>Indication of binding sites for the MSP2 specific monoclonal antibodies. Const = directed against constant regions of MSP2, variable = directed against variable regions.</p

kd values for monoclonal antibodies against MSP2.

<p>Comparison of affinity response of the mAbs against recombinant MSP2-FC27 and MSP2-3D7 proteins. The error bars indicate mean ± SEM.</p

Natural logarithm of k^d variation with age in the Tanzanian population.

<p>Affinity variation with age for samples from Tanzania, shown for binding to AMA1-D10 where the break-point was determined to be 16 years of age. The solid line is the estimated fit from the linear normal model. The red dot with a line at the bottom indicates the 95% confidence interval for the breakpoint.</p

Affinity measured as k^d in 219 individuals.

<p>Pattern of affinity response (measured as k^d values) of exposed sera (n = 219) against recombinant proteins coupled to an SPR-chip. The increasing affinity (equals lower kd values) was observed in the order of MSP2-FC27, MSP2-3D7 and AMA1-D10 and the differences were statistically significant (p<0.0001), both when the individual proteins were compared to each other and compared across all three antigens (ns = p>0.05; * = p<0.05; ** = p<0.01; *** = p<0.001). The box plot values represent the 25^th percentile, median, and the 75^th percentile. The whisker range is between the 5th and 95th percentiles.</p

SPR Sensogram for one representative plasma sample.

<p>A sensogram showing association and dissociation of a representative serum sample to recombinant MSP2-FC27, MSP2-3D7 and AMA1-D10 (Fc2, Fc3 and Fc4, respectively). Fc1 was used as blank to control for non-specific binding.</p

Presence of specific alleles of parasites in the blood associated with different affinities.

<p>Affinity of Tanzanian serum samples depending on which allele of MSP2 parasite was present in the blood at the time of collection. Presence of both alleles of MSP2 is associated with the highest affinity of antibodies in the sera, and that presence of the 3D7 allele was associated with higher affinity compared to the Fc27 allele. The error bars indicate mean ± SEM, and the asterisks indicate the level of significant differences (ns = p>0.05; * = p<0.05; ** = p<0.01; *** = p<0.001).</p

Improved <i>In Vitro</i> Culture of <i>Plasmodium falciparum</i> Permits Establishment of Clinical Isolates with Preserved Multiplication, Invasion and Rosetting Phenotypes

<div><p>To be able to robustly propagate <i>P. falciparum</i> at optimal conditions <i>in vitro</i> is of fundamental importance for genotypic and phenotypic studies of both established and fresh clinical isolates. Cryo-preserved <i>P. falciparum</i> isolates from Ugandan children with severe or uncomplicated malaria were investigated for parasite phenotypes under different <i>in vitro</i> growth conditions or studied directly from the peripheral blood. The parasite cultures showed a minimal loss of parasite-mass and preserved percentage of multiple infected pRBCs to that in peripheral blood, maintained adhesive phenotypes and good outgrowth and multiplication rates when grown in suspension and supplemented with gas. In contrast, abnormal and greatly fluctuating levels of multiple infections were observed during static growth conditions and outgrowth and multiplication rates were inferior. Serum, as compared to Albumax, was found necessary for optimal presentation of PfEMP1 at the pRBC surface and/or for binding of serum proteins (immunoglobulins). Optimal <i>in vitro</i> growth conditions of <i>P. falciparum</i> therefore include orbital shaking (50 rev/min), human serum (10%) and a fixed gas composition (5% O₂, 5% CO₂, 90% N₂). We subsequently established 100% of 76 frozen patient isolates and found rosetting with schizont pRBCs in every isolate (>26% schizont rosetting rate). Rosetting during schizogony was often followed by invasion of the bound RBC as seen by regular and time-lapse microscopy as well as transmission electron microscopy. The peripheral parasitemia, the level of rosetting and the rate of multiplication correlated positively to one another for individual isolates. Rosetting was also more frequent with trophozoite and schizont pRBCs of children with severe versus uncomplicated malaria (p<0.002; p<0.004). The associations suggest that rosetting enhances the ability of the parasite to multiply within the human host.</p></div

ELISA and growth inhibition data for Ngerenya 1998 cohort.

<p>NOTES:</p>*<p>P values calculated using a Chi-squared test or Kruskal-Wallis test.</p>a<p>Median (25th–75th percentiles).</p>b<p>High ELISA responders are those with absorbance≥the 75th percentile.</p>c<p>High inhibitors to 3D7 are those with parasite growth less than the approximate median (i.e. <45% of control).</p>d<p>High inhibitors to W2mef are those with parasite growth less than the approximate median (i.e. <35% of control).</p>e<p>Strong responders are those with high GIA response to both 3D7 and W2mef. Weak responders are those with low GIA response to both 3D7 and W2mef. Intermediate responders are those remaining.</p><p>Abbreviations: ELISA, Enzyme-Linked ImmunoSorbent Assay; MSP1 42, Merozoite Surface Protein1 ; GIA, Growth Inhibition Assay. 3D7 and W2mef represent two different parasite lines.</p