Children with hemoglobin C or S trait have low serologic responses to a subset of malaria variant surface antigens

Abstract

Children with hemoglobin AC or AS have decreased susceptibility to clinical malaria. Parasite variant surface antigen (VSA) presentation on the surface of infected erythrocytes is altered in erythrocytes with hemoglobin C (Hb AC) or sickle trait (Hb AS) mutations in vitro. The protective role of incomplete or altered VSA presentation against clinical malaria in individuals with Hb AC or AS is unclear. Using a high-throughput protein microarray, we sought to use serological responses to VSAs as a measure of host exposure to VSAs among Malian children with Hb AC, Hb AS, or wildtype hemoglobin (Hb AA). In uncomplicated malaria, when compared to Hb AA children, Hb AC children had significantly lower serological responses to extracellular Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) domains but did not differ in responses to intracellular PfEMP1 domains and other VSAs, including members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family. Healthy children with Hb AC and Hb AS genotypes recognized fewer extracellular PfEMP1s compared to children with Hb AA, especially CD36-binding PfEMP1s. These reduced serologic responses may reflect reduced VSA presentation or lower parasite exposure in children with Hb AC or AS and provide insights into mechanisms of protection.

Keywords: Hemoglobin C; Hemoglobin S; Malaria; Mali; Plasmodium falciparum; Protein microarray.

Figure 1.. Heat map of seroreactivity to 169 variant surface antigen fragments for Malian children who had uncomplicated malaria or were healthy, separated by hemoglobin status.

One hundred sixty PfEMP1 fragments, three repetitive interspersed family (RIFIN), and six subtelomeric variable open reading frame (STEVOR) fragments derived from the 3D7 reference genome were printed on a protein microarray and probed with sera from Malian children and North American, malaria naïve controls. (A) Antibody reactivity following uncomplicated malaria infections for sera from Malian children with Hb AA (left, n=53), Hb AC (center, n=14), and North American controls (n = 11). (B) Antibody reactivity in sera from smear-negative, healthy Malian children with Hb AA (left, n=56), Hb AC (center, n=5), and Hb AS (right, n=8), and North American malaria naïve controls (n =11). We quantified the fluorescence intensity signifying the magnitude of antibody response of a serum sample to each microarray protein; the color represents the intensity as indicated in the key. Rows represent the signal intensities to each protein fragment, and columns represent the signal intensity for each serum sample. PfEMP1 fragments are grouped by host receptor binding and domain classification. Individual serum samples are ordered by decreasing mean fluorescence intensity within blood type subgroup. “CD36” is a cell surface receptor present in vascular endothelium. Both “non-CD36-binding” and “CD36-binding” fragments refer to extracellular PfEMP1 domains.

Figure 2.. Hb AC sera has reduced serorecognition and seroreactivity to particular PfEMP1s compared to Hb AA sera for children with uncomplicated malaria illness and for healthy, smear-negative children.

(A) In uncomplicated malaria comparisons, sera of children with Hb AA (red) reacted more intensely to a subset of PfEMP1 fragments than sera of children with Hb AC (blue). Error bars reflect standard errors of the mean. Significant differences in seroreactivity for eachprotein fragment between hemoglobin phenotypes were determined by Wilcoxon rank-sum tests . (B) Percentage of cohort serorecognition for 169 variant surface antigen (VSA) fragments among Malian children with Hb AA (blue; N=53) or Hb AC (orange; N=14) with uncomplicated malaria. A subject’s individual serorecognition of a fragment was defined as a fluorescence intensity greater than or equal to two standard deviations above the mean of North American controls. P-values are indicated in black on the secondary y axis (Chi-squared test). (C and D) Serorecognition breadth of malaria proteins are indicated for Malian children who had (C) uncomplicated malaria or were (D) healthy and smear-negative, grouped by hemoglobin status (* indicates P < 0.05; ** indicates P < 0.005; *** indicates P < 0.0005; Chi-squared test). Group serorecognition of a protein fragment was defined as a mean fluorescence intensity of all Malian children within a cohort that was significantly greater than that of the malaria-naïve control group, based on a two sample Kolmogorov-Smirnov test. The y-axis indicates the percentage of protein fragments for each antigen subgroup on the microarray that were serorecognized by a hemoglobin group. Comparisons of breadth of serorecognition between different hemoglobin groups were made with a chi-squared test.