Children with cerebral malaria or severe malarial anaemia lack immunity to distinct variant surface antigen subsets

Abstract

Variant surface antigens (VSAs) play a critical role in severe malaria pathogenesis. Defining gaps, or "lacunae", in immunity to these Plasmodium falciparum antigens in children with severe malaria would improve our understanding of vulnerability to severe malaria and how protective immunity develops. Using a protein microarray with 179 antigen variants from three VSA families as well as more than 300 variants of three other blood stage P. falciparum antigens, reactivity was measured in sera from Malian children with cerebral malaria or severe malarial anaemia and age-matched controls. Sera from children with severe malaria recognized fewer extracellular PfEMP1 fragments and were less reactive to specific fragments compared to controls. Following recovery from severe malaria, convalescent sera had increased reactivity to certain non-CD36 binding PfEMP1s, but not other malaria antigens. Sera from children with severe malarial anaemia reacted to fewer VSAs than did sera from children with cerebral malaria, and both of these groups had lacunae in their seroreactivity profiles in common with children who had both cerebral malaria and severe malarial anaemia. This microarray-based approach may identify a subset of VSAs that could inform the development of a vaccine to prevent severe disease or a diagnostic test to predict at-risk children.

Figure 1

Breadth of recognition of P. falciparum antigens by study group. Percentage of recognized P. falciparum antigens for sera from each severe malaria group compared to their corresponding control groups. *Indicates a significant difference in percentage of recognized fragments between sera from a particular control group and the corresponding severe malaria group (P < 0.05, McNemar’s test). No statistical comparisons were made for RIFINs and STEVORs, as each of these VSA groups included fewer than 10 fragments.

Figure 2

Differentially seroreactive P. falciparum antigens for sera from uncomplicated malaria controls versus severe malaria subjects. Sera from uncomplicated malaria controls reacted more intensely to 107, 19, and 57 fragments versus sera from subjects with cerebral malaria, severe malarial anaemia, and both cerebral malaria and severe malarial anaemia, respectively.

Figure 3

Differentially seroreactive P. falciparum antigens for sera from healthy controls versus severe malaria subjects. Sera from healthy controls reacted more intensely to 26, two, and 11 fragments versus sera from subjects with cerebral malaria, severe malarial anaemia, and both cerebral malaria and severe malarial anaemia, respectively. Non-CD36-binding PfEMP1 fragments are designated with an “S”. *Indicates P < 0.05; **P < 0.01, and ***P < 0.001 (Wilcoxon’s signed-rank test).

Figure 4

Differentially seroreactive P. falciparum antigens in convalescent versus acute sera comparisons for severe malaria subjects. Convalescent sera reacted more intensely to four and eight fragments versus acute sera of subjects with cerebral malaria and severe malarial anaemia, respectively. Non-CD36-binding PfEMP1 fragments are designated with an “S”. *Indicates P < 0.05; **P < 0.01, and ***P < 0.001 (Wilcoxon’s signed-rank test).

Figure 5

Heat map of lacunae shared across different acute cerebral malaria sera comparisons. P. falciparum antigen lacunae were grouped by recurrence across different comparisons with acute sera from cerebral malaria subjects. Each column displays the seroreactivity profile of one serum sample, and each row displays the seroreactivity profile of an individual protein fragment. Grey color indicates no seroreactivity, black is low-to-moderate seroreactivity, and red denotes high seroreactivity to probed fragments. Lacunae present across more than one comparison are designated by a triangle.

Figure 6

Heat map of lacunae shared across different acute severe malarial anaemia sera comparisons. P. falciparum antigen lacunae were grouped by recurrence across different comparisons with acute sera from severe malarial anaemia subjects. Each column displays the seroreactivity profile of one serum sample, and each row displays the seroreactivity profile of an individual protein fragment. Grey color indicates no seroreactivity, black is low-to-moderate seroreactivity, and red denotes high seroreactivity to probed fragments. Lacunae present across more than one comparison are designated by a triangle.

Figure 7

Heat map of lacunae shared across different acute sera comparisons of CM + SMA subjects. P. falciparum antigen lacunae were grouped by recurrence across different comparisons with acute sera from subjects with cerebral malaria and severe malarial anaemia. Each column displays the seroreactivity profile of one serum sample, and each row displays the seroreactivity profile of an individual protein fragment. Grey color indicates no seroreactivity, black is low-to-moderate seroreactivity, and red denotes high seroreactivity to probed fragments. Lacunae present across more than one comparison are designated by a triangle.