Francis M. Ndungu

Immunity to malaria: more questions than answers

Malaria is one of the main health problems facing developing countries today. At present, preventative and treatment strategies are continuously hampered by the issues of the ever-emerging parasite resistance to newly introduced drugs, considerable costs and logistical problems. The main hope for changing this situation would be the development of effective malaria vaccines. An important part of this process is understanding the mechanisms of naturally acquired immunity to malaria. This review will highlight key aspects of immunity to malaria, about which surprisingly little is known and which will prove critical in the search for effective malaria vaccines.

Plasmodium falciparum-Infected Erythrocytes: Agglutination by Diverse Kenyan Plasma Is Associated with Severe Disease and Young Host Age

The variant surface antigens (VSAs) of Plasmodium falciparum-infected red blood cells are potentially important targets of naturally acquired immunity to malaria. Natural infections induce agglutinating antibodies specific to the VSA variants expressed by the infecting parasites. Previously, when different parasite isolates were tested against a panel of heterologous plasma from Kenyan children, the proportion of plasma that agglutinated the parasites (the agglutination frequency [AF]) was highly variable among isolates, suggesting the existence of rare and prevalent variants. Here, the AF of 115 isolates from Kenyan children were compared. The results show that the AF of isolates causing severe malaria were significantly higher than those of isolates causing mild malaria; and AF decreased significantly with the increasing age of the infected child. We propose that parasites causing severe disease tend to express a subset of VSA variants that are preferentially associated with infections of children with low immunity.

Chronic Exposure to Plasmodium falciparum Is Associated with Phenotypic Evidence of B and T Cell Exhaustion

Naturally acquired immunity to malaria develops slowly, requiring several years of repeated exposure to be effective. The cellular and molecular factors underlying this observation are only partially understood. Recent studies suggest that chronic Plasmodium falciparum exposure may induce functional exhaustion of lymphocytes, potentially impeding optimal control of infection. However, it remains unclear whether the “atypical” memory B cells (MBCs) and “exhausted” CD4 T cells described in humans exposed to endemic malaria are driven by P. falciparum per se or by other factors commonly associated with malaria, such as coinfections and malnutrition. To address this critical question we took advantage of a “natural” experiment near Kilifi, Kenya, and compared profiles of B and T cells of children living in a rural community where P. falciparum transmission is ongoing to the profiles of age-matched children living under similar conditions in a nearby community where P. falciparum transmission ceased 5 y prior to this study. We found that continuous exposure to P. falciparum drives the expansion of atypical MBCs. Persistent P. falciparum exposure was associated with an increased frequency of CD4 T cells expressing phenotypic markers of exhaustion, both programmed cell death-1 (PD-1) alone and PD-1 in combination with lymphocyte-activation gene-3 (LAG-3). This expansion of PD-1–expressing and PD-1/LAG-3–coexpressing CD4 T cells was largely confined to CD45RA+ CD4 T cells. The percentage of CD45RA+CD27+ CD4 T cells coexpressing PD-1 and LAG-3 was inversely correlated with frequencies of activated and classical MBCs. Taken together, these results suggest that P. falciparum infection per se drives the expansion of atypical MBCs and phenotypically exhausted CD4 T cells, which has been reported in other endemic areas.

Oxidative stress and erythrocyte damage in Kenyan children with severe Plasmodium falciparum malaria

Anaemia causes significant morbidity in children with Plasmodium falciparum malaria, but the mechanism(s) are unclear. During malarial infection, increased reactive oxygen species (ROS) are generated that may contribute to erythrocyte damage and anaemia. This study measured the concentrations of α‐tocopherol in plasma and erythrocyte membranes, and the percentage polyunsaturated fatty acid composition (%PUFA) (an indirect marker of ROS damage) in erythrocyte membranes in children with severe P. falciparum malaria from Kilifi, Kenya, and asymptomatic children from the same district. Malarial subjects were stratified into complicated malaria and malaria anaemia. Results demonstrated significant reductions in erythrocyte membrane α‐tocopherol concentration (1·63 ± 0·16 versus 3·38 ± 0·18 μmol/mg protein; P < 0·001) and total %PUFA (30·7 ± 0·49 versus 32·8 ± 0·44% P < 0·005) for the malarial subjects (non‐stratified) compared with controls. Malarial subjects showed a significant positive correlation between membrane α‐tocopherol and haemoglobin concentrations (P < 0·005 r = 0·63 complicated malaria group; P < 0·05 r = 0·36 non‐stratified data). There were no significant differences in plasma α‐tocopherol concentration between malaria patients and controls. In conclusion, malarial infection may be associated with oxidative damage and reduced α‐tocopherol reserve in the erythrocyte membrane, suggesting that local antioxidant depletion may contribute to erythrocyte loss in severe malaria. Erythrocyte membrane α‐tocopherol appeared a better indicator of ROS exposure than plasma.

B Cell Memory to 3 Plasmodium falciparum Blood-Stage Antigens in a Malaria-Endemic Area

To gain insight into why antibody responses to malarial antigens tend to be short lived, we studied antigen-specific memory B cells from donors in an area where malaria is endemic. We compared antibody and memory B cell responses to tetanus toxoid with those to 3 Plasmodium falciparum candidate vaccine antigens: the C-terminal portion of merozoite surface protein 1 (MSP1(19)), apical membrane antigen 1 (AMA1), and the cysteine-rich interdomain region 1 alpha (CIDR1 alpha ) of a protein from the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. These data are the first to be generated on memory B cells in children who are in the process of acquiring antimalarial immunity, and they reveal defects in B cell memory to P. falciparum antigens. Compared with the results for tetanus toxoid, more donors who were positive for antibody to AMA1 and CIDR1 alpha were negative for memory B cells. These data imply that some exposures to malaria do not result in the establishment of stable populations of circulating antigen-specific memory B cells, suggesting possible mechanisms for the short-lived nature of many anti-malarial antibody responses.

Plasmodium falciparum Infections Are Associated with Agglutinating Antibodies to Parasite-Infected Erythrocyte Surface Antigens among Healthy Kenyan Children

Antibodies to the highly diverse variant surface antigens (VSAs) expressed on Plasmodium falciparum-infected erythrocytes are thought to play a role in the development of naturally acquired immunity to malaria. It has been suggested that children gradually acquire immunity through the piecemeal acquisition of antibodies to a large number of VSAs over several years of exposure. However, in a cross-sectional survey of Kenyan children before the malaria-transmission season, the proportion of children with antibodies recognizing randomly sampled VSAs was found to be strikingly higher among children with microscopically detectable P. falciparum infection, compared with those without detectable infection. We suggest that parasitization status may be an important consideration in longitudinal assessments of the protective role of some anti-parasite immune responses and support this suggestion with data from a prospective study of VSA antibodies in a group of children who subsequently had severe malaria.

Naturally acquired immunoglobulin (Ig)G subclass antibodies to crude asexual Plasmodium falciparum lysates: evidence for association with protection for IgG1 and disease for IgG2

There is longstanding evidence for a role of immunoglobulin (Ig)G in protection against malarial disease and infection. IgG1 and IgG3 have been shown to be particularly efficient at associating with monocytes in potentially protective mechanisms (i.e. antibody‐dependent cellular inhibition, opsonization and phagocytosis). Conversely, there is some evidence that IgG2 (and possibly IgG4) antibodies may be antagonistic to this protection. The protective effect of IgG subclass antibody activity present before the beginning of a malaria transmission season (preseason antibody levels) against severe malaria has not been tested in longitudinal studies. We measured IgG class and subclass antibody levels specific to crude Plasmodium falciparum lysates by enzyme linked immunosorbent assay in a case–control study of 76 children on the coast of Kenya. The mean optical density values for both IgG class and subclass antibodies were not significantly different between the children who developed severe malaria and those who remained healthy during an observation period of two malaria transmission seasons. However, elevated levels of IgG1 in relation to levels of IgG2 and IgG4 antibodies were associated with protection from severe malaria (P = 0·02). Conversely, elevated levels of IgG2 in relation to IgG1 and IgG3 antibodies were associated with a higher risk of developing severe malaria (P = 0·006).

Memory B cells are a more reliable archive for historical antimalarial responses than plasma antibodies in no-longer exposed children

Humans respond to foreign antigen by generating plasma Abs and memory B cells (MBCs). The Ab response then declines, sometimes to below the limit of detection. In contrast, MBCs are generally thought to be long-lived. We tested and compared Plasmodium falciparum (Pf)-specific Ab and MBC responses in two populations of children: (i) previously exposed children who had documented Pf infections several years ago, but minimal exposure since then; and (ii) persistently exposed children living in a separate but nearby endemic area. We found that although Pf-specific plasma Abs were lower in previously exposed children compared with persistently exposed children, their cognate MBCs were maintained at similar frequencies. We conclude that serological analysis by itself would greatly underestimate the true memory of Pf-specific Ab responses in previously exposed children living in areas where Pf transmission has been reduced or eliminated.

Functional Memory B Cells and Long-Lived Plasma Cells Are Generated after a Single Plasmodium chabaudi Infection in Mice

Antibodies have long been shown to play a critical role in naturally acquired immunity to malaria, but it has been suggested that Plasmodium-specific antibodies in humans may not be long lived. The cellular mechanisms underlying B cell and antibody responses are difficult to study in human infections; therefore, we have investigated the kinetics, duration and characteristics of the Plasmodium-specific memory B cell response in an infection of P. chabaudi in mice. Memory B cells and plasma cells specific for the C-terminal region of Merozoite Surface Protein 1 were detectable for more than eight months following primary infection. Furthermore, a classical memory response comprised predominantly of the T-cell dependent isotypes IgG2c, IgG2b and IgG1 was elicited upon rechallenge with the homologous parasite, confirming the generation of functional memory B cells. Using cyclophosphamide treatment to discriminate between long-lived and short-lived plasma cells, we demonstrated long-lived cells secreting Plasmodium-specific IgG in both bone marrow and in spleens of infected mice. The presence of these long-lived cells was independent of the presence of chronic infection, as removal of parasites with anti-malarial drugs had no impact on their numbers. Thus, in this model of malaria, both functional Plasmodium-specific memory B cells and long-lived plasma cells can be generated, suggesting that defects in generating these cell populations may not be the reason for generating short-lived antibody responses.

A LAIR1 insertion generates broadly reactive antibodies against malaria variant antigens.

Plasmodium falciparum antigens expressed on the surface of infected erythrocytes are important targets of naturally acquired immunity against malaria, but their high number and variability provide the pathogen with a powerful means of escape from host antibodies. Although broadly reactive antibodies against these antigens could be useful as therapeutics and in vaccine design, their identification has proven elusive. Here we report the isolation of human monoclonal antibodies that recognize erythrocytes infected by different P. falciparum isolates and opsonize these cells by binding to members of the RIFIN family. These antibodies acquired broad reactivity through a novel mechanism of insertion of a large DNA fragment between the V and DJ segments. The insert, which is both necessary and sufficient for binding to RIFINs, encodes the entire 98 amino acid collagen-binding domain of LAIR1, an immunoglobulin superfamily inhibitory receptor encoded on chromosome 19. In each of the two donors studied, the antibodies are produced by a single expanded B-cell clone and carry distinct somatic mutations in the LAIR1 domain that abolish binding to collagen and increase binding to infected erythrocytes. These findings illustrate, with a biologically relevant example, a novel mechanism of antibody diversification by interchromosomal DNA transposition and demonstrate the existence of conserved epitopes that may be suitable candidates for the development of a malaria vaccine.