To study protection by anti-PfEMP1 antibodies it was necessary to focus on a group of children who were at various stages of developing a full repertoire of anti-PfEMP1 responses. As the period over which the antibody repertoire develops will vary with local rate of exposure, it was important first to determine the age range to study. To this end, 200 serum samples (40 from each of five age classes: 1–2, 3–4, 5–6, 7–8 and 9–12 years) were taken from children in March 1993. Each was tested for its ability to agglutinate four parasites (C10, A4, W1008 and W1027). As shown in , the prevalence of antibody responses to all isolates rose between 1 and 5 years of age. This agrees closely with the age range over which naturally acquired immunity to malaria develops in this part of Kenya (data not shown). As expected, responses to the different isolates were independent and specific, since the number of individuals in each age group that recognized between zero and four isolates () has a diagonal structure that fits very closely that predicted by a binomial distribution (not shown).
Fig. 1 Age-specific recognition of four P. falciparum isolates by children in the Kilifi area. C10 and A4 are subclones derived from laboratory line IT04 (ref. 18); W1008 and W1027 are wild isolates from Kilifi. a, The proportion of each age class with agglutinating (more ...)
Defining the role of parasite antigens in naturally acquired immunity has been problematic. The ubiquity of asymptomatic infection in those age groups susceptible to disease combined with the difficulty of determining the immune status of any individual means that relating any particular immune response to clinical immunity is only possible in large, prospective studies17
. With this in mind we recruited a cohort of 4783 children aged between 1 and 5 years old. Blood samples were taken from the children in a cross-sectional survey at the beginning of the long rainy season during May 1995. Over the following 8 months (which included two periods of malaria transmission), 65 parasite isolates were collected from children of the cohort who returned to the hospital with clinical malaria. Parasites were collected from patients with both severe and mild malaria; 34 were outpatients and 31 were admitted to the hospital. Since only immature parasites that do not express PfEMP1 are present in peripheral blood, parasite isolates were grown in vitro
until they were mature trophozoites. Because the repertoire of PfEMP1 variants that can be expressed by a single genotype is large and the rate of antigenic switching is high, parasites sampled at any point in time may constitute complex mixtures of different variant types18
. Thus, to assess the antibody response to these proteins, we used agglutination assays to measure response to the whole parasite population present at the time of sampling. Assays were performed to test for recognition of each isolate by the corresponding infected child’s own serum collected in the initial cross-sectional survey (index) and plasma collected at the time of acute illness (acute). To determine the expected prevalence of antibodies in the population, each parasite was also assayed against sera from 20 age-matched controls also drawn from the samples collected in May 1995. Data from the 65 parasite isolates were pooled.
A marked difference was seen in the frequency with which parasites causing disease were recognized by index sera compared with sera from age-matched controls from the same community (). This difference cannot be accounted for by differences in prior exposure, as the parasite rates in the two groups were almost identical at the time of sampling (). Since the parasite rate was found to be rising with age within the controls and has therefore not reached saturation (data not shown), this measurement is likely to provide a reasonable estimate of exposure. We further tested the possibility that there were, nonetheless, differences in exposure on a more local scale by categorizing the controls into 54 residential zones, defined previously in the 1991 national census, each containing approximately 100 homesteads and 850 people. The overall rates of positive responses in controls drawn from the zones of children who presented as cases and those living in zones from which no case presented were compared. Although one might expect higher overall exposure in the zones from which cases were drawn, no difference was detected ().
Agglutination of parasite infected red cells by sera from clinical isolates and controls
We considered the possibility that the lower prevalence of variant-specific antibodies in index sera was the result of a generalized lower response to the infected erythrocyte surface rather than the specific absence of a response to the infecting isolate. To test this possibility, 52 of the index sera were tested for their ability to agglutinate a single, heterologous wild isolate, 1759. To maximize the chances of detecting a reduced prevalence of antibody, each index serum was compared with three controls matched both by age and zone of residence within the study area. There was no significant difference between the two groups (), suggesting that the index sera have a specific reduction in the prevalence of antibodies directed against the parasites subsequently causing clinical disease in these children. The almost exclusive appearance during clinical disease of parasite variants that correspond to gaps in each child’s developing repertoire of anti-PfEMP1 antibodies suggests that preexisting antibodies provide protection against parasite variants to which they are directed.
Previous studies have described the development of strong, variant-specific anti-PfEMP1 responses over the weeks following a clinical episode12,15
. As shown in , such responses apparently evolve rapidly. Although there was no increase in the prevalence of antibodies to parasite 1759 at the time of disease, there was already a significant increase in the prevalence of antibodies specific to the infecting isolates. Since the acute plasma and parasites were sampled several hours before the parasites would express PfEMP1 in vivo
, the coexistence of parasite variants and corresponding antibody in vitro
reflects a situation that will occur in vivo
as the parasites mature, immediately before immune selection. Rapid production of antibody to the infecting isolate, together with a high rate of PfEMP1 antigen switching as previously observed in vitro18
, could result in a close dynamic relationship between the emergence of new parasite variants and their removal by the immune system.
Taken together, the variant-specific protection provided by anti-PfEMP1 antibodies, their rapid induction in response to infection and the expansion of the antibody repertoire during the period in life when disease immunity is known to be developing support the idea that these antibodies play an important role in the acquisition of immunity to malaria in children. It is possible that antibodies to nonvariant epitopes on the red cell surface are also involved in immunity, particularly in adults19
. However, although such antibodies have been isolated from immune adult serum12
, the agglutinating antibody response in adults remains predominantly variant specific14
, suggesting that variant-specific antibodies may play a continuing role in the maintenance of immunity throughout life.