Comprehensive, longitudinal blood sampling during the time children are highly susceptible to malaria along with detailed entomologic data enabled us to consider factors contributing to the COI, parasite populations within the child's successive infections, and the development of immunity. We estimated the overall COI by the number of K1 and Mad20 MSP-1 Block2 alleles within an infection (COIKM) and the presence of the repeatless MSP-1 Block2 RO allele to test if COIKM (or overall COI) was associated with either genotype-specific or genotype-transcending resistance to parasitemias of >500/μl or febrile parasitemia of any density.
The first phase of our study was to determine factors impacting the detectable COIKM in this population. Our results suggest that superinfection occurring within a very short time period (<2 weeks) and/or mosquitoes inoculating more than one parasite genotype in one bite contributed to the different COIKM and different genotypes detected within an individual over successive months more so than superinfection occurring over a period of 1 month. Three observations led us to this conclusion. First, the moderate village-based differences (~2-fold) in EIR had a greater impact on COIKM than did vast differences in EIRs with transmission season (~10-fold). Second, parasitemias following a successful antimalaria treatment had an average COIKM similar to parasitemias following untreated parasitemias of COIKM < 5. Third, K1 “genotypes” were often not redetected in subsequent blood samples.
A child not accumulating a higher COIKM
over successive months and having months where few parasite genotypes were detected suggests that it is possible for children to clear parasite genotypes (to below detection level) by innate or specific immunity. In a previous Tanzanian study, the disappearance of alleles within days in asymptomatic parasitemias of <1,000/μl was attributed to sequestration (28
). Sequestration and detection limits are relevant factors, especially at such low-density parasitemia. However, the large volume of blood (>200 μl of packed RBCs) used in this study, the infrequent redetection of genotypes in three successive infections, and lack of accumulating COIKM
over successive months suggest that children could clear parasite genotypes to below a detectable level and that the parasitemias and genotypes detected in the following 2 months were largely attributable to new inoculation (approximate EIR of >0.5 infected bites/person/night).
Interestingly, we found that although, as expected, children whose sickle cell hemoglobin genes were homozygous (SS) or heterozygous (AS) for the sickle cell genotype trait were more capable of resisting parasitemia of >500/μl, their mean COIKM
was not significantly lower than that of AA children. This observation is in agreement with Ntoumi et al., who found a higher COI in 7- to 14-year-olds with the AS genotype versus the AA genotype (32
). It seems unlikely that AS or SS children are more susceptible to infectious bites with more parasite genotypes. In comparison to AA children, AS and SS children in the ABCP appear more likely to resist parasitemia of >500/μl but not more likely to be aparasitemic (Branch et al., unpublished data). It is possible that AA children are more capable of clearing parasite genotypes or that parasites are more likely to sequester at levels below PCR detection in AA children in comparison to AS or SS children.
We found that children >2 years old had a higher COIKM
and a reduced ability to resist parasitemias of >500/μl for >2 months in comparison to children 0.5 to 1 year old. This contrasts with the conventional wisdom that, not considering the first 6 months, when maternal antibodies appear to provide protection, the ability of the host to control or clear malaria infections should correlate with exposure-acquired immunity (and/or age-acquired immunity, as discussed by Baird et al. [5
]). In agreement with our findings, others have found a higher COI in 2- to 4-year-old children in comparison to children <2 (25
). This led to the hypothesis that the fever often observed in infants could clear parasitemias, while the developing specific immune response in 2- to 4-year olds limits but does not completely clear parasite genotypes below detectable levels (25
). The studies summarized above did not have data available on antimalaria drug treatment. Here, we find the first evidence, independent of antimalaria drug treatment, transmission, and host-related differences in susceptibility, that febrile illness was associated with a greater likelihood of clearing and resisting subsequent parasitemia.
The facts that the COIKM
was not associated with parasite density and children >2 years old were less likely to resist parasitemias of >500/μl for longer than 2 months support some density-dependent regulation of parasitemias in older children (10
). However, we found that, regardless of age, the previous parasitemia's COIKM
, transmission intensity, drug treatment, parasite density, and sickle cell genotype, >30% of the time children with a COIKM
of ≤3 could resist subsequent parasitemia of >500/μl for >2 months.
We considered the ability to resist subsequent parasitemia of >500/μl (or any febrile parasitemia) in two ways: the mean time of resistance and the ability to resist for ≥2 months. The former enabled us to consider the data without preset thresholds, while the latter enabled us to consider the child's having opportunity for subsequent infection in this highly endemic transmission area.
Resistance was measured as parasitemia and was not specific to any particular parasite genotype. The fact that K1 alleles often were not redetected for >2 months evokes speculation of genotype (allele)-specific immunity (22
). However, our result can also be explained by the low probability of redetecting any particular genotype by chance alone. The resistance to parasitemia, regardless of genotype, suggests that genotype-transcending immunity (targeting antigenic determinants shared among most parasite genotypes) was more effective following low COIKM
infections with the RO parasite genotype.
Children's sickle cell trait enabled a measure of comparison of the estimated effect of COIKM and RO on detected resistance in the following months. A decrease of 2 in the detected COIKM was predictive of a fourfold-increased ability to resist parasitemia of >500/μl (and any febrile parasitemia) in the following 2 months. This was greater than the predicted twofold increase in resistance seen in AS children versus AA children.
The fact that MSP-1 is associated with protection, especially the highly conserved MSP-1 19-kDa fragment (19KD) (11
), is a major reason to consider MSP-1 Block2 (and loci in genetic disequilbrium with MSP-1 Block2) specifically rather than as only a marker for overall COI. There is extensive genetic disequilibrium in MSP-1, resulting in Block2 genotyping's not being indicative of the allelic types present in other regions of the gene (13
). However, the exception is linkage disequilibrium between MSP-1 19KD and MSP-1 Block2 (39
). The findings that the K1 allelic type was most negatively correlated with resistance and the presence of the repeatless RO parasite type was associated with greater resistance suggest that the antigenic diversity of MSP-1 Block2 (or linked antigenic diversity) impacts the development of resistance to subsequent parasitemia, but cannot rule out the impact of overall COI on the association. For instance, the K1 genotypes might be a better indication of overall antigenic diversity, since it is more polymorphic in this population, or the K1 genotypes might be more effective at antagonizing or distracting an effective immune response to Block2 or other antigenic determinants (2
Data investigating anti-Block2's role in protection have been conflicting (7
). While some anti-Block2 antibodies are cross-reactive within main families, others are specific to differences between the main families (7
). Some studies suggest that high anti-Block2 antibody levels are positively associated with susceptibility to parasitemia and are not associated with protection (24
). In contrast, a recent study found that children with both K1 and Mad20 anti-Block2 antibodies at the end of the low-transmission season were more likely to manifest signs of clinical malaria within the following 6 months than individuals without anti-Block2 antibodies at the end of the transmission season (14
). It is possible, however, that anti-MSP-1 Block2 antibodies are only a marker of recent parasitemia and, therefore, a marker of short-lived antibodies to other malaria antigens (especially MSP-1 19KD) (7
). This would result in an association with Block2 antibodies regardless of their specific role in protection.
If anti-Block2 antibodies can impart significant protection, our results suggest that an effective response was impaired in high COIKM
, as predicted by the smoke screen hypothesis (2
), possibly facilitated by immunologic antagonism (34
). However, evoking this form of immune evasion as an explanation for susceptibility to parasitemia of >500/μl requires these postulated immune evasion mechanisms to be generalized and not specific to a particular genotype.
The association between the RO parasite and subsequent resistance, in agreement with earlier reports (1
), is interesting. The resistance seen following an infection with RO would be expected if RO was linked to a particular 19KD allele or an allele that did not block effective anti-19KD antibodies (42
). In addition, linkage disequilibrium with other genes might result in RO parasites having different growth rates and/or erythrocyte specificity (39
). Finally, and most speculatively, the lack of repeats in the RO Block2 region (and extremely limited linkage disequlibrium in P. falciparum
) might induce a more effective immune response.
Speculation abounds regarding the role of repetitive antigens in immune evasion. It can be postulated that T-independent antibody responses to the repetitive Block2 antigens (7
) could result in a cytokine environment nonconducive to T-dependent antibody responses to adjacent determinants. This was first suggested and studied regarding the circumsporozoite protein repeats (40
), but it was thought that the smaller Block2 would not have great potential for altering the cytokine environment. However, one must consider the natural infections where the host's immune response is exposed to many different parasite genotypes at one time (mixed infections) (33
). The within-host diversity would amplify the number of B-cell clones responding within a given infection. It is important to note that homeostasis in the immune response and diverse immunogenic antigens might distract or exceed a regulation threshold (21
) and/or soluble Block2 might impede germinal center formation (36
). Our results suggest that infections lacking the repeatless RO parasite genotype are more vulnerable to such evasion mechanisms.
Either impediment of a protective immune response to Block2 or indirect immune evasion mechanisms as described above might explain the apparent diversifying selection on MSP1 Block2 (45) and the prevalence of mixed infections (33
). Some of this speculation can be directed to consider why genotype-transcending immunity, targeting conserved antigenic determinants, is delayed during natural malaria infections.
In summary, this study has provided information on dynamics of parasitemia in children's first few years of life. We have found that independent of febrile illness, age, transmission, and previous months' parasitemia history, the COIKM is negatively correlated with resistance to parasitemia of >500/μl in the following months. This correlation was detectable within an individual child's history of successive infections. From a public health perspective, this observation suggests that intervention control and prevention strategies that reduce the complexity of infection may lead to faster development of natural immunity that protects against high-density parasitemia and clinical manifestations of illness.