It has become very clear that substantial genetic differentiation exists between parasite populations in Asian and African isolates (Volkman 2007
; Neafsey 2008
). Here we report the assessment of a new structural polymorphism, a large deletion in the P. falciparum
erythrocyte invasion ligand PfRh2b, a dominant invasion ligand involved in sialic-acid independent invasion of erythrocytes. In addition to showing that it is widespread throughout the world, we further provide evidence that it elicits a humoral response which could be the selective pressure driving its frequency.
Using a population genetic analysis, we show that although the PfRh2b deletion is present worldwide, it appears to vary in frequency significantly in different populations. Our results show that the frequency of the deletion is high in all study sites with the exception of Thailand and Brazil. When calculating Fst
for the PfRh2b deletion globally, the Fst
value was very comparable to that observed for polymorphisms in Pfs48/45, which have been previously shown to be under directional selection (Conway 2000
; Escalante 2002
). There was no evidence from the inter-population variance that the PfRh2b polymorphism is under balancing selection globally. However, Fst
calculation using only Senegalese sites revealed a value very close to zero (0.009), a similar result is found when only African sites are analyzed. This low Fst
indicates little genetic differentiation, or divergence among African populations. Indeed, similar values were seen for all of the allelic polymorphisms considered in our study, suggesting the presence of significant gene-flow within the continent, similar to previous observations (Awadalla 2001
The dramatic difference in the PfRh2b deletion frequencies in Brazil and Thailand population compared with the other populations invites the speculation the genetic background of the host in these populations may influence the allele frequencies, due to the presence of different erythrocyte receptor polymorphisms. Alternatively, the PfRh2b deletion may have originated in Africa, and could be at low frequency in Asian and American populations because it is has only recently arrived in those locations. We have some evidence that the PfRh2b deletion is associated with the use of protease-sensitive erythrocyte receptors for invasion (Lantos 2009
). Previous studies have shown that parasite lines from different geographic areas express PfRh proteins variantly (Taylor 2002
; Duraisingh 2003
; Nery 2006
; Bei 2007
). This variation in expression may also possibly contribute to the fact that the PfRh2b polymorphism does not appear to be under balancing selection since alternative ligands can compensate for polymorphism in PfRh2b.
The PfRh2b deletion was previously described in only one lab strain, T9/96, from Thailand, and more recently in Senegalese isolates (Jennings 2007
). The high prevalence of the deletion in many global populations may suggest that appearance of the deletion in the field is not recent; however, to test this hypothesis we need to measure the distribution of the PfRh2b deletion using matched archival and recent samples from the same region, or follow the prevalence in specific sites over time. The absence of the deletion in other lab strains, most of which were established in the 1980s, may on the other hand suggest that the deletion has spread recently.
We investigated if specific immune responses exist against the PfRh2b C-terminal region that harbors the PfRh2b deletion polymorphism. We hypothesize that the parasite has the ability to utilize two potential mechanisms for evading the immune response: variant expression and polymorphism. To test these hypotheses, we measured the IgG response to PfRh2b using sera from Senegal and Tanzania. The presence of an antibody response against the invasion ligand PfRh2b may be significant as these antibodies may be inhibitory to erythrocyte invasion. We found that antibodies against PfRh2b exist in Senegalese and Tanzania sera. The percentage of positive antibody responses by cut-off is similar in both sites, and the acquisition of antibody is correlated with increasing patient age. Such findings imply that this locus is recognized by the humoral immune response, which may serve as a selective force driving the frequency of the PfRh2b deletion if the deletion allele invokes a weaker response than the wild-type allele. Allele specificity of the immune reactivity, measured by competitive ELISA using the PfRh2b wildtype and deletion polymorphism, would further strengthen the hypothesis of immune selection. It has yet to be determined whether immune responses to the PfRh2b unique domain are inhibitory and whether the deletion contributes to immune evasion.
Our results show global variation in the genetic distribution of the PfRh2b deletion and Pfs48/45 polymorphisms, while the frequency distribution of the deletion seems similar in Africa, it is quite divergent worldwide relative to polymorphisms in antigens known to be subject to balancing selection, implying that this polymorphism may not be subject to strong balancing selection, or may be exposed to variable selection pressures across sites. Alternatively, the PfRh2b deletion may exhibit higher divergence among worldwide populations than the Msp-2 and EBA-175 polymorphisms because it is a younger allele, and has not had as much time to spread throughout the range of the parasite populations. We consider this unlikely due to the observation that the PfRh2b deletion is found at frequencies comparable to the Msp-2 and EBA-175 polymorphisms in African and Malaysian populations, whereas a younger allele would more likely be present at lower frequencies. Our findings imply that inclusion of antigens with such profiles may be efficacious in some, but not all countries, resulting in limited vaccine utility. In contrast, Msp-2 and EBA-175 are also involved in erythrocyte invasion, yet in our study, the EBA-175 and Msp-2 dimorphisms exhibit minimal genetic differentiation globally. This strongly suggests that they are under balancing selection, a criteria for inclusion in a universally applicable vaccine.
This population genetic study demonstrates that focusing on large sequence dimorphisms and deletions as vaccine candidates may prove to be an effective strategy for vaccine design. These large regions may inherently define discrete domains that can elicit protective antibody responses (Conway 1997
). Using such molecular population genetic analyses to predict a region of selection, and validating this region using molecular and immunological tools, is an effective approach to identifying novel antigenic regions. This could be exploited in vaccine development, particularly by including both dimorphic regions in combination.
In our study we focus on a comparison of different antigens that are presumably all under selection either by humoral immunity or by red blood cell polymorphisms. We have chosen to focus on antigen polymorphisms for which data previously exists as controls for the measurement of genetic differentiation, known to be either under balancing selection (Msp-2) or directional selection (Pfs48/45). We realize that an expanded study comparing these antigen loci with SNPs in putative neutral loci or in drug-resistance loci may be informative (Anderson 2005
In conclusion, it has been suggested that a good vaccine candidate would incorporate a region of a protein with very few allelic forms under strong balancing selection (Conway 1997
). Our results suggest that in contrast to the EBA-175 and Msp-2 dimorphisms, the PfRh2b deletion may not be under balancing selection globally. However, the C-terminal region of PfRh2b harboring the PfRh2b deletion does elicit immune responses that may exert directional selection on the polymorphism. Further studies will determine whether naturally acquired antibodies to PfRh2b in endemic populations can functionally inhibit invasion. To our knowledge, large deletions on the order of 500bp have not been previously identified in other Plasmodium antigens; however small deletions on the order of 39bp are present in Msp-3 (McColl 1997
). It will be of interest in the future to determine whether these deleted regions in other antigens are also more genetically differentiated between populations, suggesting that in general, large sequence deletions arise as a mechanism to avoid frequency-dependent immune selection.