The lack of DARC among almost all individuals of West African origin has been suggested to be due to natural selection induced by P. vivax 
. This idea, however, is controversial and P. vivax
has been observed in some populations lacking DARC, indicating that the parasite may be able to use other host cell receptors for invasion if DARC is not present 
The presence or absence of DARC has been associated with resistance and susceptibility to a range of infectious and non-infectious diseases 
. Individuals that lack DARC (Duffy negative, Fy−) are known to be ‘resistant’ to P. vivax
erythrocytic infection 
. They are, however, still susceptible to the sporozoite-induced liver stage infection for which DARC is not required. Our earlier study and those of others revealed that humoral and cellular immune responses to P. vivax
pre-erythrocytic antigens such as CSP are present in Fy− individuals 
. Among Fy+ individuals, however, the influence of each allele on the acquisition of immune response against vivax antigens has yet to be fully explored.
The expression levels of erythroid-specific DARC varied with erythrocyte age and between different FY+ genotypes 
. DARC expression was 2-fold higher in FY*A/FY*A homozygotes than in FY*A/FY*Anull heterozygotes among people living in Papua New Guinea, and higher DARC expression was associated with the higher prevalence of P. vivax
infection seen in FY*A/FY*A compared to FY*A/FY*Anull subjects 
. This was caused by the significant reduction of the adherence of Duffy binding protein to the erythrocyte in individuals who carried the FY*A/FY*Anull allele 
. Individuals with the FY*B/FY*B genotype had a higher risk of P. vivax
infection than those with FY*B/FY*Bnull genotype in malaria-endemic regions of Brazil 
. Moreover, DARC expression has been found to be lower in the FY*B/FY*B than in FY*A/FY*A and FY*A/FY*B genotypes 
. Hence, apart from the different levels of FY expression, the specific qualities and subsequent functions of the DARC upon binding to the vivax
Duffy binding protein and post invasion of erythrocytes, as well as its role in innate immunity as a receptor for chemokines may all contribute to the resistance and susceptibility to vivax infection. We were motivated to investigate the influence of different Duffy positive alleles on the acquisition of anti-P. vivax
immunity among individuals living in malaria-endemic regions.
First, we demonstrated that broad antibody responses were detected by ELISA to both P. vivax
and P. falciparum
in individuals living in the two malaria endemic areas in Colombia that we studied (). The higher prevalence of recognition of at least one P. vivax
antigen (44%, n
103) compared to recognition of the corresponding P. falciparum
antigen(s) (27%, n
64) probably reflects the higher level of P. vivax
transmission in these areas compared to that of P. falciparum 
. However, the overall low prevalence of recognition of the individual antigens reflects the relatively low level of malaria transmission in these areas and the fact that our selection criteria excluded individuals with active infections.
The high level of recognition of PvCSP regardless of the presence or absence of DARC had previously been recorded among individuals living on the Pacific coast of Colombia 
and in Brazil 
. There was no significant difference in the frequency of antibodies to the sporozoite and liver-stage antigen PvCSP among Fy+ and Fy− individuals (p
0.2996). Since sporozoites do not use DARC to infect hepatocytes, Fy+ and Fy− individuals should be equally susceptible to sporozoite infection 
To determine whether the acquired antibody responses associated with “natural” protection, we focused our studies on donors that had developed acquired antibody responses but who had undetectable parasitemia by blood smear at the time of enrollment and no history of malaria symptoms in the past 12 months. Long-lasting antibodies and memory B cell responses in other low malaria transmission endemic areas have also been reported to be associated with naturally acquired protection 
. As we show in , the antibody responses to PvMSP1 differed significantly between Fy+ and Fy− individuals in both frequency (p
0.005) and magnitude (p
0.014) (). This observation is consistent with the requirement for DARC for P. vivax
erythrocytic infection 
. We suspect that the very few Fy− donors in the current study that did have detectable anti-PvMSP1 antibodies had been transiently exposed to PvMSP1 after the release of merozoites from infected hepatocytes into the blood. These merozoites would have been cleared rapidly from the circulation because they would be unable to invade the Fy− erythrocytes in these individuals.
The major finding in this study was within the Fy+ group ( and –
), in which the frequencies and magnitudes of the antibody responses to P. vivax
erythrocytic antigens were significantly higher in individuals possessing a single negative allele (FY*A/FY*Bnull and FY*B/FY*Bnull) than in double positives (FY*A/FY*B and FY*B/FY*B). This is exactly opposite of the expected level of DARC expression 
and susceptibility to P. vivax
. It is known that the elevated DARC expression in double-positive individuals confers a higher risk of P. vivax
infection in comparison to those with one negative gene (FY*A/FY*Bnull, FY*B/FY*Bnull) 
. Furthermore, active erythrocytic malaria infections have been reported to induce immune suppression that prevents the host from mounting an effective immune response against the blood stage parasites and other co-infecting agents 
. This immune suppression ranges from inhibition of dendritic cell maturation 
, to inhibition of the generation of specific CD4 T cells 
and apoptosis of specific CD4 T cells 
. Thus, it is likely that the high susceptibility to P. vivax
blood stage infection and concomitant high P. vivax
erythrocytic parasite load in FY*A/FY*B and FY*B/FY*B double-positive individuals may contribute to the suppression of antibody responses against erythrocytic antigens when compared with FY*A/FY*Bnull and FY*B/FY*Bnull individuals. The higher antibody levels observed in those with a single negative FY allele may also limit parasite load during subsequent infections that may in turn reduce or prevent the immune suppression induced by erythrocytic parasites. Finally, based on DARC's role as a sink for excess pro-inflammatory cytokines 
, high levels of DARC expression in FY double-positives may reduce the surplus of pro-inflammatory cytokines and curb the severity of symptoms; alternatively, DARC may down-regulate the immune responses that control the erythrocytic parasitemia.
This difference in immune recognition between FY*A/FY*B and FY*B/FY*B individuals and FY*A/FY*Bnull and FY*B/FY*Bnull individuals observed with PvMSP1 was confirmed with a second P. vivax blood stage antigen, PvDBP, which is involved in P. vivax erythrocytic invasion. Our observations that the antibody responses to two P. vivax blood stage antigens are higher in single positive than in double-positive individuals leads us to speculate that immune responses to multiple erythrocytic antigens in hosts with low parasite load could act synergistically against erythrocytic parasite invasion and development, providing clinical protection against subsequent reinfection.
In order to determine whether the differential frequencies of recognition to blood versus sporozoite antigens by FY genotypes were restricted to P. vivax, we investigated the recognition of PfCSP and PfMSP1 in the same study population (, and ). As expected, no such variation in antibody response against P. falciparum antigens with FY genotype was observed.
Finally, we wanted to know whether any specific Ig subtypes are responsible for the naturally acquired antibodies that are associated with individual DARC genotypes. IgM responses to malaria are more likely to be detected early after infection and are expected to switch from the IgM isotype to the cytophilic isotypes IgG1 and/or IgG3, a switch that has been associated with clinical control of erythrocytic parasites 
. In some endemic areas the IgG1 response to PvMSP1 is higher, whereas in other areas the IgG3 response dominates 
. In this study, strong IgM and IgG responses to PvMSP1 were detected in the same Fy+ individuals (), indicating that the IgM response did not compromise the induction and development of IgG1 and IgG3 responses to this antigen. The magnitude of IgG1 response to PvMSP1 was 3 times greater than the IgG1 response to PvDBP. However, the frequency of IgG3 responses to PvMSP1 was significantly lower in FY*A/FY*B than in FY*A/FY*Bnull (p
0.015) individuals, as were the IgG1 responses to PvDBP in FY*B/FY*B compared to FY*B/FY*Bnull individuals (p
0.035). Therefore, the lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax
erythrocytic antigens in humans with the double positive FY*A/FY*B and FY*B/FY*B genotypes, respectively (). However, the immune mechanism by which differential DARC expression manipulates the specific IgM and IgG subclass profiles associated with clinical protection need to be determined.
In summary, we observed that frequency and magnitude of antibodies specific for P. vivax erythrocytic antigens varied with the host DARC genotype. Donors with genotypes associated with higher levels of DARC expression and higher susceptibility to P. vivax infection were found to have lesser frequencies and lower magnitudes of specific antibodies against PvMSP1 and PvDBP. IgG3 and IgG1 may account for the decreased responses to P. vivax erythrocytic antigens in humans with FY*A/FY*B or FY*B/FY*B genotypes. This supports the notion that one of the primary mechanisms by which P. vivax evades host immunity is through DARC indirectly down-regulating humoral responses against erythrocytic invasion and development. These results represent an important advance in our understanding of blood-stage immunity to P. vivax that will inform the rational design and development of effective vaccines to control P. vivax malaria.