This study is the first to comprehensively analyse the profile of naturally acquired antibodies against the two major families of P. falciparum merozoite ligands in asymptomatic vs symptomatic populations living in a malaria hypoendemic area. Sera from P. falciparum-infected individuals from the Peruvian Amazon were tested against all PfRh and EBL family members known to be involved in P. falciparum invasion. This study’s main finding is that antibody responses against the EBL and PfRh proteins were significantly higher in asymptomatic than symptomatic parasitaemic individuals, suggesting a potential association with the development of clinical immunity. Whether these anti-invasion ligand antibodies directly mediate protective immunity or are simply statistically associated with clinical immunity is beyond the scope of this pilot study. Significant differences in the total IgG responses were observed against EBA-175, EBA-181, PfRh2b and MSP119. IgG1 responses against EBA-181, PfRh2a and PfRh2b were also significantly higher in the asymptomatic individuals. Furthermore, elevated total IgG antibody responses against PfRh1, PfRh2a, PfRh2b, PfRh5, EBA-175, EBA-181 and MSP119 proteins were positively associated with decreased parasitaemia. IgG1 response against EBA-181, PfRh2a and PfRh2b and IgG3 response for PfRh2a were also negatively correlated with parasitaemia. Thus, these results suggest that total and IgG subclass-specific responses to distinct merozoite antigens are significantly associated with protection from high-density parasitaemia and symptomatic malaria.
IgG responses to some but not all EBL antigens has been associated with protection against symptomatic P. falciparum in some malaria endemic areas (reviewed in ). Naturally acquired antibodies to region II of EBA-175 have been found to increase with age in a naturally exposed population in Kenya and such antibodies were capable of inhibiting binding of EBA-175RII to erythrocytes as a correlate of clinical immunity in this population affected by holoendemic malaria . Nonetheless, significant associations between anti-EBA-175 antibody levels and protection from high-density parasitaemia and clinical disease has been inconsistent among studies [20–23] although experimental studies using sera from rabbits vaccinated with region II of EBA-175 were able to block invasion by > 50% of P. falciparum laboratory strains from diverse geographic origin [24, 25]. Studies that measure responses to the other EBL antigens in endemic areas outside of Africa are scarcer [5, 9, 19]. A comparative study by Ford et al., showed that the antibody responses against MSP119 and EBLs (EBA-175, EBA-140, EBA-181) in individuals living in a hypoendemic malaria region of Brazil were much lower thanin individuals living in a hyperendemic area of Cameroon .
Immune responses against PfRh invasion ligands have been less studied [4, 26, 27]. Association between antibody responses against PfRh ligands and outcome of a clinical disease has been reported only for PfRh2a and PfRh2b . Antibody responses against PfRh4, PfRh5 and the common region of PfRh2 were reported for subjects in Kenya without taking into consideration the clinical or parasitological status of the studied individuals , and responses against PfRh2b was also evaluated in subjects from Senegal and Tanzania . In children from Papua New Guinea, high levels of total IgG as well as IgG1 and IgG3 antibody responses against the PfRh2a and PfRh2b antigens were found to be strongly associated with protection from symptomatic malaria and high-density parasitaemia. Notably, these antibody responses were similar to or greater than the ones generated against the leading vaccines candidates MSP119, MSP-2 and AMA-1 , but, it must be noted, that different regions of the EBAs and the PfRH proteins were studied so it may be difficult to make direct comparisons . Antibody responses against PfPh5 have been found at much lower rates in naturally infected individuals but laboratory studies of anti-PfRh5 antibodies induced by vaccination with recombinant protein appeared to induce strain-transcending growth inhibitory antibodies [25, 27]. The focus on PfRh invasion ligands as potential vaccine antigens was augmented by the recent promising invasion inhibitory results obtained using antibodies generated against the combination of EBL and PfRh proteins; EBA-175, PfRh2 and PfRh4 , and EBA-175 and PfRh5 . The ability of antibodies against the full length PfRh5 (RH5FL) protein to inhibit the growth of all strains of parasites in vitro more effectively than antibodies induced by other vaccine candidates, such as AMA-1 and MSP-1, increased the appeal of using PfRh5 as an anti-blood-stage malaria vaccine .
Three studies have previously examined the presence of antibodies against PfRh ligands [4, 26, 27], but not against PfRh1 in an endemic population. Notably, total IgG response to PfRh1 in the Peruvian population was similar in Sym and Asy individuals based on seroprevalence, but the magnitude of the response was significantly higher in the Asy group and inversely associated with levels of parasitaemia. A high seroprevalence for PfRh2a (75%) regardless of the clinical status (Asy and Sym) was observed in the Peruvian individuals similar to what was reported in children from Papua New Guinea (94%) . However, in Peruvian subjects, significant differences were observed in the prevalence of IgG1 and IgG3 responders in the Asy group (IgG1 88% vs. 38% and IgG3 75% vs. 30%). In contrast, the response against PfRh2b in the Peruvians (31%) was higher than that reported in individuals with malaria in Senegal (8.9%) and Tanzania (5.6%) and lower than those observed in Papua New Guinea (85%) [4, 26]. However, higher total IgG and IgG1 responses against the PfRh2b ligand were associated with low parasitaemia, similar to observed in Papua New Guinea . Seroprevalence for PfRh4 in the Peruvian infected individuals was lower than observed in adults from Kenya (50% vs. 70%) , and presently it appears that these antigen specific responses are not associated with protection against clinical malaria. In this study only IgG1 and IgG3 subtypes were measured and analysed because these subtypes are associated with Fc binding and are potentially associated with functional antibody-dependent cellular cytotoxicity [29, 30]. Nonetheless, whether such anti-invasion ligand antibodies might function through ADCI, steric hindering or by another mechanism remains to be determined. Although PfRh5 was suggested to be essential for erythrocyte invasion [31, 32] and the target for vaccine-induced antibodies , only few individuals from Peru had antibodies against PfRh5 (11%), which is comparable to what was reported in Kenya (16%) . The reason for the lack of anti-PfRh5 antibody in naturally exposed individuals is still unclear. The mechanism by which this essential invasion ligand escapes immune recognition thus requires further investigation.