Background
Erythrocyte invasion by
Plasmodium falciparum is a complex process including attachment, reorientation, penetration, and formation of a parasitophorous vacuole. Several merozoite proteins that have a role during the initial steps of attachment and invasion have been extensively studied, including members of the Merozoite Surface Protein family (MSP), AMA-1, Erythrocyte Binding-Like proteins (EBL: EBA-175, EBA-181, EBA-140 and EBL-1), and the Reticulocyte Binding-Like or Reticulocyte Homologue proteins (RBL or PfRh: PfRh1, PfRh2a, PfRh2b, PfRh4 and PfRh5) [
1]. Many of the invasion ligands are currently being evaluated or developed as candidate vaccine antigens for inclusion in an anti-erythrocytic-stage malaria vaccine [
2]. Antibodies that inhibit merozoite attachment and invasion, and thus subsequent development and propagation within the red blood cells (RBC), are believed to be important in mediating naturally acquired immunity as well as immunity generated by parasite blood stage vaccine candidates [
3]. Moreover, the cytophilic IgG1 and IgG3 antibody isotype subclasses have been reported to be associated with protective responses generated against invasion ligands [
4‐
6], by enabling the activation of complement and antibody-dependent phagocytosis and consequently parasite clearance [
7]. However, it remains unclear which merozoite invasion ligand antigens might be the most important targets of naturally acquired clinical immunity, and whether the importance of such antigens are of regional specificity or globally relevance [
2].
Malaria in the Amazonian region is hypoendemic and characterized by a low transmission [
8]. The malaria infections are most commonly caused by
Plasmodium vivax, but
P. falciparum is still responsible for the major cases of severe malaria, and these infections continue to persist even though control measures are in place [
9]. Previous studies in this region have demonstrated that clinical immunity to malaria is manifested by the presence of individuals with asymptomatic parasitaemia, which is not infrequent [
8,
10]. Importantly, asymptomatic parasitaemia has major implications for public health, particularly in maintaining transmission including the introduction or reintroduction of parasites in endemic regions that stopped having malaria. Understanding the immune mechanisms by which infected humans control parasitaemia in the absence of symptoms has important implications for developing anti-malarial vaccine strategies [
10]. In individuals living in areas of intense
P. falciparum transmission clinical immunity to symptomatic malaria is thought to be acquired only after repeated exposure [
2]. In contrast, studies have demonstrated in Indonesia and in Amazonia that acquisition of clinical immunity can be rapid (within two years), especially in adults, and may require few infections [
9‐
15]. This observation clearly indicates that non-sterilizing but effective clinical anti-malarial immunity develops in low transmission regions [
9].
Given the epidemiological observations indicating clinical immunity against P. falciparum, this study aimed to test the hypothesis that antibody responses against P. falciparum invasion ligands belonging to both EBL and PfRh protein families might differ between symptomatic (Sym) and asymptomatic (Asy) individuals living in the low-transmission region of the Peruvian Amazon, and hence potentially contributing to explaining mechanisms of clinical immunity observed in the Asy individuals. Recombinant P. falciparum proteins corresponding to the known EBL and PfRh invasion ligands were used to determine the total IgG and IgG isotype-specific antibody responses in both study groups.
Discussion
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 [
1]). 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 [
20]. 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 MSP1
19 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 [
19].
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 [
4]. 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 [
27], and responses against PfRh2b was also evaluated in subjects from Senegal and Tanzania [
26]. 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 MSP1
19, MSP-2 and AMA-1 [
4], 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 [
4]. 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 [
28], and EBA-175 and PfRh5 [
25]. 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 [
27].
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%) [
4]. 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 [
4]. Seroprevalence for PfRh4 in the Peruvian infected individuals was lower than observed in adults from Kenya (50%
vs. 70%) [
27], 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 F
c 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 [
27], only few individuals from Peru had antibodies against PfRh5 (11%), which is comparable to what was reported in Kenya (16%) [
27]. 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.
Acknowledgments
We thank Marilis Rodriguez, Blood-Borne Parasites, New York Blood Center, for the production of the recombinant ligands we used in this study. We thank Deepak Gaur [International Centre for Genetic Engineering and Biotechnology (ICGEB) New Delhi, India] and Louis Miller (NIH, USA) for kindly providing recombinant protein PfRh4. We thank David Narum and Louis Miller from (NIH, USA) for kindly providing recombinant proteins EBA-175 and EBA-140. The following reagent was obtained through the MR4 as part of the BEI Resources Repository, NIAID, NIH: Plasmodium falciparum yPfMSP1-19(Q-TSR)VK1, MRA-55, deposited by DC Kaslow. The authors are also very grateful to the local health personnel in all sites of this study and the participating patients. We thank Paula Maguina, UC San Diego for scientific and logistical contributions essential to carrying out this study. This work was supported by the Peru/Brazil International Center of Excellence in Malaria Research U19AI089681 (NIH/NIAID, United States Public Health Service, USA), the NIH/Fogarty International Center Global Infectious Diseases Training Grant D43TW007120 (IMTAvH-UCSD), and the New York Blood Center.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KT, DG, JMV and SL participated in the design of the study. EV, KT, VN, JB collected the samples. MLP, NT and CL designed or carried out the ELISA studies. EV, MLP, JMV and SL drafted the manuscript. All authors read and approved the final manuscript.