Characterization of anti-EBA175RIII-V in asymptomatic adults and children living in communities in the Greater Accra Region of Ghana with varying malaria transmission intensities

The asexual stages of Plasmodium falciparum (P. falciparum) are partly responsible for the pathology associated with malaria and subsequently are the focus of malaria treatment regimens as well as the focus of malaria vaccine research. The merozoite is the only extracellular stage of the parasites erythrocytic life-cycle, making merozoite surface antigens promising malaria vaccine candidates. One such candidate is the erythrocyte binding antigen (EBA) 175 (EBA 175, Pf3D7_0731500), PfEBA-175 has been shown to play a key role during the fast cascade of interactions between the parasite and host molecules before the merozoite completely invades the erythrocyte by binding to sialic acid residues on glycophorin A on the red blood cell during merozoite invasion [1]. Of the 6 extracellular domains of EBA175, region 2 (RII), which comprises of two cysteine rich domains F1 and F2 [2] as well as RIII-V, which comprises of the dimorphic region 3 (RIII) as well as the highly conserved regions 4 (RIV) and 5 (RV) [2] have been implicated as vaccine candidate antigens.

Antibodies induced against diverse antigenic components of the erythrocytic parasite are important mediators of anti-disease immunity [3]. Some known functions of antibodies induced against the asexual parasite include preventing merozoites from invading new erythrocytes (inhibition of invasion), preventing cytoadherence of infected erythrocytes to endothelial cells as well as interfering with the normal function of monocytes and macrophages [4, 5]. Targeting the merozoite before they invade erythrocytes can serve as a means to truncate the infection. The ability of antibodies against the merozoite to prevent erythrocyte invasion has been demonstrated through in vivo human passive transfer assays [6, 7] and in vitro erythrocyte invasion inhibition assays [8] several years ago. Antibodies specific for EBA175 RIII-V have been shown to be associated with protection from malaria in symptomatic cases [9]. Also, Healer et al., [10] in immunization studies have shown that antibodies induced by a recombinant RIII-V inhibit merozoites invasion.

Repeated exposure to malaria parasites has been suggested as a necessary requirement for the maintenance of anti-parasite immunity as it has been demonstrated in a number of studies that antibodies against merozoite antigens are relatively short-lived in the absence of a new infections [11‐14]. This has been confirmed in some community studies where people with current infections had higher merozoite antibody levels than those without [15‐17]. However, a few studies including one by Wipasa et al., noticed that both antibody and memory B cell responses to malaria antigens remained steady over long periods in the absence of an infection [18]. Some studies have suggested that merozoite antibody levels show a direct correlation with malaria transmission intensity in malaria endemic settings and are higher in high transmission settings [19, 20].

The cytophilic immunoglobulins, IgG1 and IgG3 have been associated with parasite repression directly, or opsonization indirectly [21]. IgG1 and IgG3 antibodies to merozoite antigens generally have short half-lives [12]. The half-lives of IgG subclass responses against EBA175 are generally short lived, however, the half-lives of IgG1 and IgG3 have been noted to be longer lived and more prevalent than those of IgG2 and IgG4 [21, 22]. IgG1 and IgG3 responses against EBA175 have also been associated with lower parasitaemia in a high transmission setting [21] as well as a seasonal transmission setting of Papua New Guinea [23].

The process of antibody selection that occurs during humoral immune response maturation, results in the production of antibodies with increased avidity [24, 25]. Antibody properties, including high avidity, have been suggested to key determinants of protective immunity against malaria [26‐28]. High Avidity to whole schizont extract as well as to a number of specific P. falciparum antigens, have been shown to correlate with protection from malaria [29‐31]. The avidity of antibodies against MSP1 has been observed to increase after a recent P. falciparum infection [29], however some reports have implicated reduced antibody affinity maturation and antibody avidity to a recent malaria infection [32] and excessive stimulation of B cells in high parasite prevalence settings [33]. The avidity of antibodies to P. falciparum antigens has been found to be lower in areas of high malaria transmission intensity than in areas with lower transmission [34].

This study sought to determine differences in the characteristics of antibody responses to EBA175RIII-V in adults and children living in high and low malaria parasite prevalence settings.

The age range for the 161 study participants from Obom was 6 to 70 years, while that of the 169 participants from Asutsuare was 2 and 75 years (Table 1). Asymptomatic P. falciparum parasite carriage, as determined by light microscopy, was higher in Obom (57 of the 160 participants or 35.6%) compared to Asutsuare (6 of the 169 participants, representing or 3.5%). There was thus a 10-fold difference in parasite carriage between the two study communities (Table 1). Bed net ownership was low in both communities, only 11 and 2 participants in Obom and Asutsuare respectively claimed to own bed nets.

Rabbit serum containing polyclonal antibodies against Region III-V of EBA175 have been found to directly inhibit P. falciparum merozoite invasion [10]. Naturally induced antibodies against Region III-V of EBA175 have also been suggested to be indicative of strong protection from symptomatic malaria [45], however the role antibodies against EBA175RIII-V play in asymptomatic malaria has not been fully evaluated. Data from the assessment of the magnitude and avidity of antibody responses to this antigen across different age groups will potentially be relevant for the interpretation of natural and vaccine induced immune responses to this antigen.

In order to determine possible differences in natural antibody responses to Region III-V of EBA175, a cross sectional survey was carried out in Obom, which has been reported to be a high parasite prevalence community [46] and in Asutsuare, where parasite prevalence and malaria transmission intensity is known to be very low [35]. The study enrolled a range of children and adults to determine possible differences in age related antibody responses. This study confirmed the existence of very low P. falciparum parasite prevalence in Asutsuare (Table 1) as has previously been reported [35]. The number of asymptomatic individuals in Asutsuare however may be higher than the 3.5% if Plasmodium parasites were detected by molecular methods. Especially as a higher number of submicroscopic parasite infections are detected in asymptomatic individuals due to the increased sensitivity of the detection technique [47]. Bed net ownership was generally low in both communities. Ownership in Obom was higher than in Asutsuare, most likely because residents of Asutsuare encounter very low frequencies of malaria that they no longer think it is important to implement personal malaria control interventions relative to inhabitants of Obom, where malaria transmission is high.

Although only 3.5% of the participants from Asutsuare had microscopic densities of parasites, almost 60% of them were seropositive for anti-EBA175RIII-V antibodies. Similarly, although only about 35% of the participants from Obom were identified by microscopy as harboring P. falciparum parasites, over 80% had IgG antibodies against EBA175RIII-V. These collectively suggest the possible presence of parasites at densities below the detection limit of microscopy in some of the participants or that some of the participants had just recently cleared a P. falciparum infection, as antibodies against EBA175 have been suggested to be relatively short-lived [21].

The significantly higher IgG responses recorded in Obom than in Asutsuare (Fig. 2a) is indicative of the possible requirement of an active infection to induce antibodies against EBA175RIII-V as has been previously suggested, especially as these antibodies are relatively short-lived [21]. IgG subclass responses in both sites were predominantly cytophilic (IgG1 and IgG3) (Fig. 3), which supports a number of previous reports [9, 21, 48, 49]. However responses to IgG2 and IgG4 existed, although at much lower quantities and has been suggested to be due to the much shorter half-lives of IgG2 and IgG4 compared to IgG1 and IgG3 [21]. IgG subclass antibody levels were significantly higher in Obom than in Asutsuare, which was not surprising as total IgG levels were also significantly higher in Obom than in Asutsuare.

In Obom, the high transmission setting, IgG, IgG1 and IgG3 responses to EBA175RIII-V positively correlated with age (Figs. 2a, 3a&c). This supports data from a previous study conducted in a high transmission setting in Nigeria [21] and that of another study in highly asymptomatic children from a moderately seasonal setting Papua New Guinea [9]. High IgG1 and IgG3 responses for other merozoite antigens such as MSP1 [50] and MSP2 [51] have also been documented. A previous study did not find any correlation between IgG2 and IgG4 responses against EBA175 and age [21], however in this study, IgG2 responses correlated positively (Fig. 3b) with age in responses measured in the high transmission setting, Obom. The lack of age associated antibody responses in Asutsuare (Figs. 2a & 3) may be due to the very low prevalence of parasites observed in the community (Table 1) as parasite exposure has been found to be necessary for mounting immune responses against malaria antigens.

The relative antibody avidities measured in the participants from Asutsuare, the low transmission setting were significantly higher than those measured in Obom (Fig. 2b). A similar observation has been reported for antibody responses to this same antigen conducted in children living in the same high transmission setting and a different low transmission setting in Ghana [42] as well as for antibody responses to a different merozoite antigen, MSP1₁₉ [34] and could be due to the presence of fewer and less diverse parasite clones circulating in low transmission zones [52]. Although high parasite diversity and frequency of infection is anticipated in high transmission settings, the diversity of RIII-V of EBA175 was recently suggested to be relatively similar in parasites circulating in Obom (the high transmission setting) and Abura [42], a community with low parasite transmission intensity, similar to Asutsuare. Generally, an increase in exposure to diverse parasites strains/isolates will result in heterologous exposure, which could lead to reduced affinity maturation and the production of antibodies with lower avidities as measured in antibody responses in Obom. A recent report on the avidity of naturally induced antibodies against EBA175RIII-V in children living in southern Ghana similarly reported reduced avidity of antibodies from children living in the high transmission setting compared to the low transmission setting [42].

The likely short longevity of naturally induced antibodies against EBA175RIII-V is contrary to the dynamics of antibodies against MSP1–₁₉, which have been found to persist for several years after the clearance of P. falciparum parasites [53, 54] and thus may be a suitable candidate to use as a serological marker to monitor changes in malaria transmission intensity.