The significantly higher total IgG prevalence and level detected in both study sites of the present study for GMZ2 than either of its two component domains indicates the enhanced antigenicity of GMZ2. The findings suggest that people in endemic areas have high antibody levels to the GLURP and MSP3 components of GMZ2 antigen, which is part of the GMZ2 vaccine candidate, in the absence of microscopically detectable infection. The higher total IgG and subclass levels against GLURP-R0 than MSP3 in both study sites indicates the better antigenicity of GLURP-R0 compared to MSP3. The findings are in line with data from pre-clinical and clinical evaluations of the GMZ2 vaccine candidate. Pre-clinical studies in mice  and monkeys  documented higher total IgG prevalence and level against GMZ2 compared to GLURP-R0 and MSP3. The same studies showed that total IgG prevalence and level were significantly higher for GLURP-R0 than MSP3.
Initial clinical trials in malaria-naïve  and naturally exposed individuals  to assess safety and immunogenicity of GMZ2 have shown stronger anti-GMZ2 responses than the responses to its component antigens, with GLURP-R0 being relatively more immunogenic than MSP3. Studies that examined anti-GLURP-R0 and -MSP3 natural responses separately had recorded varying total IgG prevalence for the two antigens. In one such report, anti-MSP3 total IgG prevalence was higher than that for GLURP-R0 in a hyper-endemic area in Myanmar . Also, a study from Venezuelan Amazon showed higher total IgG prevalence against GLURP-R0 than MSP3 .
The inter-individual variability in antibody response to a specific antigen observed among the study participants was also reported from other malaria endemic areas . In the present study an individual who showed high response for MSP3 did not have necessarily high response for GLURP-R0 suggesting that no predisposing host factors are responsible for induction of high antibodies; it may be due to different antigen characteristics.
Currently, knowledge on the durability of antibody responses to P. falciparum vaccine antigens and how they are maintained remains poorly defined. In this study, in the context of heavily reduced transmission, strong immune responses were recorded for all of the antigens in most study participants. This suggests the stability of malaria immunity. The data provided additional evidence in support of earlier observations that antibodies can last for years in the absence of significant boost infections [18, 19].
After one year, antibody level to GMZ2 remained high in the first clinical trial in malaria-naïve adults although it was lower compared to the level on day 84 . In the absence of boost infection, anti-GMZ2 antibodies in immunized adults and children lasted six to 12 months  and then started waning. When natural MSP3- and GLURP-R0-specific IgG subclass levels were considered, no major change in the levels of specific IgG1 against the two antigens before and after five years was reported . The report however indicated that the protection status of some individuals was associated with IgG3 to GLURP-R0 at one time point and IgG3 to MSP3 after five years suggesting changes between individuals in their response to the antigens and the dynamics (or complementing effect) of responses to these antigens over time.
The present study suggests that both antigens, GLURP-R0 and MSP3, are important for the induction of protective antibodies against malaria from natural infections providing additional rational for combining the two antigens in a hybrid vaccine formulation. Thus GMZ2 has high potential as a candidate vaccine compared to either GLURP-R0 or MSP3 candidate vaccine antigens. However, it remains unclear whether the GMZ2 vaccines would be effective in the heterogeneous epidemiological settings of Ethiopia as there is evidence that vaccine-induced antibodies may not be as potent as those naturally acquired . But the induction of functional antibodies, cytophilic subclasses, following natural exposure signals that the GMZ2 vaccine would likely be effective at least through boosting the pre-existing antibodies. The role of challenge infections in boosting vaccine stimulated-antibodies for parasitaemia control was noticed in pre-clinical study in monkeys . Furthermore, there is evidence from Phase I clinical trial in naturally exposed adults that the GMZ2 vaccine boosted pre-existing natural immunity . In some studies anti-GLURP antibodies were associated with protection , in others not . The association of antibody levels to MSP3 with protection has also been described .
The overall dominance of cytophilic IgG subclasses over the noncytophilic shows the functionality of GLURP-R0-specific IgG subclasses explaining the ‘better protected’ status of Shewa Robit participants who were asymptomatic and microscopy negative. The dominance of MSP3-specific IgG1 and IgG3 isotypes in Boditi sera goes in agreement with studies from other settings [21, 24]. In both study sites, IgG subclass antibody level to GLURP-R0 was significantly higher than that to MSP3 for all corresponding subclasses characterized in most individuals indicating the increased relative antigenicity and protective potential of GLURP-R0 compared to MSP3.
Factors that control the preferential induction of polarized subclass responses are not well understood in vivo. Cytokines and B cell activators are involved in inducing Ig class switching in a model system . It was suggested that some bacterial antigens are known to induce IgG2 expression  and allergens or helminths stimulate IgG4 and IgE induction . Further, polysaccharides such as dextran or levan are known to contribute towards IgG2 elevation  especially in response to other parasitic diseases like schistosomiasis . Malaria-specific subclass pattern and relative proportions may differ from population to population depending on various factors such as level of malaria transmission, human genetics and age, and type of antigen epitope involved .
The age-related increase of antibody levels observed for the antigens in Shewa Robit is in agreement with other studies [22, 31, 32]. Although not significant, antibody levels to the antigens in the study were higher in smear-negative group for P. falciparum infection in Boditi sera. This suggests that smear-negative individuals were relatively protected due to their high antibody levels. The prevalence of IgG to MSP2 serogroup antigens in a seasonal transmission area correlated positively with presence of parasitaemia at the time of sampling . Such reports may suggest the influence of recent boost infections that resulted in relatively elevated antibody responses in one hand and the lower protective efficacy of the antibodies in parasite control on the other. Nonetheless, in another study, no relationship was observed between antibody responses to GLURP-R0 and GLURP-R2 and presence or absence of P. falciparum in blood and the level of parasitaemia .
The significantly lower falciparum-specific antibody level noticed in Boditi than Shewa Robit may be due to genetic differences between the two population groups, parasite strain variation in the localities, or level of malaria transmission. Boditi is a highland fringe at an altitude of 2,059 m above sea level (masl). The seasonality of malaria in highlands is more pronounced with characteristic lower immunity compared to warm hotbeds. A classical serological survey conducted in Ethiopian highlands indicated that high P. falciparum-specific responses were obtained for people living at elevations of 1,828 masl and less compared to those at 1,920 or above . Treatment-seeking behaviour and access to prompt effective treatment schemes, possession and appropriate usage of insecticide-treated bed net (ITN), vicinity to mosquito breeding sites, regularity in indoor residual spray (IRS); and the overall variations of control methods may account for heterogeneity in exposure to infective mosquito bites, making the level of boost infections more infrequent thereby possibly contributing towards relatively lower antibody levels in Boditi study participants. In Shewa Robit (at an elevation of 1,380 masl) an irrigated tobacco farmland created ideal pools of mosquito breeding sites. This can be a potential source of submicroscopic, subpatent infections that maintained higher immunity compared to Boditi. The Shewa Robit study participants who had stronger functional antibodies were asymptomatic and had no microscopically detectable infection suggesting that they were relatively better protected compared to the Boditi.