Background
Malaria is one of the largest causes of morbidity and mortality in tropical and subtropical regions of the world [
1].
Plasmodium falciparum had an incidence of 24.69% (12,878 cases) in 2017 in the Peruvian Amazon [
2], and it is considered one the most lethal specie of
Plasmodium that affect humans due to its drug resistance and ability to potentially cause severe malaria. Humoral immunity and IgG antibodies play a critical role in combating infection through their ability to reduce parasitaemia and clinical symptoms [
3‐
11]. Cytophilic subclasses of IgG (IgG1 and IgG3) have been considered the most important antibodies in the development of immunity to malaria, as these subclasses are capable of mediating the activation of leukocytes via their binding to FcγRI and FcγRIII. Together, the predominance of these subclasses is associated with lower risks of malaria-related complications in malaria-endemic areas [
12‐
18].
However, the development of immunity to malaria depends on the balance between cytophilic (IgG1 and IgG3) and non-cytophilic IgG antibodies (IgG4), which interfere with the binding of Fcγ receptors with cytophilic antibodies, complicating the immune response [
5,
19,
20]. Noticeably, in the presence of the H131 variant in the FcgRIIA receptor, IgG2 has a cytophilic role, whereas the R131 variant does not bind IgG2 [
21‐
23].
Excreted-secreted antigens are fundamental pieces in the host-parasite interaction and are utilized by various parasites to modulate the immune response of the host [
24‐
26]. These antigens have been recognized for their use in serological diagnostics [
27‐
29] and immunizations [
30,
31] in many different parasitic species. The excreted-secreted antigens of
P. falciparum (
Pf-ESAs) have been shown to be involved in the process of erythrocyte invasion, the activation of antibodies and lymphocytes, and in complications of infection (reviewed in [
24]). Notably, their importance in immunological development in Madagascar was evaluated by Chumpitazi et al. [
32], who reported that IgG1 responses specific for
Pf-ESAs were associated with clinical protection, in contrast to
Pf-ESA-specific IgG4 responses, which had the opposite effect.
In Peru, the transmission and incidence of malaria is much different than what is observed in Africa, and the Peruvian Amazon is a zone of low malaria transmission with a low prevalence of
P. falciparum. Nevertheless, transmission throughout the region remains persistent due to a large number of asymptomatic infections [
33‐
35]. Such subclinical cases are the product of the development of immunity to repeated infection, with Clark et al. [
36] estimating that one
P. falciparum infection can elicit the production of enough IgG antibodies to persist for over 5 months. However, it remains to be seen whether the responses of the individual IgG subclasses may be different.
Efforts toward malaria elimination and eradication goals have changed global malaria epidemiology, resulting in a substantial decline in global malaria morbidity and mortality [
1]. This study analysed the role of IgG subclasses in controlling malaria disease to help advance malaria vaccine development, which should target geographical areas of low transmission. Therefore, this study characterized the subclasses’ response to
P. falciparum excreted-secreted antigens in symptomatic and asymptomatic carriers within a community in a low-transmission area of the Peruvian Amazon.
Discussion
Understanding the nature of immunity to malaria is essential for controlling and eradicating the disease. This is especially true in areas of low transmission, such as the Peruvian Amazon, where despite the low prevalence of
P. falciparum, the ability to develop protective immunity has been demonstrated [
33‐
36]. Indeed, the high incidence of asymptomatic cases and lack of complications associated with symptomatic infections help facilitate sustained transmission of malaria in the region, thereby complicating control efforts.
These results indicate that the levels of whole IgG are higher in asymptomatic carriers (P < 0.05), similar to results from other studies [
3‐
6,
9,
10,
20,
44‐
48], which showed that higher levels of IgG antibodies were associated with protection against the complications of malaria. Additionally, these carriers have a stronger correlation between IgG1 and IgG3 (Table
3), a common association in communities of low malaria transmission [
16].
Both carriers (symptomatic and asymptomatic) show a predominance of IgG3 and IgG1 responses (Fig.
1) in comparison to other studies that used exoantigens, such as Chumpitazi et al. [
32] and Ferreira et al. [
49], who observed a predominance of IgG2 in their populations; it is important to clarify that these studies only analysed the whole population without discrimination of symptomatology. These results reflect the heterogeneity of the antibody response in malaria infections, as described previously [
15,
49‐
51].
Although asymptomatic antibodies are characterized by higher titres of cytophilic antibodies [
5,
10,
20,
45,
46], differences in the titres of cytophilic antibodies between both carriers were not observed (Fig.
2), in contrast to reports by Scopel et al. [
52] and Braga et al. [
45] for IgG3, which was present at higher levels in both carriers (661.6 and 636.2) (Table
2). Medeiros et al. [
6] did not observe differences in the responses to the recombinant antigens MSP1-19kD and MSP3-3D7. A stronger correlation was observed between IgG2 and IgG3 (Table
3), which is especially notable because these subclasses had a negative correlation with parasitaemia only in symptomatic carriers (Fig.
3). Although IgG2 was not present at high levels in this study (Table
2 and Fig.
1), it can provide protection, as observed in other studies [
22,
32,
49,
53]. However, to elucidate its activity, the authors suggest evaluating the Zungarococha population for an H131 polymorphism that may be related to the protective activities of IgG2.
These results can explain why symptomatic carriers in the Zungarococha community do not show severe malaria complications (neurological symptoms, severe anemia, or respiratory distress), as observed by Branch et al. [
33] and did not have cases of hyperparasitemia (> 100,000 parasites/μl), which is consistent with the observed median parasitaemia of 2735 parasites/µl (Table
1).
As Medeiros et al. [
6] have suggested, it can be assumed that symptomatic carriers may be in the process of acquiring clinical immunity to malaria and will become asymptomatic in the future. Part of this process of acquiring clinical protection requires a balance between cytophilic and non-cytophilic responses. However, this process is not at all complete in symptomatic carriers, as the results reflect higher antibody levels and seroprevalence of IgG4 in symptomatic carriers. The binding of IgG4 to an antigen blocks the recognition of the antigen by cytophilic IgG and, therefore, the activation of effector cells through Fc receptors [
4,
22,
51,
54‐
56]. This imbalance in the major mechanism controlling malaria symptoms is reflected in the fever, chills, nausea, and body aches observed in Zungarococha settlers [
33].
The principal limitation of this study was the number of samples (n = 57), limiting factors in the measure of co-variables related to nutritional status (Table
1) and lack of specific demographic and clinical information to increase the number of samples (Additional file
2).
But in these circumstances, a high cytophilic antibody response was observed in this community, and symptomatic carriers were characterized by the IgG4 response. Further studies utilizing larger sample sizes will be required to confirm these results, especially the role of IgG4 in clinical symptoms, elucidating its role as a marker of susceptibility that helps to identify clinically relevant patients in Zungarococha. In addition, such studies could clarify the pro-inflammatory response in these patients and help to elucidate the factors involved in the regulation of the immune response in both asymptomatic and symptomatic carriers. Such efforts would ideally evaluate the IgG subclass responses during both the seasons of highest and lowest transmission to determine the temporal effects on antibody titers and to identify the duration of each immune response.
Notably, the
Pf-ESAs elicited elevated seropositivity for two cytophilic antibodies, resulting in a response similar to those antigens used by Garraud et al. [
57]. This serves to reinforce their importance in the development of protective immunity as well as their possible role in vaccine development. Furthermore, as
P. falciparum shows a high genetic diversity in the study area [
58], wild strains may present different antigens, as was described in Anders et al. [
59], so that is important analyse antibodies responses to
Pf-ESAs obtained from wild strains isolated in Zungarococha community. Also, serological techniques, such as Western blots would allow us to determine the protein profiles of
Pf-ESAs for each antibody subclass, similar to studies by Olesen et al. [
20] and Ouevray et al. [
60]. This could help us to determine the differences in immunity between asymptomatic and symptomatic individuals and to identify possible markers, thereby improving the understanding of the changes associated with the development of immunological protection against clinical disease.
Conclusion
The Peruvian Amazon requires more research to help understand the immune response to malaria in populations with a high prevalence of asymptomatic and low-density infections. This is the first study that characterizes the IgG subclass response in the Peruvian Amazon, and these results show that populations from regions with low malaria transmission can develop an appropriate cytophilic response by IgG subclass antibodies, while symptomatic carriers require non-cytophilic responses to develop protective immunity. Additionally, these findings can contribute to a better understanding of immunity in populations exposed to malaria transmission that could be beneficial in the development and testing of Pf-ESA-based vaccines.
Further studies are needed to evaluate whether the IgG subclass responses are better markers of protective immunity than total IgG responses and to understand their role in protection, especially against other antigens that are considered to have a potential use in vaccination.
Authors’ contributions
VPC and RSL conceived the study. MEVC and RSL performed the in vitro cultivation and serological test. AVC and RSL analysed the results. AVC, VPC and RSL wrote the first draft of the paper. SD, JM and HS contributed to the writing of the paper. All authors read and approved the final manuscript.