Background
According to the World Health Organization, there were an estimated 200 million cases of malaria in 2014 and an estimated 584,000 resulting deaths worldwide [
1]. Among the five species that can cause malaria in humans,
Plasmodium vivax is the most widely distributed, accounting for most of the cases of malaria in South and Southeast Asia, Latin America and Oceania; there are an estimated 2.5 billion people living in areas at risk of transmission of the disease [
1,
2]. Furthermore, although vivax malaria has not been considered life-threatening for a long time, reports of severe cases associated with
P. vivax have been increasingly numerous [
3].
The blood stage of the
Plasmodium lifecycle is responsible for the pathology associated with malaria. In this stage, merozoites released from schizont-infected erythrocytes invade non-infected erythrocytes, resulting in their destruction and the release of more merozoites into the bloodstream. During this brief extracellular period, these free merozoites are exposed to the host immune system, and proteins that are critical for parasite invasion are, therefore, important targets for the development of malaria vaccines. Merozoite surface proteins (MSPs) are among the most studied, especially MSP-1, which is necessary for merozoite attachment to erythrocytes [
4] and normal parasite development [
5].
The most widely accepted structure of the
P. vivax merozoite surface protein 1 (PvMSP-1) gene indicates that it consists of six polymorphic blocks (blocks 2, 4, 6, 8, 10 and 12) flanked by seven conserved blocks (blocks 1, 3, 5, 7, 9, 11 and 13) [
6]. By analysing the primary structure of PvMSP-1, several seroepidemiological studies have been performed to evaluate its immunogenic potential [
7‐
11]. Although the C-terminal portion of the protein (PvMSP-1
19) is the most immunogenic [
7,
9,
12], a number of studies showed high prevalence of IgG against N-terminal PvMSP-1 in individuals exposed to
P. vivax [
9,
10,
13,
14]. Furthermore, specific IgG3 antibodies against the N-terminal portion of PvMSP-1 have been associated with clinical protection in two riverine communities of the Brazilian Amazon [
9,
11], similar to that observed in
P. falciparum infection, where persistence of antibodies IgG3 against N-terminal of MSP-1 was related with prolongation time without malaria [
15]. In fact, antibodies specific for the asexual blood stage are thought to be involved in clinical protection against malaria vivax. Longitudinal cohort studies have shown correlations between magnitude of antibody responses to
P. vivax merozoite antigens and protection from malaria [
11,
16‐
18]. Due to the inability in maintaining
P. vivax in continuous in vitro culture, it is difficult to define the role of antibodies to this species, but few evidences support that it may be related to inhibition of merozoite invasion [
19,
20]. Furthermore, complement and FcR mediated mechanisms seem to be important in antibody-mediated protection [
21].
The development of an adequate immune response depends on the fine regulation of lymphocyte activation. For this, in general, lymphocytes require two activation signals. The first signal is antigen-specific, whereas the second signal, called co-stimulation, is generated by the interaction between the surface molecules of T cells and those of antigen-presenting cells, including B cells. The interaction between CD28 and its ligands, CD80 and CD86, provides the strongest costimulatory signal for T-cell proliferation, whereas CTLA-4 is a negative regulator that plays a key role in T cell homeostasis and in central tolerance [
22]. Another member of the CD28 family, Inducible co-stimulator (ICOS), is an important immune regulatory molecule that participates in T-cell activation and T-cell dependent B-cell responses [
23,
24]. CD40 is presented on the surface of B-cells and the CD40-CD40L interaction is the major costimulatory signal for B cells to mount a humoral response [
25]. B lymphocyte stimulator (BLYS) is produced mainly by innate immune cells and is needed to provide signals for B cell survival and proliferation [
26]. Considering the importance of these molecules in development of immune response and because there are currently multiple lines of evidence showing that the genes involved in the immune response can influence antibody production during malaria infection [
27‐
34], the authors hypothesised that polymorphisms in the genes of the co-stimulatory molecules CD28, CTLA-4, ICOS, CD86, CD40 and BLYS are involved in the magnitude of the naturally acquired antibody-driven response against N-terminal PvMSP-1 in individuals infected with
P. vivax in the Brazilian Amazon.
Discussion
The main strategies used for malaria control are based on prompt diagnosis and treatment and on vector control. However, new resistant parasite strains arise as new drugs are applied, and vector control is also encountering great challenges due to the growing resistance to insecticides, thus justifying research on the development of a vaccine that is effective against malaria. Characterisation of the naturally acquired immune response in different populations is a useful tool for the identification of molecules that can be targeted by anti-malarial vaccines.
The present study was to evaluate the naturally acquired immune response against the N-terminal portion of PvMSP-1. Although the C-terminal portion (MSP-1
19) is considered to be the most immunogenic region of the protein [
7,
9,
12], there is evidence suggesting that antibodies targeting the N-terminal portion of MSP-1 provide clinical protection during infections with both
P. falciparum [
46,
47] and
P. vivax [
9,
11]. The results of the IgG-mediated humoral immune response showed that ICB2-5 was detected by just over half of the studied individuals (50.6 %). This prevalence of responders is similar to that found in individuals infected with
P. vivax in other places of the Brazilian Amazon [
8,
11]. A small percentage (~10 %) of the individuals who reported having had several previous episodes of malaria exhibited high IgM levels, but no IgG was evidenced, suggesting that class switching from IgM to IgG may be impaired. This observation has been reported in previous studies [
8,
48], and Soares et al. [
8] suggested that this impaired switch from IgM to IgG may be related to deficient CD40/CD40-L interactions. Thus, the authors evaluated whether co-stimulatory molecule gene polymorphisms are involved in the delayed class switch from IgM to IgG. However, no association was observed.
Regarding IgG subclasses, higher levels of IgG1 were found compared to the other IgG subclasses, which is in contrast to previous studies showing a predominance of IgG3 specific for ICB2-5 [
8,
9,
11]. Although there is still no consensus regarding the role of antibody subclasses in clinical protection, it has been suggested that only the cytophilic subclasses, i.e., IgG1 and IgG3, are protective [
49,
50]. Two longitudinal studies performed in the Brazilian Amazon, specifically, one in Portuchuello, near Porto Velho, and the other in a community of Rio Pardo, Amazonas state, have observed that ICB2-5-specific antibodies were associated with clinical protection against malaria caused by
P. vivax and that IgG3 was detected in all asymptomatic individuals, whereas most symptomatic patients exhibited no IgG3 [
9,
11]. If the above results were to be extrapolated to Goianésia do Pará, the site of this study, the fact that a passive collection was performed on patients exhibiting symptoms could explain the low IgG3 levels found. Furthermore, some studies have also shown that IgG2 may be associated with protection. Deloron
et al. [
51] found an association between high IgG2 levels and low risk of acquiring an infection by
P. falciparum. Although the design of the present study does not allow for the association of the prevalence and levels of clinically protective antibodies, higher IgG2 levels were observed in subjects who reported having had fewer cases of malaria.
The development of an immune response against
Plasmodium species is a complex process, and one of the main issues is understanding why individuals differ in their immune responses against the parasite. Thus, the objective of the present study was to investigate the influence of co-stimulatory molecule gene polymorphisms on the production of antibodies specific for an important
P. vivax vaccine candidate antigen. The most important result was that
CD28 rs3116496 was associated with levels of IgG1 specific for ICB2-5. In addition to the important role of CD28 in T cell activation, the binding of this receptor to its ligands CD80 and CD86 on the surface of B cells provides bidirectional signals that appear to be important for IgG production by B cells [
52]. Thus, CD28 may be involved in the immune response against malaria. In fact,
CD28 knockout mice infected with
Plasmodium chabaudi were unable to resolve the infection, maintaining low levels of parasitaemia for weeks after infection [
53,
54]. Furthermore, treatment of wild type mice with monoclonal anti-CD86 antibodies abolished IL-4 production and was significantly associated with reduced levels of
P. chabaudi-specific IgG1 [
55].
In the present study, individuals exhibiting the
T allele for rs3116496 in
CD28 were found to be associated with reduced IgG1 levels. Although the biological functions of this SNP, which is located in the third intron of the gene, are still unknown, it is located near a splice site, at which point mutations can induce abnormal splicing, thus affecting protein expression [
56]. The relationship between rs3116496 in
CD28 and susceptibility to several diseases has already been evaluated, and significant associations have been found in type 1 diabetes [
57], cervical [
58] and breast cancer [
59], and rheumatoid arthritis [
60]. However, the role of this polymorphism in malaria has not yet been assessed, and even if the presence of the
T allele, which is associated with lower levels of ICB2-5-specific IgG1, were implicated in higher susceptibility to vivax malaria, further elucidation would still be necessary.
Although it seems likely that immunity against malaria is affected by several genes, the influence of combined polymorphisms is rarely ever investigated. The present study performed analyses of interactions between the SNP pairs and found that
CD28 rs35593994 and
BLYS rs9514828 together were associated with levels of IgM specific for ICB2-5, especially in individuals with
GA and
TT genotypes, respectively. Specifically, they were associated with a ~180 % increase in IgM levels compared to that of wild type genotype individuals. Although the biological mechanisms underlying this interaction are still unknown, CD28 and BLyS may be involved in the production of memory B cells and antibody isotype class switching [
61,
62]. Both polymorphisms, i.e.,
CD28 rs35593994 and
BLYS rs9514828, are located in the 5′ regulatory region of the gene; thus, differential expression of these genes may change the regulation of the B cells involved in the production of ICB2-5-specific antibodies. Liu et al. [
63] have shown that despite the generation of memory B cells as a response to vaccination with MSP-1
19, the function of these cells was nullified due to a lack of BLyS expression in dendritic cells from mice infected with
P. yoelii.
Although it was observed the influence of these two SNPs in the antibody response to ICB2-5, most of the evaluated polymorphisms did not show any significant differences. In fact, variations in the immune response to malaria can be attributed to several factors, including the environment, previous exposure to malaria and immunogenicity of antigen. Thus, as a complex trait, it is likely that many polymorphisms have small effect in malaria immune response, but large sample size is required to detect it.