Background
Malaria in pregnancy (MiP) causes significant maternal, fetal, and neonatal mortality and is a major health issue globally [
1]. Parasite accumulation in the placenta is a key feature of MiP following infection with
Plasmodium falciparum [
2,
3] but is not prominent with other human infecting
Plasmodium species. This largely results from the selective binding of pRBCs to chondroitin sulfate A (CSA) expressed on syncytiotrophoblasts [
4,
5], and other binding interactions may play secondary roles [
6,
7]. This is mediated by a
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variant surface antigen, VAR2CSA, encoded by the
var multigene family [
5,
8]. The risk of MiP is greatest in primigravid women and generally decreases with successive pregnancies in malaria endemic areas due in part to the acquisition of protective antibodies directed against placental-binding
P. falciparum infected red blood cells (pRBCs) [
9]. Antibodies to placental-binding pRBCs and VAR2CSA have been associated with improved outcomes in some studies, although associations have not been entirely consistent [
10]. How antibodies to pRBCs and VAR2CSA function in protective immunity to MiP and improved birth outcomes is not fully understood [
10], and these key knowledge gaps are restricting advancement of MiP vaccines. Existing data shows that antibodies may act via inhibition of placental adhesion of pRBCs [
11] and promoting phagocytosis of pRBCs [
12]. However, a recent systematic review found that data on associations between these antibody functions and protection from the consequences of MiP are limited and variable [
10] and data suggest these mechanisms may not fully explain immunity to MiP. VAR2CSA is a leading vaccine candidate for MiP with two VAR2CSA-based vaccines having completed phase I trials [
13,
14], which highlights the importance of a strong understanding of immunity to inform further vaccine design and development for MiP.
Antibodies to placental-binding pRBCs and VAR2CSA are dominated by IgG1 and IgG3 subclasses [
15,
16], which have the potential to fix and activate human complement against infecting pathogens [
17]. Complement activation via the classical pathway is initiated by binding of complement C1q to antigen-antibody complexes. This leads to a cascade of activation of other complement components including C3 and culminates in the formation of the C5-9 membrane attack complex (MAC) [
18]. Complement fixation can mediate protective functions through several mechanisms: (i) MAC formation can lead to cell lysis or death, (ii) complement components C3 and C5 can promote phagocytosis by monocytes and neutrophils through interactions with complement receptors expressed on those cells, and (iii) binding of complement components to a pathogen surface may also have direct inhibitory or neutralizing activity [
18‐
20]. Antibody-mediated complement fixation against
P. falciparum has been implicated in immunity in non-pregnant individuals, targeting merozoites [
21,
22], sporozoites [
23,
24], and gametocytes [
25]. While there is a potential role for antibody-mediated complement fixation in immunity against MiP, this has not been established and red blood cells (RBCs) are known to express complement regulatory proteins that can inhibit complement activation and confer resistance to lysis [
26]. Older studies have reported antibody-mediated complement fixation on the surface of mature pigmented trophozoite stage pRBCs, but these did not assess different functional activities or associations with protection [
26,
27]. Further, MiP has also been associated with excessive complement activation and production of inflammatory products mediating adverse outcomes [
28]. Therefore, the role of complement fixation in preventing or reducing placental infection remains unclear, and there has been little investigation of its potential role in immunity.
We hypothesized that acquired antibodies against VAR2CSA in some pregnant women may fix complement on placental binding pRBCs, which may contribute to the control or prevention of placental parasitemia. Using a prospective longitudinal cohort study of malaria-exposed pregnant women from PNG, we investigated antibody-complement interactions in immunity to MiP. We investigated antigenic targets of complement fixation on the surface of pRBCs using genetically modified pRBCs and recombinant VAR2CSA proteins and evaluated functional mechanisms of complement-fixing antibodies. We examined whether antibody-mediated complement fixation on pRBCs is associated with reduced risk of placental infection.
Discussion
Antibodies are thought to mediate protection against placental infection by P. falciparum. However, the mechanisms mediating immunity are not fully understood. Here, we reveal a new mechanism in immunity to malaria in pregnancy, which may contribute to reducing the risk of placental parasitemia. We show that acquired antibodies among pregnant women can mediate complement fixation on placental-binding P. falciparum pRBCs. Using genetically modified P. falciparum pRBCs and recombinant antigens, we found that antibody-mediated complement-fixation predominantly targeted PfEMP1 (VAR2CSA) expressed on the surface of pRBCs. Importantly, higher complement-fixing antibodies were associated with a reduced risk of placental parasitemia and resulted in enhanced inhibition of pRBC binding to CSA suggesting this mechanism contributes to immunity to MiP.
We demonstrated the ability of acquired antibodies to fix and activate complement using several approaches. Antibodies could fix C1q, the first step in the classical pathway and C3, indicating complement activation. This was demonstrated by labeling complement components on pRBCs by flow cytometry, and by Western blotting of pRBC protein extracts, as well as using recombinant VAR2CSA domains. Complement fixation among antibodies from malaria-exposed pregnant women was significantly higher than antibodies from non-exposed donors and correlated with IgG reactivity. Furthermore, complement-fixing antibodies were generally higher among multigravid than primigravid women, consistent with the reported acquisition of immunity to MiP. Consistent with data from Africa [
15,
16], IgG1 (80% seropositivity) and IgG3 (37% seropositivity) dominated responses in PNG women and moderately correlated with complement-fixing antibodies. We observed some complement C3 fixation when using malaria non-exposed donor antibodies or non-opsonized pRBCs, suggesting some activation via the antibody-independent alternate pathway; however, complement fixation was always much higher in the presence of antibodies from pregnant women supporting a greater role for the classical pathway. Significant polymorphisms occur in VAR2CSA, which can impact on binding of acquired IgG [
37,
48,
49]. Previously we established that the CS2 isolate used in this study is well recognized by malaria-exposed pregnant women in PNG, and IgG reactivity to CS2 pRBCs strongly correlated with IgG reactivity to a placental-binding clinical isolate from PNG [
37,
50].
Interestingly, there was no clear evidence of enhanced MAC formation on pRBCs by antibodies, even though antibodies promoted C1q and C3 fixation. Consistent with this, complement did not result in pRBC lysis or inhibition of growth. Complement regulatory proteins expressed on RBCs (e.g., CD59) likely prevent effective MAC formation, as suggested by older studies [
26]. Our findings highlight the complexities of adaptive humoral immunity. While host regulatory mechanisms designed to protect RBCs may prevent MAC formation and lysis of pRBCs, antibody-complement interactions can still mediate effects through C1q and C3 fixation in acquired immunity targeting pRBCs.
Two earlier studies showed evidence of complement fixation by antibodies among non-pregnant individuals on pRBCs with known binding to CD36 and ICAM-1, which are endothelial receptors [
26,
27]. However, the acquisition and targets of these antibodies were not assessed, nor were associations with protective immunity. In contrast to our findings, one recent study evaluated purified IgG from a pool of plasma samples from malaria-exposed pregnant Ghanaian women and a VAR2CSA monoclonal antibody [
51]. Complement fixation was detected on recombinant VAR2CSA, but not on the surface of pRBCs. However, the acquisition and functions of antibodies, or associations with clinical features or outcomes, were not assessed. The detection of limited complement fixation on pRBCs in that study, compared to our results, may reflect differences in the sensitivity of different assays and the reagents used, and in that study, the authors used 1% fresh serum as a source of complement (we used 25% serum in complement-fixation assays with pRBCs). Additionally, using a pool of samples would contribute to a reduced ability to detect complement-fixing antibodies if the prevalence of such antibodies is low in the population.
Importantly, complement fixing antibodies at enrolment were prospectively associated with a reduced risk of placental parasitaemia at delivery, suggesting this immune mechanism may contribute to reducing parasitemia. Our further exploratory analysis of associations with active and chronic infection subgroups (defined by placental histology) found similar associations. This protective association was only evident among women with
P. falciparum infection at baseline, suggesting that women who mount higher complement-fixing antibodies when challenged with infection more effectively control and clear infection. Among women without infection at enrolment, there was no significant association between complement fixing antibodies to pRBCs and risk of placental infection. Where malaria transmission is heterogenous, only a proportion of the population are exposed to infection. One approach to address this is to stratify the analysis of associations with protection by infection status at baseline [
52]. Other studies investigating associations between antibodies and protection against malaria in children [
53,
54] or pregnant women [
55] have used this approach, revealing protective associations only in the infected group who have documented exposure to malaria. A potential limitation of our study is that women commenced in the study in mid-pregnancy (most were second trimester), even though we recruited pregnant women at their first antenatal clinic visit. Presenting to first antenatal clinic in the second trimester is commonly observed in PNG [
56,
57]. This means that we cannot fully account for malaria exposure history in their pregnancy. A further limitation is that the placental tissue was available only for 77% women. However, we did not observe major difference in demographic or clinical features of women who did or did not have placental tissue collected [
29]. Ideally, a future study would enroll women early in pregnancy with frequent follow-up during pregnancy and employ a larger sample size to further investigate associations we observed here, including the relative roles of different antibodies and the influence of gravidity.
We found that complement significantly enhanced inhibition of pRBC adhesion to CSA by acquired antibodies, suggesting a potential mechanism mediating protective effects of complement-fixing antibodies. Additionally, it is well established that complement enhances phagocytosis through interactions with complement receptors expressed on monocytes and neutrophils, particularly via C3b [
19,
58]. Therefore, higher complement fixation by antibodies is likely to contribute to enhanced clearance of pRBCs. Anti-VAR2CSA antibodies have been proposed to protect against MiP possibly through inhibition of
P. falciparum binding in the placenta along with enhanced phagocytosis [
11,
15,
59,
60]. Previous studies, however, have largely examined these functional mechanisms in the absence of complement. The initial step of the classical pathway of complement activation involving C1q appeared essential and sufficient in mediating inhibition enhancement. However, it should be noted that there is evidence that C1q can bind directly to CSA [
61], emphasizing the need for appropriate controls when assessing this effect. Others have shown complement can enhance inhibition of
P. falciparum merozoite and sporozoite invasion [
21,
23] and phagocytosis by monocytes [
58]. Prior studies of viruses have shown that binding of C1q by antibodies can enhance virus neutralization, without the need for additional complement components [
20,
62]. In contrast to protective roles of antibody-complement interactions, widespread complement activation has also been implicated in MiP pathology. Higher plasma C5a was associated with adverse birth outcomes including low birth weight, fetal growth restriction, and preterm birth [
28]. Excess C5a is thought to mediate pathogenesis via inflammation and skews angiogenic profiles critical for normal placental vascularization and development towards an anti-angiogenic profile that promotes fetal growth restriction [
28]. Therefore, there is a balance between antibody-mediated complement activity on the surface of pRBCs that may be protective, versus widespread systemic complement activation that may be detrimental [
63]. We propose that acquisition of antibodies with the right specificity and properties leads to effective complement-fixing activity on the pRBC surface that reduces placental infection through mechanisms such as pRBC placental binding inhibition and enhanced phagocytic clearance. However, in the absence of protective antibodies, infection goes unchecked leading to high-density placental infections triggering excessive complement activation contributing to poor birth outcomes. Further studies to understand protective versus detrimental responses would be valuable.
While
P. falciparum expresses multiple antigens on the surface of pRBCs that can be targeted by antibodies [
47], we found that complement-fixing antibodies predominantly target PfEMP1. We used genetically modified CS2
P. falciparum in which PfSBP1 has been disrupted to inhibit PfEMP1 surface expression [
64]. That there was still some complement fixation on pRBCs lacking PfEMP1 expression suggests that other surface expressed antigens might be secondary targets of complement-fixing antibodies, and these may include repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) proteins [
65] and warrant future investigation. C3 fixation against CS2 SBP1KO pRBCs might also be the result of complement activation via the antibody-independent alternate pathway. We further demonstrated that VAR2CSA is a target of complement-fixing antibodies using recombinant DBL3 and DBL5 domains, which are prominent targets of acquired antibodies associated with protection from MiP [
40,
66]. It would be valuable to investigate the complement fixing activity of antibodies induced by vaccines based on VAR2CSA in recent clinical trials [
13,
14].
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