Background
Immunity to the pre-erythrocytic stage of
Plasmodium is considered the most promising target for malaria vaccine development. The resulting protection is typically sterile, i.e., it prevents blood-stage infection and, thus, the onset of symptoms and blocks transmission of the parasites to other individuals. Most sporozoites egress from the skin into either the lymphatics or the blood stream after being injected into the skin by the mosquito during a blood meal (reviewed in [
1]). The main target of anti-sporozoite antibodies is the circumsporozoite protein (CSP), which is the most abundantly expressed protein on the surface of the sporozoite. CSP has been the leading vaccine antigen for decades, albeit with variable success depending on the vaccine platform [
2‐
5]. RTS,S/AS01, currently the lead recombinant vaccine candidate against malaria, is based on a pseudoparticle consisting of the hepatitis B surface antigen and a large fragment of the CSP, namely the central repeat region and the C-terminus of the protein. While only a few correlates of protection are known for most of the human vaccines (reviewed in [
6]), it is becoming increasingly apparent that antibodies to the repeat region in RTS,S are associated with protection against malaria [
7]. Whether or not they are only surrogate markers or true correlates of protection remains to be determined, and the mechanisms by which sporozoite-specific antibodies may mediate protection is still not known. There have been significant advancements in the understanding of antibody-mediated immune functions in the last few years. Until recently, the main emphasis was placed on measuring the magnitude of an antigen-specific antibody response. This does not take into account the quality of the humoral response in the form of antibody avidity and isotype, as well as epitope specificity. Functional antibody assays can address the question whether immune complexes bind to cellular receptors and trigger phagocytosis. This process results in the uptake, degradation of antigenic/pathogenic material and subsequent antigen-presentation to adaptive immune cells [
8,
9].
Although it has been shown that anti-CSP repeat region antibodies are necessary for the protection elicited by RTS,S/AS01, subsequent clinical trials have shown that the magnitude of the anti-CSP repeat region antibody response is only weakly associated with protection [
7,
10‐
13]. One explanation for this apparent discrepancy is that the quantity of anti-CSP repeat region antibodies in an antibody response may only serve as a surrogate marker for its functional capacity to neutralize the
Plasmodium parasite. One possible hypothesis is that protection induced by the RTS,S vaccine is mediated by opsonization and phagocytosis. The uptake of opsonized parasites by phagocytic cells can lead to several possible outcomes, including phagocytosis, destruction of the parasite, followed by antigen presentation to T lymphocytes, or phagosomal escape of the parasite, which then resides in the phagocytic cell. The latter would constitute an immune escape mechanism. Although opsonization and phagocytosis have, to date, been poorly characterized in pre-erythrocytic stage immunity, this has been studied previously for blood-stage parasites and found to be associated with natural immunity to clinical malaria [
14,
15], underscoring its potential role in protection for malaria vaccines.
The aim of the present study was to directly measure antibody-mediated opsonic phagocytic activity and identify how the fine specificity of the antibody response, defined as the relative response to individual epitope regions of the CS antigen, is associated with opsonization activity and protection. Towards that end, a highly sensitive, high-throughput assay to measure the phagocytic activity mediated by antibodies was developed and applied to human sera from subjects vaccinated with the RTS,S vaccine. The flow cytometry-based assay uses antigen-coated fluorescent beads to determine the frequency and intensity of phagocytosis by immune cells. This assay represents a significant improvement over a previous attempt to quantify CSP antibody-mediated opsonization due to its increased sensitivity, which allows for the identification of distinct populations of cells based on their phagocytic activity. Furthermore, unlike previous studies that measure opsonization using fluorescently labelled parasites [
15,
16], the method described here can be performed by any laboratory without the need to have access to parasites.
The present study characterizes sera from subjects immunized in a phase 2 study using the standard RTS,S/AS01-vaccination regimen (three doses in four-week intervals). The efficacy of the trial, and the basic immunological evaluation have previously been reported [
10]. The first step in the analysis was to measure the ability of the RTS,S/AS01-induced antibodies to opsonize CSP-coated fluorescent beads and mediate uptake by phagocytic cells. Next, the data was stratified based on the protective status of the individuals following the controlled challenge with infectious mosquitoes. No significant difference in overall phagocytic activity with respect to protection was observed. However, relative phagocytic activity, measured as ‘opsonization index’, was found to be associated with protection—protected subjects, surprisingly, had
lower opsonization efficiency, than non-protected subjects. An in-depth analysis of the fine specificity of RTS,S/AS01-induced antibodies suggested that it was antibodies targeting the C-terminal region of CSP, and not the repeat region, that were associated with phagocytic activity, and that antibody responses from protected individuals in this study were characterized by higher repeat-specific antibody titres and lower opsonization activity. Given the limited sample size in this study (n = 20), further research on the role of phagocytic activity on protection in the RTS,S-induced antibody response is warranted, however, this study demonstrates how the combined analysis of multiple in vitro functional assays can yield insight into the mechanisms that underlie the biological activity of vaccine-induced antibody responses.
Discussion
RTS,S/AS01 has been evaluated in several trials around the world and is on track to become the first licensed malaria vaccine in the world. As is the case for most other vaccines, no definite functional correlate of protection is known for RTS,S/AS01; most of the uncertainty related to other vaccines is associated with the contribution of cellular responses to protection [
6]. In case of RTS,S/AS01-mediated protection, CD4
+ responses and anti-CSP antibodies have both been identified as surrogate markers of protection with antibodies [
7]. This provides a strong rationale for further analysing the biological activity of these vaccine-induced responses.
The present study demonstrates for the first time the relationship between antibody titres to various regions of the CSP, the contribution of epitope specific antibodies to the biological function (i.e., phagocytic activity in this study) and the underlying protective status of the samples. The current study has generated several key findings: (1) ELISA titres to the CSP central repeat region were significantly higher in protected individuals; (2) no differences in the overall ELISA titres to C-terminus or full-length CSP were detected; (3) the assessment of the opsonization titre per se did not yield a statistically significant difference between protected and non-protected individuals; (4) calculation of the opsonization index (which is a function of phagocytic activity and ELISA titres) allowed the distinction between protected and non-protected subjects; (5) unexpectedly, the opsonization index was inversely correlated with protection; (6) phagocytic activity is mediated by C-terminal antibodies and not repeat specific antibodies; (7) the predictive value of repeat specific ELISA titres or the ratio of PF16:NANP titres is lower than when using the opsonization index; and, (8) the ability to predict protection is highest when ELISA titres are combined with the opsonization index. One study has previously attempted to assess the phagocytic activity of RTS,S-induced antibodies, and although they reported a modest association between phagocytic activity and protection, their findings were limited by poor sensitivity of the assay [
21]. Specifically, the low sensitivity in that study required a user-defined cut-off to be used to distinguish phagocytic from non-phagocytic cells. Beyond differences in methodology and reported opsonization measures, differences in the respective RTS,S clinical trials used in this previous study [
21] and the current study, such as with immunization regimen and adjuvant conditions, preclude a direct comparison of the results.
The observation that repeat-specific antibodies were not contributing to the phagocytic activity measured in immunized subjects is a novel finding that requires further investigations into the underlying cause. The findings also indicate that a deeper understanding of the role of C-terminal antibodies in protection is needed. Studies in pre-clinical models have revealed that CSP C-terminal antibodies can play a crucial role in protection [
22]. Previous work by the authors, employing a molecular adjuvant based on the complement factor C3d, demonstrated that the loss of C-terminal specificity in the overall humoral response to CSP greatly impaired protective efficacy [
22]. The C-terminus of CSP contains several important functional elements, such as adhesion motifs for complement, thrombospondin and properdin. The properdin binding sequence, found in all
Plasmodium species, may modulate susceptibility to infection [
23‐
25]. The C-terminus has also been implicated in the initial entry of the sporozoites into hepatocytes [
26] and, therefore, antibodies against this CSP-region play a role in protection [
22,
27,
28]. A separate study by the authors has demonstrated that the parasites target this region of the CSP in an attempt to deviate the immune response [
22], thus further supporting the hypothesis that this is a crucial region for the function and ultimately survival of the parasite. Therefore, an in-depth analysis of the biological function of C-terminal antibodies is needed before drawing conclusions regarding the overall importance in vaccine-induced protection.
The finding that phagocytosis is inversely correlated with protection was unexpected and merits further study with a larger sample size. The question remains: What is the biological consequence of the antibody-mediated uptake of the sporozoite into the phagocyte? Additional studies addressing this question are needed to determine whether
Plasmodium employs similar immune escape strategies as has been reported for
Leishmania (reviewed in [
29]). Metacyclic promastigotes of
Leishmania express a specialized protein, namely gp63, which quickly converts the complement factor C3b to C3bi, resulting in a preferential interaction between the opsonized promastigote and Complement receptor 3 (CR3) (rather than Complement receptor 1) [
30]. The binding to CR3 still results in phagocytosis, but prevents the oxidative burst response during phagocytosis [
31].
Beyond the limited sample size, there are a number of limitations of the present study in assessing the biological phagocytic activity of the RTS,S antibody response. There is mounting evidence that in vitro assays have only a limited ability to forecast the precise Fc effector mechanisms engaged by opsonizing antibodies since these assays are typically performed with monocytic cells [
15,
16]. In contrast, in vivo, there is a wide range of immune cells competing with each other for the binding of immune complexes [
32]. Most leukocytes express several Fc receptors and these can be classified based on their biological activity: type I receptors that provide an activating signal for the cell, while type II receptors provide a modulatory response [
33]. Which Fc receptors are engaged by immune complexes is not solely governed by the isotype of the antibody, but also by the conformational state of the antibody (i.e., the glycosylation pattern). Moreover, some of these receptors are differentially expressed on immune cells, e.g., NK cells express only type I receptors and are, therefore, activated when immune complexes bind. In contrast, B cell express only type II receptors and, thus, binding of immune complexes to the Fc receptor without simultaneous ligation of the BCR results in a pro-apoptotic signal [
34]. Moreover, high antibody titres and immune complexes can result in immune dysfunction by interfering with effector functions [
35]. Finally, given the role of epitope density and arrangement in opsonophagocytosis, it is possible that there are differences in CSP presentation or density in sporozoites and in CSP-coated beads.
Although the current study cannot determine the ultimate biological consequences for sporozoite opsonization, it provides opsonization as a surrogate marker of protective immunity based on measuring the phagocytic activity mediated by vaccine (RTS,S)-induced antibodies, specifically antibodies targeting the CSP C-terminus. Typically, surrogate markers of protection are indicative of a protective response, but the activity measured is per se not involved in the mediation of protection [
6]. Future studies may address the discrepancies between in vitro and in vivo assay systems by utilizing peripheral blood mononuclear cells of each study subject to measure the phagocytic activity within these individuals, thus accounting for the heterogeneity of phagocytic cells as well as any polymorphisms in the Fc receptors of the respective subjects.
Authors’ contributions
SC performed the data analysis and compiled the manuscript; CFO performed the serological assessment of the samples; SD provided the reagents for the assay and edited the manuscript; FL and EJ co-designed the experiments and provided scientific discussion; RP provided institutional support, JR, AW, NW, and EJ consulted on the analysis and edited the manuscript; EBL designed the experiments, developed and performed phagocytosis assays. All authors read and approved the final manuscript.