Background
Natural immunity against malaria is based on the presence of antibodies directed against the blood stage parasite, as demonstrated by passive transfer experiments of immunoglobulins [
1‐
3]. The mode of action of blood stage-specific antibodies depends on their antigen-specificity: they can bind to merozoites, opsonize and target them towards phagocytic cells of the host [
4], or prevent invasion of new erythrocytes [
5]. Once infected, antibodies against the asexual blood stage antigen Pf332 inhibit the intra-erythrocytic development of
Plasmodium falciparum [
6‐
8]. Furthermore, antibodies against free parasitic glycosylphosphatidylinositol (GPI) can control the severity of disease by neutralizing toxic components released from ruptured infected erythrocytes [
9,
10].
The major merozoite surface protein -1 (MSP-1) was identified in immune complexes from merozoite lysates, which provided the rationale for developing vaccines against this antigen [
11]. MSP-1 undergoes two successive proteolytic cleavage events [
12]. The second processing event occurs immediately before invasion, resulting in the cleavage of the p42 molecule into a p33 and a p19 fragment. The p19 fragment remains attached to the merozoite surface through a GPI anchor [
13] and is comprised of two epidermal growth factor (EGF)-like domains [
14], which may have a role in the invading complex. Serological studies have provided significant evidence to suggest that immune responses directed against the C-terminus of MSP-1 (MSP-1
19 and MSP-1
42) are associated with immunity in preclinical models [
15‐
18], as well as from individuals residing in endemic areas [
16,
19].
In the course of characterizing immune responses induced by MSP-1 vaccines, it was recognized that: (1) proteins produced by various expression systems differ in their immunogenicity and ability to induce anti-parasite activities [
20,
21]; (2) not all MSP-1-based vaccines induce protective immunity [
22] and; (3) the degree of inhibition was dependent on the method chosen to measure invasion- and growth inhibition mediated by anti-MSP-1p42 and that only the methods that are based on cell viability and/or metabolic activity are able to accurately measure growth inhibition, while all the methods tested were equally able to detect invasion inhibition [
23]. Thus, selecting the appropriate method permits differentiation between the various mechanisms by which antibodies affect parasite development.
The current study was designed to investigate the effect of anti-MSP-1p42 antibodies on intra-erythrocytic parasite development and their consequences on parasite viability and infectivity. Depending on the parasite clone, anti-MSP-1p42 antibodies prevented schizont rupturing by stalling or arresting intra-erythrocytic parasite development likely through direct interactions with intra-erythrocytic parasites within the parasitophorous vacuole. The nature of the inhibitory effect was strongly affected not only by the parasite clone, but also by the fine-specificity of the antibodies. Only antibodies that bound the p19 subunit, but not the EGF-like domain 1 or 2 subunits, displayed growth inhibitory activities indicating that protective epitopes are dependent on the tertiary structure of the molecule.
Several important lessons can be gained from these findings: (1) various allele-specific effector mechanisms can develop to the same blood stage antigen; (2) while
in vitro assays provide a useful tool to determine vaccine efficacy, such assays need to take into account the predominant antibody-mediated effector mechanisms against a particular clone; (3) inhibitory anti-MSP-1 specific antibodies map to epitopes formed through the "properly" folded p19 subunit and not to its sub-domains [
24,
25]; (4) with the exclusion of potential effector cells, only macromolecules of a defined size have access to the intra-erythrocytic parasite either through the putative "parasitophorous duct" or through the "leaky" erythrocytic membrane [
6,
26], and thus MSP-1-specific antibodies could be used as carriers and targeting moieties for anti-malarial drugs.
Discussion
There is significant data in the literature supporting the development of MSP-1p42 as a blood-stage vaccine candidate primarily as antibodies directed against this antigen have been shown to protect against homologous challenge in
Aotus monkey studies, albeit using strong adjuvants like complete Freund's/incomplete Freund's [
15], as well as shown to correlate with reduced parasite burden and clinical disease from human sero-epidemiological studies (reviewed in [
36]). Thus, it is crucial to understand the potential mechanisms underlying the anti-parasitic activities for this target antigen in order to design MSP-1-based vaccines with maximal vaccine efficacy. In two recent studies which compare various MSP-1-based vaccine candidates with regards to their immunogenicity and ability to induce antibodies that mediate anti-parasitic activity [
20,
21], the differences observed in antibody binding and functional activities induced were attributed to either the immunogens ability to induce more "biologically" relevant activities against the parasite or from the effects on the immunogens conformation as a result of the expression or purification systems utilized. This is not surprising as the quality of the immune response induced may be influenced by the quality of the protein produced. To avoid issues of expression and purification process differences, the recombinant MSP-1p42 molecules used as immunogens in the current study were all expressed similarly from
E. coli and purified as soluble proteins [
15,
28,
29]. Where directly comparable, no apparent differences were observed in the anti-parasitic activities induced by either the single codon mutated MSP-1p42 (FMP003) and full gene codon harmonized MSP-1p42 (FMP010) in FA or Montanide ISA 720 as adjuvant. Therefore, for at least the two adjuvants tested, the mode of action of the functional activities induced in rabbits was not adjuvant-dependent.
The present study demonstrates that antibodies directed against MSP-1p42 play a complex role during invasion and intra-erythrocytic maturation (growth) of parasites. Using comparative studies of
P. falciparum FVO, CAMP/FUP and 3D7 parasite cultures, we made several observations: first, that the quality of the response of MSP-1p42 specific antibodies is independent of the immunogen and adjuvant used (Figure
1); second, that the "type" of response against the parasite, whether invasion or growth inhibitory, was dependent on the assay clone, for instance, invasion- and growth inhibition was observed for 3D7 and CAMP/FUP parasites while only invasion inhibition was observed for FVO parasites (Figures
1,
6); third, that only antibodies raised against MSP-1p42 or affinity-purified using either the 3D7 or FVO p19 fragments had any inhibitory activity, while antibodies affinity-purified using either the EGF-like domain 1 or 2 did not (Figure
6); fourth, that the MSP-1p19 specific antibodies component exhibited homologous allele-specific inhibitory activities that were not observed for antibodies to the total MSP-1p42; and finally, that antibodies were able to enter viable infected erythrocytes and bind to intra-erythrocytic parasites, and that likely through this action, inhibit either further development or prevent rupture of maturing schizonts (Figures
2,
5,
6).
The present study revealed various reaction patterns of blood stage parasites in response to MSP-1p42 specific antibodies: invasion inhibition mediated by preventing schizont maturation ("stalled schizonts"), schizont rupturing and agglutination of merozoites prior to or upon their release (Figure
2). The observation that anti-MSP-1p42 specific antibodies cannot prevent the rupture of 3D7 schizonts (Figure
2) is not due to a general inability of the schizonts to be stalled as anti-AMA-1 specific antibodies induced this phenotype as well (Bergmann-Leitner, unpublished observations). Preventing schizonts from rupturing permanently affected the schizonts, as no spike in parasitaemia at any later time points could be detected suggesting that the observed effect was likely not due to a transient phenotype. Growth inhibition or retardation becomes evident by progressive losses in the ability to stain DNA with HE and reductions in the levels of pLDH measured in the cultures in the presence of immune serum. Moreover, growth retardation was observed when pRBC cultures treated with either control or immune serum were compared for their DNA content as a measurement of parasite maturation (Figure
5). As suggested by the HE-staining, 3D7 parasites were more affected by the phenomenon of growth retardation than were FVO parasites. However, measuring the DNA content as a marker for maturation does not necessarily indicate that the effect of immune antibodies is persistent, i.e., even though the maturation is slowed these cultures retained similar multiplication rates as control cultures. Thus currently, the only feasible measures of growth inhibition are those methods that are based on parasite viability such as HE-staining of DNA and measurement of pLDH [
23].
In an effort to better understand how MSP-1p42 specific antibodies affect intra-erythrocytic parasite development, such that schizonts can no longer rupture, it was first corroborated that antibodies, regardless of their specificity, can enter infected RBCs (Figure
4), as shown previously for other macromolecules, such as beads having diameters up to 80 nm [
26,
33], iron chelators [
37], antisense oligonucleotides [
38], plasmids [
39] and antibodies [
6]. In the case where these antibodies are unable to prevent invasion of erythrocytes, antibodies that are bound to parasites may be dragged into the newly invaded RBC and remain bound to the ring stage parasite during the early phases of development immediately after invasion (unpublished observation, and [
40]). A recent report demonstrates that the p19 fragment of MSP-1 may be involved in the formation of the food vacuole [
41]. It needs to be determined how antibody binding to p19 during and after invasion affects the role of the molecule during this crucial developmental step. In the later development it is possible that the antibody-bound MSP-1 on intra-erythrocytic parasites interferes with the ability to cause the phenotypic changes on FVO and CAMP/FUP schizonts that lead to rupture. Finally, this study addressed the question of anti-MSP-1p42 specific antibodies fine specificities and their role in mediating the various phenotypes of invasion and growth inhibition that were detected. To this end, antibodies were affinity-purified using either recombinant MSP-1p19 or the EGF-like domain 1 or 2 (representative of the 3D7 or FVO allele) contained within the p19 fragment (Figure
6). Flow cytometric analysis of parasite cultures treated with the various antibody preparations derived from rabbit immune serum revealed that only antibodies raised against the MSP-1p42 or affinity purified antibodies specific for MSP-1p19 had any inhibitory activity. These results are consistent with the findings obtained from naturally-exposed Western Kenyan adults' immune sera where using transgenic
P. falciparum expressing MSP-1-p19 orthologs, the majority of the inhibitory activity was directed to the MSP-1p19 portion of the molecule [
19,
35]. Affinity purified anti-MSP-1 antibodies using EGF-like domain 1 and 2 proteins did not capture antibodies that had any inhibitory function indicating that the crucial epitopes are formed by both domains or that the epitope is larger than just one of the domains alone. This finding is in agreement with epitope mapping studies for the growth inhibitory mAb, namely 12.10 [
42], where both domains were required for mAb binding [
25,
43]. Interestingly, MSP-1p19-purified antibodies appeared to be more allele-specific with regard to invasion inhibition than the total MSP-1p42 antibody population as MSP-1p19 (3D7) antibodies were only able to inhibit 3D7 parasites and to a lesser extent CAMP/FUP parasites and not FVO parasites. Similarly, MSP-1p19 (FVO)-purified antibodies only inhibited invasion of FVO parasites, while the effect on 3D7 and CAMP/FUP parasites was solely growth-inhibitory. Thus the mechanism of invasion inhibition may be more allele-specific than the mechanism of growth inhibition.
Acknowledgements
The authors would like to thank Dr. Michael Zidanic (WRAIR, Silver Spring, MD) for the confocal microscopy analysis and Dr. Mark Wilson (NIH, Bethesda, MD) for critically reviewing and editing the manuscript. The work was initiated under the guidance of Dr. Jeffrey A. Lyon.
This work was supported by the United States Agency for International Development, Project Number 936-6001, Award Number AAG-P-00-98-00006, Award Number AAG-P-00-98-00005, and by the United States Army Medical Research and Materiel Command.
Authors' contributions
ESB-L drafted the manuscript, co-designed the study, conducted most of the experiments and performed growth inhibition assays. EHD assisted in growth inhibition assays, kinetic experiments and performed the affinity purifications. EA designed the study, directed the work and edited the manuscript. All authors have read and approved the final manuscript.