Abs (antibodies) are complex glycoproteins that play a crucial role in protective immunity to malaria, but their effectiveness in mediating resistance can be enhanced by genetically engineered modifications that improve on nature. These Abs also aid investigation of immune mechanisms operating to control the disease and are valuable tools in developing neutralization assays for vaccine design. This review explores how this might be achieved.
Malaria, which rivals HIV/AIDS as the world's most deadly infection, kills a child every 5 s . The success of passive immunization against malaria indicates that Ig-based therapies are potential alternatives following increasing parasite drug resistance. The complexity of the malaria genome/proteome, especially concerning the biology of proteins seen during an immune response, has hampered the development of effective vaccines whilst highlighting the current lack of knowledge as to the protective mechanisms mediated by Abs (antibodies). Abs or their fragments are the paradigm for the design of high-affinity protein-based binding reagents. Ab-based reagents now represent approx. 25% of all proteins undergoing clinical trials . Technology for Ab design has taken huge strides forward and research into these important molecules has undergone a major revival with the realization that these molecules lie at the interface between innate and adaptive immunity [3,4]. As a consequence, at least 11 Abs have FDA (Federal Drug Administration) approval for use and at least a further 400 are in clinical trials worldwide, the majority for the treatment of cancer, allograft rejection or autoimmune disease . Despite these triumphs, none are in development for malaria, principally because they are perceived as being too expensive for a disease mainly afflicting poor and marginalized populations, but also because knowledge of the optimal effector mechanisms recruited by Abs to control malaria is limited. An improved comprehension of the structure and function of the different Ab classes in the context of malaria will guide the development of immuno-optimal reagents to control the disease. This review explores how this might be achieved.
Ab EFFECTOR FUNCTION
There are nine recognized human Ig (IgM, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE and IgD) classes that vary significantly both in structure and function (Figure 1), and excellent reviews exist for each Ab class [6–10]. The Fab′2 region of the molecule is concerned with binding Ag (antigen), whereas the Fc region mediates effector functions, although this is an oversimplification, since receptors that bind Fab are known to also mediate effector function . The ‘typical’ Ab is composed of two pairs of identical light (κ or λ) and heavy chains (μ, γ1, γ2, γ3, γ4, α1, α2, ϵ and δ), each containing variable and constant domains (Figure 1). The variable domains, named because of their hypervariable or CDRs (complementarity determining regions), each contribute three loops that interact with an Ag. Except in the case of polymeric forms, the four chain basic structure consists of two light and two heavy chains held together by covalent and non-covalent bonds that orientate the molecule with bilateral asymmetry (Figure 1). The primary function of an Ab is to bind Ag, which in some cases can have a direct effect, for example by neutralizing malaria toxin or by preventing parasite attachment and entry into erythrocytes. In general, however, binding to Ag is without significance unless secondary effector functions are recruited, including FcRs (Fc-receptors), PRRs (pattern recognition receptors) and components of the complement cascade. The realization that Abs are key immune modulators bridging innate and acquired immunity has led to the revelation that Abs can be effective against micro-organisms for which they do not mediate a direct biological effect . Abs may therefore also be effective in malaria by reducing the damage that results from inappropriate host inflammatory responses . Recent work showing that auto-Abs and Ab immune complexes (common in malaria) drive B-cell responses through the PRR TLR-9 (Toll-like receptor-9) supports such theories for malaria . Abs themselves can be viewed as PRRs, since complex N- and O-glycan structures found in the flexible hinge region can act both as bacterial adhesins or ligands for PRRs, including mannose binding lectin .
Human IgG1, IgA1 and a diabody (in red) are shown binding to
P. falciparum MSP119 (green) and recruiting FcγRIIa (blue) or FcαR (cyan) found on human immune killer cells
Abs AND IMMUNITY TO MALARIA
Immunity to malaria occurs only after many years of recurring infection. This is believed to be due to antigenic variation and the time taken by individuals to develop high-affinity Abs to invariant parts of otherwise very polymorphic Ags. Protection from malaria manifests as lessened disease symptoms and lower levels of parasites in the blood. IgG plays a crucial role in host defence against erythrocytic stages of Plasmodium, since passive transfer of IgG from immune African adults to African children killed malaria parasites [16,17]. In various animal models, transfer of mAb (monoclonal Ab) or immunization with fusion proteins encoding key Ags can protect from normally lethal Plasmodium infection (for review, see ). This immunity is primarily mediated by Abs that target the erythrocytic stage of the life cycle. Although naturally acquired Abs from clinically immune individuals belong to the IgG1 and IgG3 subclasses , there is little correlation between levels of specific Abs and resistance to infection or clinical disease, since the parasite also drives the synthesis of high-titre low-affinity Ab leading to hypergammaglobulinaemia . These low-affinity Abs seem to be directed to short highly repetitive amino acid sequences, are cross-reactive with several malarial Ags and may result from a process of immune evasion . The role of Abs in protective immunity is not completely understood, but inhibition of merozoite invasion of erythrocytes , Ab-mediated phagocytosis via FcR and complement  and an Ab-dependent cellular inhibition seem the most likely [19,23]. That the recruitment of effector cells by Fc is vital to this transfer of immunity has been elegantly demonstrated by the observation that passive transfer of Abs specific to the malarial Ag MSP1 (merozoite surface protein 1)19 cannot prevent death in FcR-deficient and -immunodeficient mouse models, although they could significantly delay its onset .
FcRs AND IMMUNITY TO MALARIA
Although the contribution of Abs in the immune response to malaria is unequivocal, the role of their cognate FcRs has been investigated less thoroughly (Figure 2). Specific FcRs have been described for each Ig class [7,25,26]. With the exception of FcϵRII (CD23), which is a C-type lectin receptor, most belong to the Ig gene superfamily, are structurally related and comprise a unique ligand-binding chain (α-chain) often complexed in their transmembrane region with partner proteins, including a dimer of the common FcR γ-chain. Effector mechanisms are signalled via ITAMs (immunoreceptor tyrosine-based activation motifs) or ITIMs (immunoreceptor tyrosine-based inhibitory motifs) present in the cytoplasmic regions of either the α- or γ-chain. Most genes encoding FcRs map to human chromosome 1, whereas curiously the FcαR gene lies in the LCR (leucocyte receptor complex) on chromosome 19 and shares homology with adjacent genes, including those encoding natural KIRs (killer cell inhibitory receptors), the ILTs (Ig-like transcripts) and GPVI (glycoprotein VI).
The human leukocyte Fc-receptors
The importance of FcR subunits in the course and outcome of parasitization is being studied in animals with FcR deletions. Although informative, these gene-deficient mouse models may not always mimic the human immune condition, due to differences in FcR biology and an apparent lack of true homologues [25,26]. Studies examining the role of FcR in immunity to parasites have made use of FcR γ-chain knockout mice . The γ-chain, a subunit common to FcγRI, FcγRIIIa, FcϵRI and FcαRI, is required for efficient cell-surface expression and signal transduction. Consequently FcRγ−/− mice are unable to elicit phagocytosis or ADCC (Ab-dependent cell-mediated cytotoxicity) reactions through these receptors. In a study using FcRγ−/− mice, a crucial role for FcR-mediated Ab-dependent phagocytosis in host resistance to blood-stage Plasmodium berghei XAT infection has been demonstrated . Before this work, experiments with P. yoelii had concluded that the protective effects of Abs probably arise from inhibition of erythrocyte invasion by merozoites rather than through FcR-mediated phagocytosis . However, this conclusion ignores two possibilities. Firstly, there might be other, as yet unidentified, FcRs involved in the observed response, and secondly, the α-chain of many FcRs may associate with signalling proteins other than the common γ-chain. With this in mind, it is interesting to note that mouse IgG3-opsonized Cryptococcus neoformans can still be phagocytosed by macrophages from FcRγ−/− mice . This effect is probably mediated via an undefined FcR without requiring γ-chain for function because, of the known FcRs, only murine FcγRI binds mouse IgG3, as demonstrated by transfection studies . In addition, FcR γ-chain-deficient mice were found to express partially functional FcγRI in more recent mouse knockouts [32,33]. It is now known that the α-chain of FcγRI can mediate MHC class II Ag presentation without active γ-chain signalling , and that the α-chain can interact with periplakin to control receptor endocytosis and IgG binding capacity . Of further note is the observation that mice deficient in the α-chain of FcγRI, and thus with macrophages that cannot bind IgG3, are protected from P. yoelii infection following passive transfer of an MSP-119-specific IgG3 mAb . Further work will be necessary to resolve the mechanism of action of IgG3 Abs.
Conclusions from FcRγ−/−-deleted mice cannot completely preclude a role for FcR in clearing malaria, because certain receptors, such as FcγRIIa and Fcα/μR, can mediate endocytosis of Ab-coated microbes in the absence of FcR γ-chain . IgA and IgM, the two classes bound by Fcα/μR, have been neglected in human malaria research principally because experimentation has been driven by murine systems. Mouse IgA is dimeric, and there is no murine equivalent of the 1–5 mg/ml monomeric IgA present in human serum. This makes IgA the second most abundant Ig in human serum after IgG1. Transgenic mice expressing human FcαRI have highlighted the importance of IgA in clearing serum pathogens, suggesting that a re-appraisal of the role of IgA in malarial infections might be timely . Although Ab-mediated protection against lethal malaria in murine models might involve triggers other than FcR, there is good evidence for the importance of these receptors in clearing human malaria [19,23]. Transgenic and knockout mice are increasingly being used in models of parasite infection, but it is important to recognize the differences between FcR systems in mice and humans. For example, there are no known murine equivalents of human FcγRIIa, FcγRIIc, FcγRIIIb and FcαRI [25,26]. Looking at FcγRIIa in particular, this human receptor has its own unique ITAM and is therefore capable of signal transduction and phagocytosis in the absence of γ-chain or associated subunits. Naturally acquired antimalarial Abs from clinically immune individuals belong to the human IgG1 and IgG3 subclasses [19,38], isotypes that are able to bind and trigger a phagocytic signal through FcγRIIa. The lack of a mouse counterpart for the interaction between this receptor and these Ab subclasses prevents proper analysis of the potential antiplasmodial effects of all receptor–ligand combinations in mouse models. Hence the divergence of FcγRII and FcγRIII genes between mice and humans might introduce difficulties in drawing conclusions from experiments in mice on human malaria parasites. The murky water has also been complicated by the recent discovery of eight human and six mouse FcRHs (Fc-R homologues), substantially increasing the size and functional potential of the FcR family . Although the ligands for FcRHs remain unknown, sequence conservation suggests that they have the potential to bind Ig-Fc, and transfection studies have suggested that FcRH4 and FcRH5 have a low affinity for aggregated IgA and IgG respectively .
FcR POLYMORPHISMS AND MALARIA
FcRs are a fertile area for dissecting Ab function in malaria, since polymorphisms in these key molecules have recently been shown to critically affect the severity and outcome of malaria in humans. Kenyan infants homozygous for the FcγRIIa-Arg131 allele are reported to be less at risk from high-density P. falciparum infection compared with children with the heterozygous Arg/His131 genotype . Although there was no significant effect of homozygosity for the alternative His131 allele on infection risk in Kenyan infants, a recent study in Gambian children has reported that this genotype was significantly associated with susceptibility to severe malaria and that the presence of the Arg131 allele, rather than the heterozygous Arg131 genotype, appeared to be important in protection from the disease . Adults homozygous for the His131 variant have been shown to be at increased risk of developing cerebral malaria , and this variant also associates with enhanced susceptibility to placental malaria in HIV-positive women, but not in HIV-negative women, in western Kenya . Both polymorphic variants can bind IgG3 and IgG1, but only the His131 genotype can bind IgG2, suggesting that IgG2 may play a detrimental role in the outcome of malaria . This is supported by in vitro studies showing that binding of IgG3/IgG1 immune complexes with monocytes confers protection using an Ab-dependent cellular inhibition assay , whereas IgG2 triggers less effective killing . Coincidently, FcγRs have been shown to bind CRP (C-reactive protein), an acute-phase protein that can also bind to the surface of P. falciparum sporozoites, potentially important in innate immunity [47,48]. The other known FcR polymorphisms, including FcγRIIIa-Val/Phe158, and those in the promoter region of FcαR remain to be investigated in relation to malaria.
RECOMBINANT CHIMAERIC HUMAN Abs AS TOOLS TO PROBE MALARIA IMMUNITY
Manipulating Ab genes allows the design of Igs with defined class and specificity, targeting protective epitopes on the parasite surface. An appropriate target for malaria is the 19 kDa C-terminal region of MSP119. This polypeptide displays limited sequence polymorphism , is expressed on the surface of parasite stages responsible for pathological symptoms  and acts as a major target of erythrocyte invasion-inhibitory Abs in individuals immune to P. falciparum malaria . There are numerous candidate vaccine Ags in addition to MSP1 (e.g. MSP3, GLURP, and AMA1) currently in clinical trials that may form the basis for targeted Ab therapies. MSP1 is used in this review as an example to illustrate the potential of Ab-based therapies, since much is known about the structure and function of this particular Ag in the context of protective epitopes seen by Abs. For an in depth analysis of Ab-based therapies in the context of other target Ags, the reader is directed to .
Since the mechanisms whereby an Ab mediates protection in malaria are unclear and as a preliminary step towards the development of passive Ab-based therapies, we grafted the variable genes from a protective mouse IgG2b mAb into expression plasmids containing human constant region genes, to generate chimaeric human IgG1 and IgA1 with specificity for an epitope on MSP119 from P. yoelii (Figure 1), for use in this lethal murine model of malaria . The engineered Abs could trigger potent FcR-mediated killing by human neutrophils, and IgA1 was consistently the most efficient Ab at mediating NADPH-mediated respiratory bursts from these cells. Highly effective killing mediated through FcαR has been shown against numerous pathogens and this receptor is now considered to have the best therapeutic potential for malaria, because of its optimal distribution on key immune cells, including granulocytes, DCs (dendritic cells) and Kupffer and NK (natural killer) cells . The FcαR is a discrete housekeeper of the immune system that mediates both anti- and pro-inflammatory functions depending on the form of IgA bound . Triggering FcαR has been shown to inhibit TNF-α (tumour necrosis factor-α) and IL-6 (interleukin 6) secretion by human monocytes , which is desirable in malaria where high TNF-α levels correlate with severe malaria and poor prognosis . Interestingly, high titres of naturally occurring Plasmodium-specific IgA have been reported both from sera  and breast milk , although preliminary work from our laboratory (M. Lazarou and R. J. Pleass, unpublished work) has been unable to detect MSP119-specific IgA in serum, presumably due to competition for binding from IgG. The recent finding that the FcαR plays a pivotal role in the immune system by mediating both anti- and pro-inflammatory functions of IgA via the γ-chain ITAM should encourage further work into understanding the role of IgA in malaria . Immature neutrophils have recently been shown to mediate tumour cell killing via IgA, but not IgG, FcR . Unlike IgM, IgG and IgE that are implicated in pathology associated with rosette formation , placental malaria  and severe malaria , IgA appears to be relatively benign in the malaria disease process, arguing strongly for its consideration in any passive Ab therapy.
Despite having comparable affinities and in vitro effector functions for MSP119, recombinant IgA1 and IgG1 were unable to protect mice from malaria, in contrast with the parental mouse IgG2b from which these Abs were derived. The failure of the engineered Abs to protect in vivo most probably stems from an inability of their human Fc regions to effectively trigger murine effector mechanisms . The impotence of IgA1 to protect mice in vivo may be explained by the lack of a murine homologue for human FcαR (CD89) and the questionable ability of IgA to fix complement. We are currently exploring the role of human IgA in mice transgenic for human FcαR and complement genes. The lack of effect with human IgG1 is less clear, but may rest in poor complement activation. Complement can play a role in killing malarial parasites, in a manner dependent on Abs and phagocytic cells, in particular neutrophils [62,63]. Since complement plays a central role in passive protection by human IgG1 anti-pneumococcal Abs in mice , it may be that merozoites are in some way resistant to this activation. Although human IgG1 is known to bind murine FcγR, it is less clear if binding leads to murine neutrophil effector activity. It is also possible that human IgG1 preferentially binds to murine FcγRIIb such that the overwhelming stimulus is inhibitory, rather than activatory. Without further dissection of the molecules involved, it is difficult to reconcile the reasons for the lack of protection offered by the human Abs.
Regardless of the mechanism involved, the inability of the human IgA1 and IgG1 versions to protect indicate that, for this epitope at least, mere blocking of MSP119 function by some form of steric hindrance is insufficient to bring about protection. Epitope specificity appears to be critical since Ab fragments, including scFvs (single-chain Fvs) and Fabs, have been shown to reduce parasitaemia . These unique reagents will permit delineation of human effector mechanisms in vivo in experiments using mice transgenic for human FcRs and complement genes. Such in vivo experiments are not possible with human malaria parasites, including P. falciparum, which are exquisitely and uniquely evolved for life in their human hosts. Although human/mouse chimaeric Abs against P. yoelii MSP119 tested in mice transgenic for human FcRs and challenged with P. yoelii is a useful and relevant model, we are also generating fully human Abs against P. falciparum. These reagents will be useful in correlating epitopes on MSP119 with protective immunity both as an aid to vaccine design, but also in neutralization assays. Until now, work on human malaria parasites has made use of Abs purified from immune sera. This situation is far from optimal, since sera contain a mixture of Abs, some with inappropriate specificities such as blocking Abs  and the potential to trigger inhibitory FcRs through ITIM signalling (Figure 2). Recombinant Abs offer reproducible standards for neutralization assays.
RECOMBINANT Ab-BASED REAGENTS AS THERAPEUTIC VACCINES
Passive therapy with inhibitory recombinant Abs may complement traditional vaccine development. Selective targeting of invariant inhibitory epitopes on MSP119 overcomes potential problems of antigenic polymorphism. Moreover, by careful choice of Ab class, it should be possible to target pertinent Ags to Ag-presenting cells, providing a ‘vaccine effect’ to limit parasite proliferation on re-infection (Figure 3) . In addition, passive immunotherapy does not require complex delivery systems or toxic adjuvants, although the latter have been shown to bolster protective immunity .
Possible mechanisms through which passively transferred antibodies mediate therapeutic vaccination and protection against malaria
One potential impediment to using parasite-specific IgG for malaria therapy might be competition with IgG of the non-specific hypergammaglobulinaemia frequently generated by parasites as a smokescreen to evade immunity . These antibodies may compete for binding to FcγRs and could explain why large doses of specific Abs are required for parasite neutralization both in vivo and in vitro . A further problem with parasite-specific IgG is that its Fc region may bind to FcγRs on platelets, B-cells or other non-cytotoxic cells, resulting in the inadvertent triggering of inhibitory FcγRIIb, which might benefit the parasite much as it has been shown in other systems (e.g. enhancing tumour cell growth ). These drawbacks to using IgG may potentially be overcome by engineering BsAbs (bispecific Abs).
BsAb reagents have been developed to redirect or enhance immune effector activity towards tumour or pathogen targets [71,72]. They work by linking the pathogen directly to host cytotoxic cells. The prototypic BsAb consists of two covalently linked Fab′ fragments, with one directed to the target molecule and the other directed to a trigger molecule (Figure 1). Originally produced by the fusion of hybridomas, they can now be made simply and cheaply by chemical conjugation or by recombinant expression systems. These reagents exist in many formats and have shown great promise in clinical settings where they have been used to kill tumour cells and pathogens [71,72]. By coupling variable domains recognizing P. yoelii MSP119 to variable domains recognizing human FcγRI, via a flexible peptide linker, we have generated a bispecific diabody designed to bind FcγRI outside its natural high-affinity binding site for IgG, thus preventing interference by irrelevant IgG . We found that simultaneous engagement of MSP119 and FcγRI by the diabody induced a potent neutrophil respiratory burst. The biphasic response was approx. 3-fold higher and of longer duration than the oxidative burst derived from equimolar amounts of antibody, suggesting that lower concentrations would be required in vivo to derive a similar therapeutic effect. In contrast with IgG1, the diabody was unable to induce phagocytosis of live merozoites. It is known that activation of NADPH oxidase by both chemicals  and antibodies  can occur in the absence of phagocytosis. Given that neutrophils normally express low levels of FcγRI (<2000 receptors/cell), compared with FcγRII (approx. 30000–60000 receptors/cell) or FcγRIII (approx. 100000–200000 receptors/cell), these results suggest that fewer receptors may need to be cross-linked for the induction of a respiratory burst than would be required for the complex molecular events associated with phagocytosis [71,72]. The reduced affinity and monovalent nature of the diabody would contrive to reduce its overall avidity, and may explain the absence of phagocytosis. Reagents that can initiate killing of malaria parasites without phagocytosis may offer certain advantages. During severe malaria, a considerable and often lethal burden is placed on both the spleen and liver, where parasites and infected erythrocytes are removed by phagocytosis . Thus therapies that can avoid unwanted liver and spleen pathology, yet kill parasites, may be very desirable.
Although passive mAbs can confer immediate protection from malaria, fast and reliable methods for producing high-affinity neutralizing mAbs for use in BsAb formats have been slower in development. Considerable progress has been made in this area, including (i) improved immortalization of human B-cells by Epstein–Barr virus for the generation of high-affinity Abs to SARS-CoV (severe acute respiratory syndrome coronavirus) , (ii) high-throughput phage, prokaryotic and fungal expression systems for selecting high-affinity human Ab-binding fragments [77–79], and (iii) immunization of transgenic mice carrying human Ig loci, followed by mAb production using hybridoma technology .
Given increasing problems with resistance to antimalarial drugs, a vaccine against malaria has become the ultimate goal. Unfortunately, its development has been beset with problems and alternative strategies to treat malaria have become of paramount importance. In this review, we have shown that passive delivery of recombinant Ab-based proteins should be considered important adjuncts to more traditional vaccine approaches, since Abs have the potential to act both as therapies and as vehicles for the optimal delivery of Ags in vaccination.
immunoreceptor tyrosine-based activation motif
immunoreceptor tyrosine-based inhibitory motif
merozoite surface protein 1
pattern recognition receptor
tumour necrosis factor-α
We thank the Medical Research Council (Career Establishment Award G0300145 to R.J.P.) and the European Union (Marie Curie Excellence Grant, Antibody Immunotherapy for Malaria 509670) for funding this work.