Malaria merozoite surface proteins (MSPs) are thought to have important roles in red blood cell (RBC) invasion and their exposure on the parasite surface makes them attractive vaccine candidates. However, their role in invasion has not been directly demonstrated and their biological functions remain unknown. One of the most abundant merozoite surface proteins is PfMSP2, a likely ancestral protein that has been maintained in the Plasmodium falciparum lineage and is a focus of vaccine development. Using CRISPR-Cas9 gene editing, we removed PfMSP2 from two different P. falciparum lines with no impact on parasite replication or phenotype in vitro, demonstrating that it is not essential for RBC invasion. Interestingly, loss of PfMSP2 led to increased inhibitory potency of antibodies targeting other merozoite proteins involved in invasion, particularly PfAMA1. In a solid-phase model, increasing concentrations of PfMSP2 protein reduced binding of different antibodies against PfAMA1 in a dose-dependent manner. These data suggest that PfMSP2 can modulate the susceptibility of merozoites to protective inhibitory antibodies. The results of this study change our understanding of the potential functions of PfMSP2 and establish a new concept in malaria where a surface protein can reduce the protective efficacy of antibodies targeting a different antigen. These findings have important implications for understanding malaria immunity and informing vaccine development.

Maintaining high-affinity antibodies after vaccination may be important for long-lasting immunity to malaria, but data on induction and kinetics of affinity is lacking. In a phase 1 malaria vaccine trial, antibody affinity increased following a second vaccination but declined substantially over 12 months, suggesting poor maintenance of high-affinity antibodies.Clinical Trials Registration Australian New Zealand Clinical Trials Registry ACTRN12607000552482. Maintaining high-affinity antibodies after vaccination may be important for long-lasting immunity to malaria, but knowledge is lacking. In a phase 1 malaria vaccine trial, affinity of vaccine antibodies declined substantially over 12 months, suggesting poor maintenance of high-affinity antibodies.

Apical membrane antigen-1 (AMA1) is a conserved malarial vaccine candidate essential for the formation of tight junctions with the rhoptry neck protein (RON) complex, enabling Plasmodium parasites to invade human erythrocytes, hepatocytes, and mosquito salivary glands. Despite its critical role, extensive surface polymorphisms in AMA1 have led to strain-specific protection, limiting the success of AMA1-based interventions beyond initial clinical trials. Here, we identify an i-body, a humanised single-domain antibody-like molecule that recognises a conserved pan-species conformational epitope in AMA1 with low nanomolar affinity and inhibits the binding of the RON2 ligand to AMA1. Structural characterisation indicates that the WD34 i-body epitope spans the centre of the conserved hydrophobic cleft in AMA1, where interacting residues are highly conserved among all Plasmodium species. Furthermore, we show that WD34 inhibits merozoite invasion of erythrocytes by multiple Plasmodium species and hepatocyte invasion by P. falciparum sporozoites. Despite a short half-life in mouse serum, we demonstrate that WD34 transiently suppressed P. berghei infections in female BALB/c mice. Our work describes the first pan-species AMA1 biologic with inhibitory activity against multiple life-cycle stages of Plasmodium. With improved pharmacokinetic characteristics, WD34 could be a potential immunotherapy against multiple species of Plasmodium.

Merozoite surface protein 2 (MSP2) is a highly abundant, GPI-anchored surface antigen on merozoites of the malaria parasite Plasmodium falciparum. It consists of highly conserved N- and C-terminal domains, and a central polymorphic region that allows all MSP2 alleles to be categorized into the 3D7 or FC27 family. Previously it has been shown that epitope accessibility differs between lipid-bound and lipid-free MSP2, suggesting that lipid interactions modulate the conformation and antigenicity in a way that may better mimic native MSP2 on the merozoite surface. Therefore, we have immunised mice with MSP2 engrafted onto liposomes using a C-terminal tether that mimics the native GPI anchor. To improve the immunogenicity of the formulated antigen, liposomes were supplemented with Pathogen Associated Molecular Pattern molecules, specifically agonists of the Toll-like receptor 4 (TLR4) or TLR2. Induced antibodies were directed mostly towards conserved epitopes, predominantly in the conserved C-terminal region of MSP2. We also found that immunisation with a combination of 3D7 and FC27 MSP2 enhanced antibody responses to conserved epitopes, and that the overall responses of mice immunised with MSP2-engrafted liposomes were comparable in magnitude to those of mice immunised with MSP2 formulated in Montanide ISA720. The antibodies elicited in mice by immunising with MSP2-engrafted liposomes recognised the native form of parasite MSP2 on western blots and were found to be cross-reactive with isolated 3D7 and FC27 merozoites when investigated by ELISA. The liposome-tethered MSP2 induced higher titres of complement-fixing antibodies to 3D7 and FC27 MSP2 than did MSP2 formulated in Montanide ISA720. Our results indicate that liposomal formulation represents a viable strategy for eliciting a strong immune response that favours conserved epitopes in MSP2 and thus a strain-transcendent immune response.

The malaria vaccine candidate merozoite surface protein 2 (MSP2) has shown promise in clinical trials and is in part responsible for a reduction in parasite densities. However, strain-specific reductions in parasitaemia suggested that polymorphic regions of MSP2 are immuno-dominant. One strategy to bypass the hurdle of strain-specificity is to bias the immune response towards the conserved regions. Two mouse monoclonal antibodies, 4D11 and 9H4, recognise the conserved C-terminal region of MSP2. Although they bind overlapping epitopes, 4D11 reacts more strongly with native MSP2, suggesting that its epitope is more accessible on the parasite surface. In this study, a structure-based vaccine design approach was applied to the intrinsically disordered antigen, MSP2, using a crystal structure of 4D11 Fv in complex with its minimal binding epitope. Molecular dynamics simulations and surface plasmon resonance informed the design of a series of constrained peptides that mimicked the 4D11-bound epitope structure. These peptides were conjugated to keyhole limpet hemocyanin and used to immunise mice, with high to moderate antibody titres being generated in all groups. The specificities of antibody responses revealed that a single point mutation can focus the antibody response towards a more favourable epitope. This structure-based approach to peptide vaccine design may be useful not only for MSP2-based malaria vaccines, but also for other intrinsically disordered antigens.

Asparaginyl endopeptidases (AEPs) are a class of enzymes commonly associated with proteolysis in the maturation of seed storage proteins. However, a subset of AEPs work preferentially as peptide ligases, coupling release of a leaving group to formation of a new peptide bond. These "ligase-type" AEPs require only short recognition motifs to ligate a range of targets, making them useful tools in peptide and protein engineering for cyclisation of peptides or ligation of separate peptides into larger products. Here we report the recombinant expression, ligase activity and cyclisation kinetics of three new AEPs from the cyclotide producing plant Oldenlandia affinis with superior kinetics to the prototypical recombinant AEP ligase OaAEP1(b). These AEPs work preferentially as ligases at both acidic and neutral pH and we term them "canonical AEP ligases" to distinguish them from other AEPs where activity preferences shift according to pH. We show that these ligases intrinsically favour ligation over hydrolysis, are highly efficient at cyclising two unrelated peptides and are compatible with organic co-solvents. Finally, we demonstrate the broad scope of recombinant AEPs in biotechnology by the backbone cyclisation of an intrinsically disordered protein, the 25 kDa malarial vaccine candidate Plasmodium falciparum merozoite surface protein 2 (MSP2).

The abundant Plasmodium falciparum merozoite surface protein MSP2, a potential malaria vaccine candidate, is an intrinsically disordered protein with some nascent secondary structure present in its conserved N-terminal region. This relatively ordered region has been implicated in both membrane interactions and amyloid-like aggregation of the protein, while the significance of the flanking-disordered region is unclear. In this study, we show that aggregation of the N-terminal conserved region of MSP2 is influenced in a length- and sequence-dependent fashion by the disordered central variable sequences. Intriguingly, MSP2 peptides containing the conserved region and the first five residues of the variable disordered regions aggregated more rapidly than a peptide corresponding to the conserved region alone. In contrast, MSP2 peptides extending 8 or 12 residues into the disordered region aggregated more slowly, consistent with the expected inhibitory effect of flanking-disordered sequences on the aggregation of amyloidogenic ordered sequences. Computational analyses indicated that the helical propensity of the ordered region of MSP2 was modulated by the adjacent disordered five residues in a sequence-dependent manner. Nuclear magnetic resonance and circular dichroism spectroscopic studies with synthetic peptides confirmed the computational predictions, emphasizing the correlation between aggregation propensity and conformation of the ordered region and the effects thereon of the adjacent disordered region. These results show that the effects of flanking-disordered sequences on a more ordered sequence may include enhancement of aggregation through modulation of the conformational properties of the more ordered sequence.

Background. Overcoming antigenic diversity is a key challenge in the development of effective Plasmodium falciparum malaria vaccines. Strategies that promote the generation of antibodies targeting conserved epitopes of vaccine antigens may provide protection against diverse parasites strains. Understanding differences between vaccine-induced and naturally acquired immunity is important to achieving this goal. Methods. We analyzed antibodies generated in a phase 1 human vaccine trial, MSP2-C1, which included 2 allelic forms of MSP2, an abundant vaccine antigen on the merozoite surface. Vaccine-induced responses were assessed for functional activity against multiple parasite strains, and cross-reactivity of antibodies was determined using competition ELISA and epitope mapping approaches. Results. Vaccination induced cytophilic antibody responses with strain-transcending opsonic phagocytosis and complement-fixing function. In contrast to antibodies acquired via natural infection, vaccine-induced antibodies were directed towards conserved epitopes at the C-terminus of MSP2, whereas naturally acquired antibodies mainly targeted polymorphic epitopes. Functional activity of C-terminal-targeted antibodies was confirmed using monoclonal antibodies that promoted opsonic phagocytosis against multiple parasite strains. Conclusion. Vaccination generated markedly different responses to polymorphic antigens than naturally acquired immunity and targeted conserved functional epitopes. Induction of antibodies targeting conserved regions of malaria antigens provides a promising vaccine strategy to overcome antigenic diversity for developing effective malaria vaccines.

Merozoite surface protein 2 (MSP2) is an intrinsically disordered antigen that is abundant on the surface of the malaria parasite Plasmodium falciparum. The two allelic families of MSP2, 3D7 and FC27, differ in their central variable regions, which are flanked by highly conserved C-terminal and N-terminal regions. In a vaccine trial, full-length 3D7 MSP2 induced a strain-specific protective immune response despite the detectable presence of conserved region antibodies. This work focuses on the conserved C-terminal region of MSP2, which includes the only disulphide bond in the protein and encompasses key epitopes recognised by the mouse monoclonal antibodies 4D11 and 9H4. Although the 4D11 and 9H4 epitopes are overlapping, immunofluorescence assays have shown that the mouse monoclonal antibody 4D11 binds to MSP2 on the merozoite surface with a much stronger signal than 9H4. Understanding the structural basis for this antigenic difference between these antibodies will help direct the design of a broad-spectrum and MSP2-based malaria vaccine. 4D11 and 9H4 were reengineered into antibody fragments [variable region fragment (Fv) and single-chain Fv (scFv)] and were validated as suitable models for their full-sized IgG counterparts by surface plasmon resonance and isothermal titration calorimetry. An alanine scan of the 13-residue epitope 3D7-MSP2207–222 identified the minimal binding epitope of 4D11 and the key residues involved in binding. A 2.2-Å crystal structure of 4D11 Fv bound to the eight-residue epitope NKENCGAA provided valuable insight into the possible conformation of the C-terminal region of MSP2 on the parasite. This work underpins continued efforts to optimise recombinant MSP2 constructs for evaluation as potential vaccine candidates.

Merozoite surface protein 2 (MSP2) is a highly abundant, GPI-anchored antigen on the malaria parasite Plasmodium falciparum. MSP2 induces an immune response in the context of natural infections and vaccine trials, and these responses are associated with protection from parasite infection. Recombinant MSP2 is highly disordered in solution but antigenic analyses suggest that it is more ordered on the merozoite surface. We have shown previously that the interaction of recombinant full-length MSP2 with lipid surfaces induces a conformational change in the conserved N-terminal region of MSP2, which contributes to epitope masking in this region. To explore the impacts of lipid interactions on the conformation and antigenicity of the conserved C-terminal region of MSP2, a construct corresponding to this domain, MSP2(172-221), was designed. NMR studies indicate that many residues in MSP2(172-221) interact with DPC micelles, including some in epitopes recognised by C-terminal-specific monoclonal antibodies, but, in contrast to the MSP2 N-terminus, there is no indication of stable helical conformation. The binding affinities of a panel of monoclonal antibodies indicate that MSP2(172-221) is antigenically similar to full-length MSP2 and show that liposome conjugation alters the antigenicity in a manner that may mimic native MSP2 on the merozoite surface. These findings highlight the impact of lipid interactions on the conformation and antigenicity of MSP2(172-221) and will assist in the design of recombinant MSP2 immunogens for use as malaria vaccine candidates.

Antibodies play a key role in acquired human immunity to Plasmodium falciparum (Pf) malaria and target merozoites to reduce or prevent blood-stage replication and the development of disease. Merozoites present a complex array of antigens to the immune system, and currently, there is only a partial understanding of the targets of protective antibodies and how responses to different antigens are acquired and boosted. We hypothesized that there would be differences in the rate of acquisition of antibodies to different antigens and how well they are boosted by infection, which impacts the acquisition of immunity. We examined responses to a range of merozoite antigens in 2 different cohorts of children and adults with different age structures and levels of malaria exposure. Overall, antibodies were associated with age, exposure, and active infection, and the repertoire of responses increased with age and active infection. However, rates of antibody acquisition varied between antigens and different regions within an antigen following exposure to malaria, supporting our hypothesis. Antigen-specific responses could be broadly classified into early response types in which antibodies were acquired early in childhood exposure and late response types that appear to require substantially more exposure for the development of substantial levels. We identified antigen-specific responses that were effectively boosted after recent infection, whereas other responses were not. These findings advance our understanding of the acquisition of human immunity to malaria and are relevant to the development of malaria vaccines targeting merozoite antigens and the selection of antigens for use in malaria surveillance.

MSP2 is an intrinsically disordered protein that is abundant on the merozoite surface and essential to the parasite Plasmodium falciparum . Naturally-acquired antibody responses to MSP2 are biased towards dimorphic sequences within the central variable region of MSP2 and have been linked to naturally-acquired protection from malaria. In a phase IIb study, an MSP2-containing vaccine induced an immune response that reduced parasitemias in a strain-specific manner. A subsequent phase I study of a vaccine that contained both dimorphic forms of MSP2 induced antibodies that exhibited functional activity in vitro . We have assessed the contribution of the conserved and variable regions of MSP2 to the generation of a strain-transcending antibody response by generating MSP2 chimeras that included conserved and variable regions of the 3D7 and FC27 alleles. Robust anti-MSP2 antibody responses targeting both conserved and variable regions were generated in mice, although the fine specificity and the balance of responses to these regions differed amongst the constructs tested. We observed significant differences in antibody subclass distribution in the responses to these chimeras. Our results suggest that chimeric MSP2 antigens can elicit a broad immune response suitable for protection against different strains of P. falciparum .

Disordered proteins are important antigens in a range of infectious diseases. Little is known, however, about the molecular details of recognition of disordered antigens by their cognate antibodies. Using a large dataset of protein antigens, we show that disordered epitopes are as likely to be recognized by antibodies as ordered epitopes. Moreover, the affinity with which antigens are recognized is, unexpectedly, only weakly dependent on the degree of disorder within the epitope. Structurally defined complexes of ordered and disordered protein antigens with their cognate antibodies reveal that disordered epitopes are smaller than their ordered counterparts, but are more efficient in their interactions with antibody. Our results demonstrate that disordered antigens are bona fide targets of antibody recognition, and that recognition of disordered epitopes is particularly sensitive to epitope variation, a finding with implications for the effects of disorder on the specificity of molecular recognition more generally.

The interaction between apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) plays a key role in the invasion of red blood cells by Plasmodium parasites. Disruption of this critical protein–protein interaction represents a promising avenue for antimalarial drug discovery. In this work, we exploited a 13-residue β-hairpin based on the C-terminal loop of RON2 to probe a conserved binding site on Plasmodium falciparum AMA1. A series of mutations was synthetically engineered into β-hairpin peptides to establish structure–activity relationships. The best mutations improved the binding affinity of the β-hairpin peptide by ~7-fold for 3D7 AMA1 and ~14-fold for FVO AMA1. We determined the crystal structures of several β-hairpin peptides in complex with AMA1 in order to define the structural features and specific interactions that contribute to improved binding affinity. The same mutations in the longer RON2sp2 peptide (residues 2027–2055 of RON2) increased the binding affinity by >30-fold for 3D7 and FVO AMA1, producing KD values of 2.1nM and 0.4nM, respectively. To our knowledge, this is the most potent strain-transcending peptide reported to date and represents a valuable tool to characterize the AMA1–RON2 interaction.

Malaria remains a significant global health burden. The development of an effective malaria vaccine remains as a major challenge with the potential to significantly reduce morbidity and mortality. While Plasmodium spp. have been shown to contain a large number of intrinsically disordered proteins (IDPs) or disordered protein regions, the relationship of protein structure to subcellular localisation and adaptive immune responses remains unclear. In this study, we employed several computational prediction algorithms to identify IDPs at the proteome level of six Plasmodium spp. and to investigate the potential impact of protein disorder on adaptive immunity against P. falciparum parasites. IDPs were shown to be particularly enriched within nuclear proteins, apical proteins, exported proteins and proteins localised to the parasitophorous vacuole. Furthermore, several leading vaccine candidates, and proteins with known roles in host-cell invasion, have extensive regions of disorder. Presentation of peptides by MHC molecules plays an important role in adaptive immune responses, and we show that IDP regions are predicted to contain relatively few MHC class I and II binding peptides owing to inherent differences in amino acid composition compared to structured domains. In contrast, linear B-cell epitopes were predicted to be enriched in IDPs. Tandem repeat regions and non-synonymous single nucleotide polymorphisms were found to be strongly associated with regions of disorder. In summary, immune responses against IDPs appear to have characteristics distinct from those against structured protein domains, with increased antibody recognition of linear epitopes but some constraints for MHC presentation and issues of polymorphisms. These findings have major implications for vaccine design, and understanding immunity to malaria.

Merozoite surface protein 2 (MSP2) is an intrinsically disordered, membrane-anchored antigen of the malaria parasite Plasmodium falciparum. MSP2 can elicit a protective, albeit strain-specific, antibody response in humans. Antibodies are generated to the conserved N- and C-terminal regions but many of these react poorly with the native antigen on the parasite surface. Here we demonstrate that recognition of a conserved N-terminal epitope by mAb 6D8 is incompatible with the membrane-bound conformation of that region, suggesting a mechanism by which native MSP2 escapes antibody recognition. Furthermore, crystal structures and NMR spectroscopy identify transient, strain-specific interactions between the 6D8 antibody and regions of MSP2 beyond the conserved epitope. These interactions account for the differential affinity of 6D8 for the two allelic families of MSP2, even though 6D8 binds to a fully conserved epitope. These results highlight unappreciated mechanisms that may modulate the specificity and efficacy of immune responses towards disordered antigens.