Defining species-specific and conserved interactions of apical membrane protein 1 during erythrocyte invasion in malaria to inform multi-species vaccines.

Plasmodium falciparum and P. vivax are the major causes of human malaria, and P. knowlesi is an important additional cause in SE Asia. Binding of apical membrane antigen 1 (AMA1) to rhoptry neck protein 2 (RON2) was thought to be essential for merozoite invasion of erythrocytes by Plasmodium spp. Our findings reveal that P. falciparum and P. vivax have diverged and show species-specific binding of AMA1 to RON2, determined by a ß-hairpin loop in RON2 and specific residues in AMA1 Loop1E. In contrast, cross-species binding of AMA1 to RON2 is retained between P. vivax and P. knowlesi. Mutation of specific amino acids in AMA1 Loop1E in P. falciparum or P. vivax ablated RON2 binding without impacting erythrocyte invasion. This indicates that the AMA1-RON2-loop interaction is not essential for invasion and additional AMA1 interactions are involved. Mutations in AMA1 that disrupt RON2 binding also enable escape of invasion inhibitory antibodies. Therefore, vaccines and therapeutics will need to be broader than targeting only the AMA1-RON2 interaction. Antibodies targeting AMA1 domain 3 had greater invasion-inhibitory activity when RON2-loop binding was ablated, suggesting this domain is a promising additional target for vaccine development. Targeting multiple AMA1 interactions involved in invasion may enable vaccines that generate more potent inhibitory antibodies and address the capacity for immune evasion. Findings on specific residues for invasion function and species divergence and conservation can inform novel vaccines and therapeutics against malaria caused by three species, including the potential for cross-species vaccines.

The direct binding of Plasmodium vivax AMA1 to erythrocytes defines a RON2-independent invasion pathway.

We used a transgenic parasite in which Plasmodium falciparum parasites were genetically modified to express Plasmodium vivax apical membrane antigen 1 (PvAMA1) protein in place of PfAMA1 to study PvAMA1-mediated invasion. In P. falciparum, AMA1 interaction with rhoptry neck protein 2 (RON2) is known to be crucial for invasion, and PfRON2 peptides (PfRON2p) blocked the invasion of PfAMA1 wild-type parasites. However, PfRON2p has no effect on the invasion of transgenic parasites expressing PvAMA1 indicating that PfRON2 had no role in the invasion of PvAMA1 transgenic parasites. Interestingly, PvRON2p blocked the invasion of PvAMA1 transgenic parasites in a dose-dependent manner. We found that recombinant PvAMA1 domains 1 and 2 (rPvAMA1) bound to reticulocytes and normocytes indicating that PvAMA1 directly interacts with erythrocytes during the invasion, and invasion blocking of PvRON2p may result from it interfering with PvAMA1 binding to erythrocytes. It was previously shown that the peptide containing Loop1a of PvAMA1 (PvAMA1 Loop1a) is also bound to reticulocytes. We found that the Loop1a peptide blocked the binding of PvAMA1 to erythrocytes. PvAMA1 Loop1a has no polymorphisms in contrast to other PvAMA1 loops and may be an attractive vaccine target. We thus present the evidence that PvAMA1 binds to erythrocytes in addition to interacting with PvRON2 suggesting that the P. vivax merozoites may exploit complex pathways during the invasion process.

Anti-Gametocyte Antigen Humoral Immunity and Gametocytemia During Treatment of Uncomplicated Falciparum Malaria: A Multi-National Study.

Introduction: Understanding the human immune response to Plasmodium falciparum gametocytes and its association with gametocytemia is essential for understanding the transmission of malaria as well as progressing transmission blocking vaccine candidates. Methods: In a multi-national clinical efficacy trial of artemisinin therapies (13 sites of varying transmission over South-East Asia and Africa), we measured Immunoglobulin G (IgG) responses to recombinant P. falciparum gametocyte antigens expressed on the gametocyte plasma membrane and leading transmission blocking vaccine candidates Pfs230 (Pfs230c and Pfs230D1M) and Pfs48/45 at enrolment in 1,114 participants with clinical falciparum malaria. Mixed effects linear and logistic regression were used to determine the association between gametocyte measures (gametocytemia and gametocyte density) and antibody outcomes at enrolment. Results: Microscopy detectable gametocytemia was observed in 11% (127/1,114) of participants at enrolment, and an additional 9% (95/1,114) over the follow-up period (up to day 42) (total 20% of participants [222/1,114]). IgG levels in response to Pfs230c, Pfs48/45 and Pfs230D1M varied across study sites at enrolment (p < 0.001), as did IgG seroprevalence for anti-Pfs230c and D1M IgG (p < 0.001), but not for anti-Pfs48/45 IgG (p = 0.159). In adjusted analyses, microscopy detectable gametocytemia at enrolment was associated with an increase in the odds of IgG seropositivity to the three gametocyte antigens (Pfs230c OR [95% CI], p: 1.70 [1.10, 2.62], 0.017; Pfs48/45: 1.45 [0.85, 2.46], 0.174; Pfs230D1M: 1.70 [1.03, 2.80], 0.037), as was higher gametocyte density at enrolment (per two-fold change in gametocyte density Pfs230c OR [95% CI], p: 1.09 [1.02, 1.17], 0.008; Pfs48/45: 1.05 [0.98, 1.13], 0.185; Pfs230D1M: 1.07 [0.99, 1.14], 0.071). Conclusion: Pfs230 and Pfs48/45 antibodies are naturally immunogenic targets associated with patent gametocytemia and increasing gametocyte density across multiple malaria endemic settings, including regions with emerging artemisinin-resistant P. falciparum.

Antibody Targets and Properties for Complement-Fixation Against the Circumsporozoite Protein in Malaria Immunity.

The Plasmodium falciparum circumsporozoite protein (CSP) forms the basis of leading subunit malaria vaccine candidates. However, the mechanisms and specific targets of immunity are poorly defined. Recent findings suggest that antibody-mediated complement-fixation and activation play an important role in immunity. Here, we investigated the regions of CSP targeted by functional complement-fixing antibodies and the antibody properties associated with this activity. We quantified IgG, IgM, and functional complement-fixing antibody responses to different regions of CSP among Kenyan adults naturally exposed to malaria (n=102) and using a series of rabbit vaccination studies. Individuals who acquired functional complement-fixing antibodies had higher IgG, IgM and IgG1 and IgG3 to CSP. Acquired complement-fixing antibodies targeted the N-terminal, central-repeat, and C-terminal regions of CSP, and positive responders had greater antibody breadth compared to those who were negative for complement-fixing antibodies (p<0.05). Using rabbit vaccinations as a model, we confirmed that IgG specific to the central-repeat and non-repeat regions of CSP could effectively fix complement. However, vaccination with near full length CSP in rabbits poorly induced antibodies to the N-terminal region compared to naturally-acquired immunity in humans. Poor induction of N-terminal antibodies was also observed in a vaccination study performed in mice. IgG and IgM to all three regions of CSP play a role in mediating complement-fixation, which has important implications for malaria vaccine development.

Community-based molecular and serological surveillance of subclinical malaria in Myanmar.

BACKGROUND: In the Greater Mekong Subregion (GMS), current malaria surveillance strategies rely on a network of village health volunteers (VHVs) reporting the results of rapid diagnostic tests (RDTs), known to miss many asymptomatic infections. Integration of more sensitive diagnostic molecular and serological measures into the VHV network may improve surveillance of residual malaria transmission in hard-to-reach areas in the region and inform targeted interventions and elimination responses. However, data on residual malaria transmission that would be captured by these measures in the VHV-led testing and treatment surveillance network in the GMS is unknown. METHODS: A total of 114 VHVs were trained to collect dried blood spots from villagers undergoing routine RDTs as part of VHV-led active and passive case detection from April 2015 to June 2016. Samples were subjected to molecular testing (quantitative polymerase chain reaction [qPCR]) to determine Plasmodium falciparum and P. vivax infection and serological testing (against P. falciparum and P. vivax antigens) to determine exposure to P. falciparum and P. vivax. RESULTS: Over 15 months, 114 VHVs performed 32,194 RDTs and collected samples for molecular (n = 13,157) and serological (n = 14,128) testing. The prevalence of molecular-detectable P. falciparum and P. vivax infection was 3.2% compared to the 0.16% prevalence of Plasmodium spp. by RDT, highlighting the large burden of infections undetected by standard surveillance. Peaks in anti-P. falciparum, but not P. vivax, merozoite IgG seroprevalence coincided with seasonal P. falciparum transmission peaks, even in those with no molecularly detectable parasites. At the individual level, antibody seropositivity was associated with reduced odds of contemporaneous P. falciparum (OR for PfCSP 0.51 [95%CI 0.35, 0.76], p = 0.001, PfAMA1 0.70 [95%CI 0.52, 0.93], p = 0.01, and PfMSP2 0.81 [95%CI 0.61, 1.08], p = 0.15), but not P. vivax infection (OR PvAMA1 1.02 [95%CI 0.73, 1.43], p = 0.89) indicating a potential role of immunity in protection against molecular-detectable P. falciparum parasitaemia. CONCLUSIONS: We demonstrated that integration and implementation of sample collection for molecular and serological surveillance into networks of VHV servicing hard-to-reach populations in the GMS is feasible, can capture significant levels of ongoing undetected seasonal malaria transmission and has the potential to supplement current routine RDT testing. Improving malaria surveillance by advancing the integration of molecular and serological techniques, through centralised testing approaches or novel point-of-contact tests, will advance progress, and tracking, towards malaria elimination goals in the GMS.

Novel Virus-Like Particle Vaccine Encoding the Circumsporozoite Protein of Plasmodium falciparum Is Immunogenic and Induces Functional Antibody Responses in Mice.

RTS,S is the leading malaria vaccine in development, but has demonstrated only moderate protective efficacy in clinical trials. RTS,S is a virus-like particle (VLP) that uses the human hepatitis B virus as scaffold to display the malaria sporozoite antigen, circumsporozoite protein (CSP). Particle formation requires four-fold excess scaffold antigen, and as a result, CSP represents only a small portion of the final vaccine construct. Alternative VLP or nanoparticle platforms that reduce the amount of scaffold antigen and increase the amount of the target CSP antigen present in particles may enhance vaccine immunogenicity and efficacy. Here, we describe the production and characterization of a novel VLP that uses the small surface antigen (dS) of duck hepatitis B virus to display CSP. The CSP-dS fusion protein successfully formed VLPs without the need for excess scaffold antigen, and thus CSP represented a larger portion of the vaccine construct. CSP-dS formed large particles approximately 31-74 nm in size and were confirmed to display CSP on the surface. CSP-dS VLPs were highly immunogenic in mice and induced antibodies to multiple regions of CSP, even when administered at a lower vaccine dosage. Vaccine-induced antibodies demonstrated relevant functional activities, including Fc-dependent interactions with complement and Fcγ-receptors, previously identified as important in malaria immunity. Further, vaccine-induced antibodies had similar properties (epitope-specificity and avidity) to monoclonal antibodies that are protective in mouse models. Our novel platform to produce VLPs without excess scaffold protein has wide implications for the future development of vaccines for malaria and other infectious diseases.

Mechanisms and targets of Fcγ-receptor mediated immunity to malaria sporozoites.

A highly protective vaccine will greatly facilitate achieving and sustaining malaria elimination. Understanding mechanisms of antibody-mediated immunity is crucial for developing vaccines with high efficacy. Here, we identify key roles in humoral immunity for Fcγ-receptor (FcγR) interactions and opsonic phagocytosis of sporozoites. We identify a major role for neutrophils in mediating phagocytic clearance of sporozoites in peripheral blood, whereas monocytes contribute a minor role. Antibodies also promote natural killer cell activity. Mechanistically, antibody interactions with FcγRIII appear essential, with FcγRIIa also required for maximum activity. All regions of the circumsporozoite protein are targets of functional antibodies against sporozoites, and N-terminal antibodies have more activity in some assays. Functional antibodies are slowly acquired following natural exposure to malaria, being present among some exposed adults, but uncommon among children. Our findings reveal targets and mechanisms of immunity that could be exploited in vaccine design to maximize efficacy.

Multifunctional Antibodies Are Induced by the RTS,S Malaria Vaccine and Associated With Protection in a Phase 1/2a Trial.

BACKGROUND: RTS,S is the leading malaria vaccine candidate but only confers partial efficacy against malaria in children. RTS,S is based on the major Plasmodium falciparum sporozoite surface antigen, circumsporozoite protein (CSP). The induction of anti-CSP antibodies is important for protection; however, it is unclear how these protective antibodies function. METHODS: We quantified the induction of functional anti-CSP antibody responses in healthy malaria-naive adults (N = 45) vaccinated with RTS,S/AS01. This included the ability to mediate effector functions via the fragment crystallizable (Fc) region, such as interacting with human complement proteins and Fcγ-receptors (FcγRs) that are expressed on immune cells, which promote various immunological functions. RESULTS: Our major findings were (1) RTS,S-induced antibodies mediated Fc-dependent effector functions, (2) functional antibodies were generally highest after the second vaccine dose, (3) functional antibodies targeted multiple regions of CSP, (4) participants with higher levels of functional antibodies had a reduced probability of developing parasitemia following homologous challenge (P < .05), and (5) nonprotected subjects had higher levels of anti-CSP IgM. CONCLUSIONS: Our data suggest a role for Fc-dependent antibody effector functions in RTS,S-induced immunity. Enhancing the induction of these functional activities may be a strategy to improve the protective efficacy of RTS,S or other malaria vaccines. CLINICAL TRIALS REGISTRATION: NCT00075049.

Structure-Activity Studies of Truncated Latrunculin Analogues with Antimalarial Activity.

Malarial parasites employ actin dynamics for motility, and any disruption to these dynamics renders the parasites unable to effectively establish infection. Therefore, actin presents a potential target for malarial drug discovery, and naturally occurring actin inhibitors such as latrunculins are a promising starting point. However, the limited availability of the natural product and the laborious route for synthesis of latrunculins have hindered their potential development as drug candidates. In this regard, we recently described novel truncated latrunculins, with superior actin binding potency and selectivity towards P. falciparum actin than the canonical latrunculin B. In this paper, we further explore the truncated latrunculin core to summarize the SAR for inhibition of malaria motility. This study helps further understand the binding pattern of these analogues in order to develop them as drug candidates for malaria.

Malaria vaccine candidates displayed on novel virus-like particles are immunogenic and induce transmission-blocking activity.

The development of effective malaria vaccines remains a global health priority. Currently, the most advanced vaccine, known as RTS,S, has only shown modest efficacy in clinical trials. Thus, the development of more efficacious vaccines by improving the formulation of RTS,S for increased efficacy or to interrupt malaria transmission are urgently needed. The RTS,S vaccine is based on the presentation of a fragment of the sporozoite antigen on the surface of virus-like particles (VLPs) based on human hepatitis B virus (HBV). In this study, we have developed and evaluated a novel VLP platform based on duck HBV (known as Metavax) for malaria vaccine development. This platform can incorporate large and complex proteins into VLPs and is produced in a Hansenula cell line compatible with cGMP vaccine production. Here, we have established the expression of leading P. falciparum malaria vaccine candidates as VLPs. This includes Pfs230 and Pfs25, which are candidate transmission-blocking vaccine antigens. We demonstrated that the VLPs effectively induce antibodies to malaria vaccine candidates with minimal induction of antibodies to the duck-HBV scaffold antigen. Antibodies to Pfs230 also recognised native protein on the surface of gametocytes, and antibodies to both Pfs230 and Pfs25 demonstrated transmission-reducing activity in standard membrane feeding assays. These results establish the potential utility of this VLP platform for malaria vaccines, which may be suitable for the development of multi-component vaccines that achieve high vaccine efficacy and transmission-blocking immunity.

Induction and decay of functional complement-fixing antibodies by the RTS,S malaria vaccine in children, and a negative impact of malaria exposure.

BACKGROUND: Leading malaria vaccine, RTS,S, is based on the circumsporozoite protein (CSP) of sporozoites. RTS,S confers partial protection against malaria in children, but efficacy wanes relatively quickly after primary immunization. Vaccine efficacy has some association with anti-CSP IgG; however, it is unclear how these antibodies function, and how functional antibodies are induced and maintained over time. Recent studies identified antibody-complement interactions as a potentially important immune mechanism against sporozoites. Here, we investigated whether RTS,S vaccine-induced antibodies could function by interacting with complement. METHODS: Serum samples were selected from children in a phase IIb trial of RTS,S/AS02A conducted at two study sites of high and low malaria transmission intensity in Manhiça, Mozambique. Samples following primary immunization and 5-year post-immunization follow-up time points were included. Vaccine-induced antibodies were characterized by isotype, subclass, and epitope specificity, and tested for the ability to fix and activate complement. We additionally developed statistical methods to model the decay and determinants of functional antibodies after vaccination. RESULTS: RTS,S vaccination induced anti-CSP antibodies that were mostly IgG1, with some IgG3, IgG2, and IgM. Complement-fixing antibodies were effectively induced by vaccination, and targeted the central repeat and C-terminal regions of CSP. Higher levels of complement-fixing antibodies were associated with IgG that equally recognized both the central repeat and C-terminal regions of CSP. Older age and higher malaria exposure were significantly associated with a poorer induction of functional antibodies. There was a marked decay in functional complement-fixing antibodies within months after vaccination, as well as decays in IgG subclasses and IgM. Statistical modeling suggested the decay in complement-fixing antibodies was mostly attributed to the waning of anti-CSP IgG1, and to a lesser extent IgG3. CONCLUSIONS: We demonstrate for the first time that RTS,S can induce complement-fixing antibodies in young malaria-exposed children. The short-lived nature of functional responses mirrors the declining vaccine efficacy of RTS,S over time. The negative influence of age and malaria exposure on functional antibodies has implications for understanding vaccine efficacy in different settings. These findings provide insights into the mechanisms and longevity of vaccine-induced immunity that will help inform the future development of highly efficacious and long-lasting malaria vaccines.

Cellular dissection of malaria parasite invasion of human erythrocytes using viable Plasmodium knowlesi merozoites.

Plasmodium knowlesi, a zoonotic parasite causing severe-to-lethal malaria disease in humans, has only recently been adapted to continuous culture with human red blood cells (RBCs). In comparison with the most virulent human malaria, Plasmodium falciparum, there are, however, few cellular tools available to study its biology, in particular direct investigation of RBC invasion by blood-stage P. knowlesi merozoites. This leaves our current understanding of biological differences across pathogenic Plasmodium spp. incomplete. Here, we report a robust method for isolating viable and invasive P. knowlesi merozoites to high purity and yield. Using this approach, we present detailed comparative dissection of merozoite invasion (using a variety of microscopy platforms) and direct assessment of kinetic differences between knowlesi and falciparum merozoites. We go on to assess the inhibitory potential of molecules targeting discrete steps of invasion in either species via a quantitative invasion inhibition assay, identifying a class of polysulfonate polymer able to efficiently inhibit invasion in both, providing a foundation for pan-Plasmodium merozoite inhibitor development. Given the close evolutionary relationship between P. knowlesi and P. vivax, the second leading cause of malaria-related morbidity, this study paves the way for inter-specific dissection of invasion by all three major pathogenic malaria species.

Functional Conservation of the AMA1 Host-Cell Invasion Ligand Between P. falciparum and P. vivax: A Novel Platform to Accelerate Vaccine and Drug Development.

Plasmodium vivax and P. falciparum malaria species have diverged significantly in receptor-ligand interactions and host-cell invasion. One protein common to both is the merozoite invasion ligand AMA1. While the general structure of AMA1 is similar between species, their sequences are divergent. Surprisingly, it was possible to genetically replace PfAMA1 with PvAMA1 in P. falciparum parasites. PvAMA1 complemented PfAMA1 function and supported invasion of erythrocytes by P. falciparum. Genetically modified P. falciparum expressing PvAMA1 evaded the invasion inhibitory effects of antibodies to PfAMA1, demonstrating species specificity of functional antibodies. We generated antibodies to recombinant PvAMA1 that effectively inhibited invasion, confirming the function of PvAMA1 in genetically modified parasites. Results indicate significant molecular flexibility in AMA1 enabling conserved function despite substantial sequence divergence across species. This provides powerful new tools to quantify the inhibitory activities of antibodies or drugs targeting PvAMA1, opening new opportunities for vaccine and therapeutic development against P. vivax.

Low Levels of Human Antibodies to Gametocyte-Infected Erythrocytes Contrasts the PfEMP1-Dominant Response to Asexual Stages in P. falciparum Malaria.

Vaccines that target Plasmodium falciparum gametocytes have the potential to reduce malaria transmission and are thus attractive targets for malaria control. However, very little is known about human immune responses to gametocytes present in human hosts. We evaluated naturally-acquired antibodies to gametocyte-infected erythrocytes (gametocyte-IEs) of different developmental stages compared to other asexual parasite stages among naturally-exposed Kenyan residents. We found that acquired antibodies strongly recognized the surface of mature asexual-IEs, but there was limited reactivity to the surface of gametocyte-IEs of different stages. We used genetically-modified P. falciparum with suppressed expression of PfEMP1, the major surface antigen of asexual-stage IEs, to demonstrate that PfEMP1 is a dominant target of antibodies to asexual-IEs, in contrast to gametocyte-IEs. Antibody reactivity to gametocyte-IEs was similar to asexual-IEs lacking PfEMP1. Significant antibody reactivity to the surface of gametocytes was observed when outside of the host erythrocyte, including recognition of the major gametocyte antigen, Pfs230. This indicates that there is a deficiency of acquired antibodies to gametocyte-IEs despite the acquisition of antibodies to gametocyte antigens and asexual IEs. Our findings suggest that the acquisition of substantial immunity to the surface of gametocyte-IEs is limited, which may facilitate immune evasion to enable malaria transmission even in the face of substantial host immunity to malaria. Further studies are needed to understand the basis for the limited acquisition of antibodies to gametocytes and whether vaccine strategies can generate substantial immunity.

Truncated Latrunculins as Actin Inhibitors Targeting Plasmodium falciparum Motility and Host Cell Invasion.

Polymerization of the cytosolic protein actin is critical to cell movement and host cell invasion by the malaria parasite, Plasmodium falciparum. Any disruption to actin polymerization dynamics will render the parasite incapable of invading a host cell and thereby unable to cause infection. Here, we explore the potential of using truncated latrunculins as potential chemotherapeutics for the treatment of malaria. Exploration of the binding interactions of the natural actin inhibitor latrunculins with actin revealed how a truncated core of the inhibitor could retain its key interaction features with actin. This truncated core was synthesized and subjected to preliminary structure-activity relationship studies to generate a focused set of analogues. Biochemical analyses of these analogues demonstrate their 6-fold increased activity compared with that of latrunculin B against P. falciparum and a 16-fold improved selectivity ex vivo. These data establish the latrunculin core as a potential focus for future structure-based drug design of chemotherapeutics against malaria.

A novel approach to identifying patterns of human invasion-inhibitory antibodies guides the design of malaria vaccines incorporating polymorphic antigens.

BACKGROUND: The polymorphic nature of many malaria vaccine candidates presents major challenges to achieving highly efficacious vaccines. Presently, there is very little knowledge on the prevalence and patterns of functional immune responses to polymorphic vaccine candidates in populations to guide vaccine design. A leading polymorphic vaccine candidate against blood-stage Plasmodium falciparum is apical membrane antigen 1 (AMA1), which is essential for erythrocyte invasion. The importance of AMA1 as a target of acquired human inhibitory antibodies, their allele specificity and prevalence in populations is unknown, but crucial for vaccine design. METHODS: P. falciparum lines expressing different AMA1 alleles were genetically engineered and used to quantify functional antibodies from two malaria-exposed populations of adults and children. The acquisition of AMA1 antibodies was also detected using enzyme-linked immunosorbent assay (ELISA) and competition ELISA (using different AMA1 alleles) from the same populations. RESULTS: We found that AMA1 was a major target of naturally acquired invasion-inhibitory antibodies that were highly prevalent in malaria-endemic populations and showed a high degree of allele specificity. Significantly, the prevalence of inhibitory antibodies to different alleles varied substantially within populations and between geographic locations. Inhibitory antibodies to three specific alleles were highly prevalent (FVO and W2mef in Papua New Guinea; FVO and XIE in Kenya), identifying them for potential vaccine inclusion. Measurement of antibodies by standard or competition ELISA was not strongly predictive of allele-specific inhibitory antibodies. The patterns of allele-specific functional antibody responses detected with our novel assays may indicate that acquired immunity is elicited towards serotypes that are prevalent in each geographic location. CONCLUSIONS: These findings provide new insights into the nature and acquisition of functional immunity to a polymorphic vaccine candidate and strategies to quantify functional immunity in populations to guide rational vaccine design.

The association between naturally acquired IgG subclass specific antibodies to the PfRH5 invasion complex and protection from Plasmodium falciparum malaria.

Understanding the targets and mechanisms of human immunity to malaria is important for advancing the development of highly efficacious vaccines and serological tools for malaria surveillance. The PfRH5 and PfRipr proteins form a complex on the surface of P. falciparum merozoites that is essential for invasion of erythrocytes and are vaccine candidates. We determined IgG subclass responses to these proteins among malaria-exposed individuals in Papua New Guinea and their association with protection from malaria in a longitudinal cohort of children. Cytophilic subclasses, IgG1 and IgG3, were predominant with limited IgG2 and IgG4, and IgG subclass-specific responses were higher in older children and those with active infection. High IgG3 to PfRH5 and PfRipr were significantly and strongly associated with reduced risk of malaria after adjusting for potential confounding factors, whereas associations for IgG1 responses were generally weaker and not statistically significant. Results further indicated that malaria exposure leads to the co-acquisition of IgG1 and IgG3 to PfRH5 and PfRipr, as well as to other PfRH invasion ligands, PfRH2 and PfRH4. These findings suggest that IgG3 responses to PfRH5 and PfRipr may play a significant role in mediating naturally-acquired immunity and support their potential as vaccine candidates and their use as antibody biomarkers of immunity.

Structure-Activity Studies of ß-Hairpin Peptide Inhibitors of the Plasmodium falciparum AMA1-RON2 Interaction.

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 ß-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 ß-hairpin peptides to establish structure-activity relationships. The best mutations improved the binding affinity of the ß-hairpin peptide by ~7-fold for 3D7 AMA1 and ~14-fold for FVO AMA1. We determined the crystal structures of several ß-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.

Merozoite surface proteins in red blood cell invasion, immunity and vaccines against malaria.

Malaria accounts for an enormous burden of disease globally, with Plasmodium falciparum accounting for the majority of malaria, and P. vivax being a second important cause, especially in Asia, the Americas and the Pacific. During infection with Plasmodium spp., the merozoite form of the parasite invades red blood cells and replicates inside them. It is during the blood-stage of infection that malaria disease occurs and, therefore, understanding merozoite invasion, host immune responses to merozoite surface antigens, and targeting merozoite surface proteins and invasion ligands by novel vaccines and therapeutics have been important areas of research. Merozoite invasion involves multiple interactions and events, and substantial processing of merozoite surface proteins occurs before, during and after invasion. The merozoite surface is highly complex, presenting a multitude of antigens to the immune system. This complexity has proved challenging to our efforts to understand merozoite invasion and malaria immunity, and to developing merozoite antigens as malaria vaccines. In recent years, there has been major progress in this field, and several merozoite surface proteins show strong potential as malaria vaccines. Our current knowledge on this topic is reviewed, highlighting recent advances and research priorities.

PfRH5 as a candidate vaccine for Plasmodium falciparum malaria.

PfRH5 has recently emerged as a strong vaccine candidate for Plasmodium falciparum malaria. Antibodies inhibit invasion of erythrocytes by merozoites and blood-stage replication, and PfRH5 is a significant target of acquired human immunity. Recent studies have shown protective efficacy of PfRH5 vaccines in a non-human primate model of malaria.