Publications by authors named "Damien R Drew"

34 Publications

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

Front Immunol 2021 17;12:641421. Epub 2021 Mar 17.

Life Sciences, Burnet Institute, Melbourne, VIC, Australia.

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.
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http://dx.doi.org/10.3389/fimmu.2021.641421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010251PMC
March 2021

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

Nat Commun 2021 03 19;12(1):1742. Epub 2021 Mar 19.

Burnet Institute for Medical Research and Public Health, Melbourne, VIC, Australia.

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.
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http://dx.doi.org/10.1038/s41467-021-21998-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979888PMC
March 2021

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

ChemMedChem 2021 Feb 10;16(4):679-693. Epub 2020 Nov 10.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.

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.
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http://dx.doi.org/10.1002/cmdc.202000399DOI Listing
February 2021

Multi-functional antibodies are induced by the RTS,S malaria vaccine and associated with protection in a phase I/IIa trial.

J Infect Dis 2020 Mar 31. Epub 2020 Mar 31.

Burnet Institute, Melbourne, Australia.

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-naïve 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 i) RTS,S-induced antibodies mediate Fc-dependent effector functions, ii) functional antibodies were generally highest after the second vaccine dose; iii) functional antibodies targeted multiple regions of CSP, iv) participants with higher levels of functional antibodies had a reduced probability of developing parasitemia following homologous challenge (p<0.05); v) non-protected subjects had higher levels of anti-CSP IgM.

Conclusions: Our data suggests 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.
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http://dx.doi.org/10.1093/infdis/jiaa144DOI Listing
March 2020

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

PLoS One 2019 10;14(9):e0221733. Epub 2019 Sep 10.

Burnet Institute, Life Sciences, Melbourne, VIC, Australia.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0221733PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736250PMC
March 2020

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

BMC Med 2019 02 25;17(1):45. Epub 2019 Feb 25.

Burnet Institute, Melbourne, Australia.

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/AS02 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.
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http://dx.doi.org/10.1186/s12916-019-1277-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6388494PMC
February 2019

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

Front Immunol 2018 14;9:3126. Epub 2019 Jan 14.

Burnet Institute for Medical Research and Public Health, Melbourne, VIC, Australia.

Vaccines that target 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 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.
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http://dx.doi.org/10.3389/fimmu.2018.03126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340286PMC
October 2019

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

Sci Rep 2018 07 5;8(1):10165. Epub 2018 Jul 5.

Department of Life Sciences, Imperial College London, Sir Alexander Fleming Building, Exhibition Road, South Kensington, London, UK.

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.
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http://dx.doi.org/10.1038/s41598-018-28457-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033891PMC
July 2018

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

J Infect Dis 2018 01;217(3):498-507

Burnet Institute, Melbourne, Australia.

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.
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http://dx.doi.org/10.1093/infdis/jix583DOI Listing
January 2018

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

J Med Chem 2016 12 2;59(24):10994-11005. Epub 2016 Dec 2.

Monash Institute of Pharmaceutical Sciences, Monash University , Parkville, Victoria 3052, Australia.

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.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7224986PMC
December 2016

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

BMC Med 2016 Sep 23;14(1):144. Epub 2016 Sep 23.

The Burnet Institute of Medical Research and Public Health, 85 Commercial Road, Melbourne, Victoria, 3004, Australia.

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.
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http://dx.doi.org/10.1186/s12916-016-0691-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5034621PMC
September 2016

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

Sci Rep 2016 09 8;6:33094. Epub 2016 Sep 8.

Centre for Biomedical Research, Burnet Institute, Melbourne, Australia.

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.
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http://dx.doi.org/10.1038/srep33094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015043PMC
September 2016

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

J Mol Biol 2016 10 14;428(20):3986-3998. Epub 2016 Jul 14.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia. Electronic address:

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 K 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.
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http://dx.doi.org/10.1016/j.jmb.2016.07.001DOI Listing
October 2016

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

FEMS Microbiol Rev 2016 05 31;40(3):343-72. Epub 2016 Jan 31.

Burnet Institute for Medical Research and Public Health, 85 Commercial Road, Melbourne, Victoria, Australia Department of Microbiology, Monash University, Clayton, Victoria, Australia Department of Medicine, University of Melbourne, Parkville, Victoria, Australia.

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.
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http://dx.doi.org/10.1093/femsre/fuw001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4852283PMC
May 2016

PfRH5 as a candidate vaccine for Plasmodium falciparum malaria.

Trends Parasitol 2015 Mar 19;31(3):87-8. Epub 2015 Feb 19.

Burnet Institute, 85 Commercial Road, Melbourne, Victoria, Australia; Department of Microbiology, Monash University, Victoria, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.pt.2015.02.001DOI Listing
March 2015

Research priorities for the development and implementation of serological tools for malaria surveillance.

F1000Prime Rep 2014 4;6:100. Epub 2014 Nov 4.

Burnet Institute 85 Commercial Road, Melbourne, Victoria 3004 Australia ; School of Population Health and Department of Medicine (RMH), University of Melbourne Victoria 3010 Australia ; Department of Microbiology, Monash University Victoria 3800 Australia.

Surveillance is a key component of control and elimination programs. Malaria surveillance has been typically reliant on case reporting by health services, entomological estimates and parasitemia (Plasmodium species) point prevalence. However, these techniques become less sensitive and relatively costly as transmission declines. There is great potential for the development and application of serological biomarkers of malaria exposure as sero-surveillance tools to strengthen malaria control and elimination. Antibodies to malaria antigens are sensitive biomarkers of population-level malaria exposure and can be used to identify hotspots of malaria transmission, estimate transmission levels, monitor changes over time or the impact of interventions on transmission, confirm malaria elimination, and monitor re-emergence of malaria. Sero-surveillance tools could be used in reference laboratories or developed as simple point-of-care tests for community-based surveillance, and different applications and target populations dictate the technical performance required from assays that are determined by properties of antigens and antibody responses. To advance the development of sero-surveillance tools for malaria elimination, major gaps in our knowledge need to be addressed through further research. These include greater knowledge of potential antigens, the sensitivity and specificity of antibody responses, and the longevity of these responses and defining antigens and antibodies that differentiate between exposure to Plasmodium falciparum and P. vivax. Additionally, a better understanding of the influence of host factors, such as age, genetics, and comorbidities on antibody responses in different populations is needed.
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http://dx.doi.org/10.12703/P6-100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4229730PMC
January 2015

Limited antigenic diversity of Plasmodium falciparum apical membrane antigen 1 supports the development of effective multi-allele vaccines.

BMC Med 2014 Oct 16;12:183. Epub 2014 Oct 16.

The Burnet Institute of Medical Research and Public Health, 85 Commercial Road, Melbourne, Victoria, 3004, Australia.

Background: Polymorphism in antigens is a common mechanism for immune evasion used by many important pathogens, and presents major challenges in vaccine development. In malaria, many key immune targets and vaccine candidates show substantial polymorphism. However, knowledge on antigenic diversity of key antigens, the impact of polymorphism on potential vaccine escape, and how sequence polymorphism relates to antigenic differences is very limited, yet crucial for vaccine development. Plasmodium falciparum apical membrane antigen 1 (AMA1) is an important target of naturally-acquired antibodies in malaria immunity and a leading vaccine candidate. However, AMA1 has extensive allelic diversity with more than 60 polymorphic amino acid residues and more than 200 haplotypes in a single population. Therefore, AMA1 serves as an excellent model to assess antigenic diversity in malaria vaccine antigens and the feasibility of multi-allele vaccine approaches. While most previous research has focused on sequence diversity and antibody responses in laboratory animals, little has been done on the cross-reactivity of human antibodies.

Methods: We aimed to determine the extent of antigenic diversity of AMA1, defined by reactivity with human antibodies, and to aid the identification of specific alleles for potential inclusion in a multi-allele vaccine. We developed an approach using a multiple-antigen-competition enzyme-linked immunosorbent assay (ELISA) to examine cross-reactivity of naturally-acquired antibodies in Papua New Guinea and Kenya, and related this to differences in AMA1 sequence.

Results: We found that adults had greater cross-reactivity of antibodies than children, although the patterns of cross-reactivity to alleles were the same. Patterns of antibody cross-reactivity were very similar between populations (Papua New Guinea and Kenya), and over time. Further, our results show that antigenic diversity of AMA1 alleles is surprisingly restricted, despite extensive sequence polymorphism. Our findings suggest that a combination of three different alleles, if selected appropriately, may be sufficient to cover the majority of antigenic diversity in polymorphic AMA1 antigens. Antigenic properties were not strongly related to existing haplotype groupings based on sequence analysis.

Conclusions: Antigenic diversity of AMA1 is limited and a vaccine including a small number of alleles might be sufficient for coverage against naturally-circulating strains, supporting a multi-allele approach for developing polymorphic antigens as malaria vaccines.
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http://dx.doi.org/10.1186/s12916-014-0183-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4212128PMC
October 2014

Use of immunodampening to overcome diversity in the malarial vaccine candidate apical membrane antigen 1.

Infect Immun 2014 Nov 25;82(11):4707-17. Epub 2014 Aug 25.

Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia

Apical membrane antigen 1 (AMA1) is a leading malarial vaccine candidate; however, its polymorphic nature may limit its success in the field. This study aimed to circumvent AMA1 diversity by dampening the antibody response to the highly polymorphic loop Id, previously identified as a major target of strain-specific, invasion-inhibitory antibodies. To achieve this, five polymorphic residues within this loop were mutated to alanine, glycine, or serine in AMA1 of the 3D7 and FVO Plasmodium falciparum strains. Initially, the corresponding antigens were displayed on the surface of bacteriophage, where the alanine and serine but not glycine mutants folded correctly. The alanine and serine AMA1 mutants were expressed in Escherichia coli, refolded in vitro, and used to immunize rabbits. Serological analyses indicated that immunization with a single mutated form of 3D7 AMA1 was sufficient to increase the cross-reactive antibody response. Targeting the corresponding residues in an FVO backbone did not achieve this outcome. The inclusion of at least one engineered form of AMA1 in a biallelic formulation resulted in an antibody response with broader reactivity against different AMA1 alleles than combining the wild-type forms of 3D7 and FVO AMA1 alleles. For one combination, this extended to an enhanced relative growth inhibition of a heterologous parasite line, although this was at the cost of reduced overall inhibitory activity. These results suggest that targeted mutagenesis of AMA1 is a promising strategy for overcoming antigenic diversity in AMA1 and reducing the number of variants required to induce an antibody response that protects against a broad range of Plasmodium falciparum AMA1 genotypes. However, optimization of the immunization regime and mutation strategy will be required for this potential to be realized.
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http://dx.doi.org/10.1128/IAI.02061-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249334PMC
November 2014

Overcoming antigenic diversity by enhancing the immunogenicity of conserved epitopes on the malaria vaccine candidate apical membrane antigen-1.

PLoS Pathog 2013 26;9(12):e1003840. Epub 2013 Dec 26.

Walter Reed Army Institute of Research, Silver Spring, Maryland, United States of America.

Malaria vaccine candidate Apical Membrane Antigen-1 (AMA1) induces protection, but only against parasite strains that are closely related to the vaccine. Overcoming the AMA1 diversity problem will require an understanding of the structural basis of cross-strain invasion inhibition. A vaccine containing four diverse allelic proteins 3D7, FVO, HB3 and W2mef (AMA1 Quadvax or QV) elicited polyclonal rabbit antibodies that similarly inhibited the invasion of four vaccine and 22 non-vaccine strains of P. falciparum. Comparing polyclonal anti-QV with antibodies against a strain-specific, monovalent, 3D7 AMA1 vaccine revealed that QV induced higher levels of broadly inhibitory antibodies which were associated with increased conserved face and domain-3 responses and reduced domain-2 response. Inhibitory monoclonal antibodies (mAb) raised against the QV reacted with a novel cross-reactive epitope at the rim of the hydrophobic trough on domain-1; this epitope mapped to the conserved face of AMA1 and it encompassed the 1e-loop. MAbs binding to the 1e-loop region (1B10, 4E8 and 4E11) were ∼10-fold more potent than previously characterized AMA1-inhibitory mAbs and a mode of action of these 1e-loop mAbs was the inhibition of AMA1 binding to its ligand RON2. Unlike the epitope of a previously characterized 3D7-specific mAb, 1F9, the 1e-loop inhibitory epitope was partially conserved across strains. Another novel mAb, 1E10, which bound to domain-3, was broadly inhibitory and it blocked the proteolytic processing of AMA1. By itself mAb 1E10 was weakly inhibitory but it synergized with a previously characterized, strain-transcending mAb, 4G2, which binds close to the hydrophobic trough on the conserved face and inhibits RON2 binding to AMA1. Novel inhibition susceptible regions and epitopes, identified here, can form the basis for improving the antigenic breadth and inhibitory response of AMA1 vaccines. Vaccination with a few diverse antigenic proteins could provide universal coverage by redirecting the immune response towards conserved epitopes.
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http://dx.doi.org/10.1371/journal.ppat.1003840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873463PMC
September 2014

Plasticity in transmission strategies of the malaria parasite, Plasmodium chabaudi: environmental and genetic effects.

Evol Appl 2013 Feb 10;6(2):365-76. Epub 2012 Oct 10.

Boyd Orr Centre for Population and Ecosystem Health, Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow Glasgow, UK ; Department of Systems and Computational Biology, Albert Einstein College of Medicine Bronx, NY, USA.

Parasites may alter their behaviour to cope with changes in the within-host environment. In particular, investment in transmission may alter in response to the availability of parasite resources or host immune responses. However, experimental and theoretical studies have drawn conflicting conclusions regarding parasites' optimal (adaptive) responses to deterioration in habitat quality. We analyse data from acute infections with six genotypes of the rodent malaria species Plasmodium chabaudi to quantify how investment in transmission (gametocytes) is influenced by the within-host environment. Using a minimum of modelling assumptions, we find that proportional investment in gametocytogenesis increases sharply with host anaemia and also increases at low parasite densities. Further, stronger dependence of investment on parasite density is associated with greater virulence of the parasite genotype. Our study provides a robust quantitative framework for studying parasites' responses to the host environment and whether these responses are adaptive, which is crucial for predicting the short-term and evolutionary impact of transmission-blocking treatments for parasitic diseases.
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http://dx.doi.org/10.1111/eva.12005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3586624PMC
February 2013

Defining the antigenic diversity of Plasmodium falciparum apical membrane antigen 1 and the requirements for a multi-allele vaccine against malaria.

PLoS One 2012 5;7(12):e51023. Epub 2012 Dec 5.

The Burnet Institute for Medical Research and Public Health, Melbourne, Australia.

Apical Membrane Antigen 1 (AMA1) is a leading malaria vaccine candidate and a target of naturally-acquired human immunity. Plasmodium falciparum AMA1 is polymorphic and in vaccine trials it induces strain-specific protection. This antigenic diversity is a major roadblock to development of AMA1 as a malaria vaccine and understanding how to overcome it is essential. To assess how AMA1 antigenic diversity limits cross-strain growth inhibition, we assembled a panel of 18 different P. falciparum isolates which are broadly representative of global AMA1 sequence diversity. Antibodies raised against four well studied AMA1 alleles (W2Mef, 3D7, HB3 and FVO) were tested for growth inhibition of the 18 different P. falciparum isolates in growth inhibition assays (GIA). All antibodies demonstrated substantial cross-inhibitory activity against different isolates and a mixture of the four different AMA1 antibodies inhibited all 18 isolates tested, suggesting significant antigenic overlap between AMA1 alleles and limited antigenic diversity of AMA1. Cross-strain inhibition by antibodies was only moderately and inconsistently correlated with the level of sequence diversity between AMA1 alleles, suggesting that sequence differences are not a strong predictor of antigenic differences or the cross-inhibitory activity of anti-allele antibodies. The importance of the highly polymorphic C1-L region for inhibitory antibodies and potential vaccine escape was assessed by generating novel transgenic P. falciparum lines for testing in GIA. While the polymorphic C1-L epitope was identified as a significant target of some growth-inhibitory antibodies, these antibodies only constituted a minor proportion of the total inhibitory antibody repertoire, suggesting that the antigenic diversity of inhibitory epitopes is limited. Our findings support the concept that a multi-allele AMA1 vaccine would give broad coverage against the diversity of AMA1 alleles and establish new tools to define polymorphisms important for vaccine escape.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0051023PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3515520PMC
May 2013

Competition and the evolution of reproductive restraint in malaria parasites.

Am Nat 2011 Mar;177(3):358-67

Institute of Evolutionary Biology, University of Edinburgh, School of Biological Sciences, United Kingdom.

All organisms must trade off resource allocation between different life processes that determine their survival and reproduction. Malaria parasites replicate asexually in the host but must produce sexual stages to transmit between hosts. Because different specialized stages are required for these functions, the division of resources between these life-history components is a key problem for natural selection to solve. Despite the medical and economic importance of these parasites, their reproductive strategies remain poorly understood and often seem counterintuitive. Here, we tested recent theory predicting that in-host competition shapes how parasites trade off investment in in-host replication relative to between-host transmission. We demonstrate, across several genotypes, that Plasmodium chabaudi parasites detect the presence of competing genotypes and facultatively respond by reducing their investment in sexual stages in the manner predicted to maximize their competitive ability. Furthermore, we show that genotypes adjust their allocation to sexual stages in line with the availability of exploitable red blood cell resources. Our findings are predicted by evolutionary theory developed to explain life-history trade-offs in more traditionally studied multicellular taxa and suggest that the answer to the long-standing question of why so few transmission stages are produced is that in most natural infections heavy investment in reproduction may compromise in-host survival.
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http://dx.doi.org/10.1086/658175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3939351PMC
March 2011

Chemotherapy, within-host ecology and the fitness of drug-resistant malaria parasites.

Evolution 2010 Oct 19;64(10):2952-68. Epub 2010 Aug 19.

Center for Infectious Disease Dynamics, Departments of Biology and Entomology, Pennsylvania State University, University Park, Pennsylvania 16827, USA.

A major determinant of the rate at which drug-resistant malaria parasites spread through a population is the ecology of resistant and sensitive parasites sharing the same host. Drug treatment can significantly alter this ecology by removing the drug-sensitive parasites, leading to competitive release of resistant parasites. Here, we test the hypothesis that the spread of resistance can be slowed by reducing drug treatment and hence restricting competitive release. Using the rodent malaria model Plasmodium chabaudi, we found that low-dose chemotherapy did reduce competitive release. A higher drug dose regimen exerted stronger positive selection on resistant parasites for no detectable clinical gain. We estimated instantaneous selection coefficients throughout the course of replicate infections to analyze the temporal pattern of the strength and direction of within-host selection. The strength of selection on resistance varied through the course of infections, even in untreated infections, but increased immediately following drug treatment, particularly in the high-dose groups. Resistance remained under positive selection for much longer than expected from the half life of the drug. Although there are many differences between mice and people, our data do raise the question whether the aggressive treatment regimens aimed at complete parasite clearance are the best resistance-management strategies for humans.
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http://dx.doi.org/10.1111/j.1558-5646.2010.01068.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3066636PMC
October 2010

Sex ratio adjustment and kin discrimination in malaria parasites.

Nature 2008 May;453(7195):609-14

Institute of Evolutionary Biology, Ashworth Laboratories, School of Biological Science, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, UK.

Malaria parasites and related Apicomplexans are the causative agents of the some of the most serious infectious diseases of humans, companion animals, livestock and wildlife. These parasites must undergo sexual reproduction to transmit from vertebrate hosts to vectors, and their sex ratios are consistently female-biased. Sex allocation theory, a cornerstone of evolutionary biology, is remarkably successful at explaining female-biased sex ratios in multicellular taxa, but has proved controversial when applied to malaria parasites. Here we show that, as predicted by theory, sex ratio is an important fitness-determining trait and Plasmodium chabaudi parasites adjust their sex allocation in response to the presence of unrelated conspecifics. This suggests that P. chabaudi parasites use kin discrimination to evaluate the genetic diversity of their infections, and they adjust their behaviour in response to environmental cues. Malaria parasites provide a novel way to test evolutionary theory, and support the generality and power of a darwinian approach.
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http://dx.doi.org/10.1038/nature06954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807728PMC
May 2008

Truncation of Plasmodium berghei merozoite surface protein 8 does not affect in vivo blood-stage development.

Mol Biochem Parasitol 2008 May 14;159(1):69-72. Epub 2008 Feb 14.

The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3050, Australia.

Merozoite surface protein 8 (MSP8) has shown promise as a vaccine candidate in the Plasmodium yoelii rodent malaria model and has a proposed role in merozoite invasion of erythrocytes. However, the temporal expression and localisation of MSP8 are unusual for a merozoite antigen. Moreover, in Plasmodium falciparum the MSP8 gene could be disrupted with no apparent effect on invitro growth. To address the invivo function of full-length MSP8, we truncated MSP8 in the rodent parasite Plasmodium berghei. PbDeltaMSP8 disruptant parasites displayed a normal blood-stage growth rate but no increase in reticulocyte preference, a phenomenon observed in P. yoelii MSP8 vaccinated mice. Expression levels of erythrocyte surface antigens were similar in P. berghei wild-type and PbDeltaMSP8-infected erythrocytes, suggesting that a parasitophorous vacuole function for MSP8 does not involve global trafficking of such antigens. These data demonstrate that a full-length membrane-associated form of PbMSP8 is not essential for blood-stage growth.
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http://dx.doi.org/10.1016/j.molbiopara.2008.01.005DOI Listing
May 2008

Development of reverse-transcription PCR techniques to analyse the density and sex ratio of gametocytes in genetically diverse Plasmodium chabaudi infections.

Mol Biochem Parasitol 2007 Dec 21;156(2):199-209. Epub 2007 Aug 21.

Institute of Evolution, Ashworth Laboratories, University of Edinburgh, Edinburgh EH9 3JT, UK.

We have developed cross-genotype and genotype-specific quantitative reverse-transcription PCR (qRT-PCR) assays to detect and quantify the number of parasites, transmission stages (gametocytes) and male gametocytes in blood stage Plasmodium chabaudi infections. Our cross-genotype assays are reliable, repeatable and generate counts that correlate strongly (R(2)s>90%) with counts expected from blood smears. Our genotype-specific assays can distinguish and quantify different stages of genetically distinct parasite clones (genotypes) in mixed infections and are as sensitive as our cross-genotype assays. Using these assays we show that gametocyte density and gametocyte sex ratios vary during infections for two genetically distinct parasite lines (genotypes) and present the first data to reveal how sex ratio is affected when each genotype experiences competition in mixed-genotype infections. Successful infection of mosquito vectors depends on both gametocyte density and their sex ratio and we discuss the implications of competition in genetically diverse infections for transmission success.
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http://dx.doi.org/10.1016/j.molbiopara.2007.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3818572PMC
December 2007

Transmission stage investment of malaria parasites in response to in-host competition.

Proc Biol Sci 2007 Oct;274(1625):2629-38

Institutes of Evolution, Immunology and Infection Research, Ashworth Laboratories, School of Biological Sciences, University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JT, UK.

Conspecific competition occurs in a multitude of organisms, particularly in parasites, where several clones are commonly sharing limited resources inside their host. In theory, increased or decreased transmission investment might maximize parasite fitness in the face of competition, but, to our knowledge, this has not been tested experimentally. We developed and used a clone-specific, stage-specific, quantitative PCR protocol to quantify Plasmodium chabaudi replication and transmission stage densities in mixed-clone infections. We co-infected mice from two strains with an avirulent and virulent parasite clone and found competitive suppression of in-host (blood-stage) parasite densities and generally corresponding reductions in transmission stage production, with the virulent clone obtaining overall competitive superiority. In response to competitive suppression, there was little evidence of any alteration in transmission stage investment, apart from a small reduction by one of the two clones in one of the two host strains. This alteration did not result in a competitive advantage, although it might have reduced the disadvantage. This study supports much of the current literature, which predicts that conspecific in-host competition will result in a competitive advantage and positive selection for virulent clones and thus the evolution of higher virulence.
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http://dx.doi.org/10.1098/rspb.2007.0873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1975767PMC
October 2007

A set of glycosylphosphatidyl inositol-anchored membrane proteins of Plasmodium falciparum is refractory to genetic deletion.

Infect Immun 2006 Jul;74(7):4330-8

Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3050, Australia.

Targeted gene disruption has proved to be a powerful approach for studying the function of important ligands involved in erythrocyte invasion by the extracellular merozoite form of the human malaria parasite, Plasmodium falciparum. Merozoite invasion proceeds via a number of seemingly independent alternate pathways, such that entry can proceed with parasites lacking particular ligand-receptor interactions. To date, most focus in this regard has been on single-pass (type 1) membrane proteins that reside in the secretory organelles. Another class of merozoite proteins likely to include ligands for erythrocyte receptors are the glycosylphosphatidyl inositol (GPI)-anchored membrane proteins that coat the parasite surface and/or reside in the apical organelles. Several of these are prominent vaccine candidates, although their functions remain unknown. Here, we systematically attempted to disrupt the genes encoding seven of the known GPI-anchored merozoite proteins of P. falciparum by using a double-crossover gene-targeting approach. Surprisingly, and in apparent contrast to other merozoite antigen classes, most of the genes (six of seven) encoding GPI-anchored merozoite proteins are refractory to genetic deletion, with the exception being the gene encoding merozoite surface protein 5 (MSP-5). No distinguishable growth rate or invasion pathway phenotype was detected for the msp-5 knockout line, although its presence as a surface-localized protein was confirmed.
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http://dx.doi.org/10.1128/IAI.00054-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1489731PMC
July 2006

Plasmodium falciparum merozoite surface protein 8 is a ring-stage membrane protein that localizes to the parasitophorous vacuole of infected erythrocytes.

Infect Immun 2005 Jul;73(7):3912-22

The Walter & Eliza Hall Institute for Medical Research, 1G Royal Parade, Parkville, VIC 3050, Australia.

To date, the following seven glycosylphosphatidylinositol (GPI)-anchored merozoite antigens have been described in Plasmodium falciparum: merozoite-associated surface protein 1 (MSP-1), MSP-2, MSP-4, MSP-5, MSP-8, MSP-10, and the rhoptry-associated membrane antigen. Of these, MSP-1, MSP-8, and MSP-10 possess a double epidermal growth factor (EGF)-like domain at the C terminus, and these modules are considered potential targets of protective immunity. In this study, we found that surprisingly, P. falciparum MSP-8 is transcribed and translated in the ring stage and is absent from the surface of merozoites. MSP-8 is the only GPI-anchored protein known to be expressed at this time. It is synthesized as a mature 80-kDa protein which is rapidly processed to a C-terminal 17-kDa species that contains the double EGF module. As determined by a combination of immunofluorescence and membrane purification approaches, it appears likely that MSP-8 initially localizes to the parasite plasma membrane in the ring stage. Although the C-terminal 17-kDa fragment is present in more mature stages, at these times it is found in the food vacuole. We successfully disrupted the MSP-8 gene in P. falciparum, a process that validated the specificity of the antibodies used in this study and also demonstrated that MSP-8 does not play a role essential to maintenance of the erythrocyte cycle. This finding, together with the observation that MSP-8 is exclusively intracellular, casts doubt over the viability of this antigen as a vaccine. However, it is still possible that MSP-8 is involved in an early parasitophorous vacuole function that is significant for pathogenesis in the human host.
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http://dx.doi.org/10.1128/IAI.73.7.3912-3922.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1168550PMC
July 2005

A common cross-species function for the double epidermal growth factor-like modules of the highly divergent plasmodium surface proteins MSP-1 and MSP-8.

J Biol Chem 2004 May 19;279(19):20147-53. Epub 2004 Feb 19.

Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Melbourne, Victoria 3050, Australia.

An understanding of structural and functional constraints on the C-terminal double epidermal growth factor (EGF)-like modules of merozoite surface protein (MSP)-1 and related proteins is of importance to the development of these molecules as malaria vaccines and drug targets. Using allelic replacement, we show that Plasmodium falciparum parasites can invade erythrocytes and grow efficiently in the absence of an MSP-1 protein with authentic MSP-1 EGF domains. In this mutant parasite line, the MSP-1 EGFs were replaced by the corresponding double EGF module from P. berghei MSP-8, the sequence of which shares only low identity with its MSP-1 counterpart. Hence, the C-terminal EGF domains of at least some Plasmodium surface proteins appear to perform the same function in asexual blood-stage development. Mapping the surface location of the few residues that are common to these functionally complementary EGF modules revealed the presence of a highly conserved pocket of potential functional significance. In contrast to MSP-8, an even more divergent double EGF module, that from the sexual stage protein PbS25, was not capable of complementing MSP-1 EGF function. More surprisingly, two chimeric double EGF modules comprising hybrids of the EGF domains from P. falciparum and P. chabaudi MSP-1 were also not capable of replacing the P. falciparum MSP-1 EGF module. Together, these data suggest that although the MSP-1 EGFs can accommodate extensive sequence diversity, there appear to be constraints that may restrict the simple accumulation of point mutations in the face of immune pressure in the field.
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http://dx.doi.org/10.1074/jbc.M401114200DOI Listing
May 2004