Erythrocyte Invasion Research Articles

The Role and Mechanism of Erythrocyte Invasion by Francisella tularensis.

Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensis invades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs) has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS) was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes), dotU, or iglC (two genes encoding T6SS machinery) severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake (Pseudechis guttatus), which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks.

Role of Francisella tularensis PhoH-Like Protein in Erythrocyte Invasion and Pathogenesis

Francisella tularensis is the cause of the disease tularemia. This bacterium usually infects the host via arthropod bites. F. tularensis is able to invade mammalian erythrocytes which increases tick colonization following a blood meal. We hypothesized that bacterial genes involved in erythrocyte invasion would be upregulated in the presence of red blood cells. mRNA profiling experiments revealed that FTL_0885 was one of the most highly induced genes in the presence of erythrocytes. We initially attempted to generate deletion and disruption mutants of FTL_0885. However, these attempts were unsuccessful likely due to the proximity FTL_0885 to a neighboring essential gene. A transposon insertion mutation was available in the homolog to FTL_0885 in a related bacterium, F. novicida. F. novicida is not a human pathogen and its ability to invade erythrocytes is poorly characterized. Therefore, we are reconstructing this mutation in the F. tularensis LVS background so that we may study the effects of this gene on erythrocyte invasion. Also, because erythrocytes may contain cues important for pathogenesis, we also were interested in determining whether the FTL_0885 mutant is attenuated. To test this, the mutant bacteria will be evaluated for their ability to replicate in host macrophages in vitro. In addition, the chicken embryo infection model will be used to evaluate in vivo pathogenesis.

Population genetic structure and adaptation of malaria parasites on the edge of endemic distribution.

To determine whether the major human malaria parasite Plasmodium falciparum exhibits fragmented population structure or local adaptation at the northern limit of its African distribution where the dry Sahel zone meets the Sahara, samples were collected from diverse locations within Mauritania over a range of ~1000 km. Microsatellite genotypes were obtained for 203 clinical infection samples from eight locations, and Illumina paired‐end sequences were obtained to yield high coverage genomewide single nucleotide polymorphism (SNP) data for 65 clinical infection samples from four locations. Most infections contained single parasite genotypes, reflecting low rates of transmission and superinfection locally, in contrast to the situation seen in population samples from countries further south. A minority of infections shared related or identical genotypes locally, indicating some repeated transmission of parasite clones without recombination. This caused some multilocus linkage disequilibrium and local divergence, but aside from the effect of repeated genotypes there was minimal differentiation between locations. Several chromosomal regions had elevated integrated haplotype scores (|iHS|) indicating recent selection, including those containing drug resistance genes. A genomewide FST scan comparison with previous sequence data from an area in West Africa with higher infection endemicity indicates that regional gene flow prevents genetic isolation, but revealed allele frequency differentiation at three drug resistance loci and an erythrocyte invasion ligand gene. Contrast of extended haplotype signatures revealed none to be unique to Mauritania. Discrete foci of infection on the edge of the Sahara are genetically highly connected to the wider continental parasite population, and local elimination would be difficult to achieve without very substantial reduction in malaria throughout the region.

Purification and antiparasitic activity of a few legume serine proteinase inhibitors: Effect on erythrocyte invasion, schizont rupture and proteolytic processing of the Plasmodium falciparum AMA1 protein

Abstract Legume proteinase inhibitors (PI/s) abrogate proteolytic activity of proteins and cause adverse effects on growth. In this study, two legume PIs – Archidendron ellipticum Trypsin Inhibitor (AeTI) and Derris trifoliata Trypsin Chymotrypsin Inhibitor (DtTCI) were purified and used along with standard, Soybean Trypsin Inhibitor (STI), either singly or in combination, as antiplasmodial agents against two Plasmodium forms (Pf 3D7/Pf FCR3). Both AeTI/DtTCI were purified to homogeneity by HPLC and showed over 98% trypsin/chymotrypsin inhibition, respectively. DtTCI severely arrested growth of both the Plasmodium forms (IC50 of 9.59 μM and 16.86 μM, respectively). In addition, combination of DtTCI/AeTI had synergistic effect against both Pf 3D7 (FIC – 0.19) and Pf FCR3 (FIC − 0.23). Combinations of DtTCI/STI and AeTI/STI showed additive effects for the parasite forms. Time-course studies indicated DtTCI and combination of DtTCI/AeTI, to be potent inhibitor of schizont rupture. Antiproteolytic action of the PIs on Pf Apical Merozoite Antigen 1 (AMA1) protein revealed that none of the inhibitors (singly/combinations) affected the primary proteolysis of PfAMA1 protein. Notably however, the normal secondary proteolysis of PfAMA1 was abrogated by both DtTCI (singly/combinations) and AeTI. Incidentally, the proteolytic processing of PfAMA1 remained unaffected following STI treatment.

The Plasmodium rhoptry associated protein complex is important for parasitophorous vacuole membrane structure and intraerythrocytic parasite growth.

Plasmodium parasites must invade erythrocytes in order to cause the disease malaria. The invasion process involves the coordinated secretion of parasite proteins from apical organelles that include the rhoptries. The rhoptry is comprised of two compartments: the neck and the bulb. Rhoptry neck proteins are involved in host cell adhesion and formation of the tight junction that forms between the invading parasite and erythrocyte, whereas the role of rhoptry bulb proteins remains ill-defined due to the lack of functional studies. In this study, we show that the rhoptry-associated protein (RAP) complex is not required for rhoptry morphology or erythrocyte invasion. Instead, post-invasion when the parasite is bounded by a parasitophorous vacuolar membrane (PVM), the RAP complex facilitates the survival of the parasite in its new intracellular environment. Consequently, conditional knockdown of members of the RAP complex leads to altered PVM structure, delayed intra-erythrocytic growth, and reduced parasitaemias in infected mice. This study provides evidence that rhoptry bulb proteins localising to the parasite-host cell interface are not simply by-products of the invasion process but contribute to the growth of Plasmodium in vivo.

The Plasmodium falciparum rhoptry protein RhopH3 plays essential roles in host cell invasion and nutrient uptake.

Merozoites of the protozoan parasite responsible for the most virulent form of malaria, Plasmodium falciparum, invade erythrocytes. Invasion involves discharge of rhoptries, specialized secretory organelles. Once intracellular, parasites induce increased nutrient uptake by generating new permeability pathways (NPP) including a Plasmodium surface anion channel (PSAC). RhopH1/Clag3, one member of the three-protein RhopH complex, is important for PSAC/NPP activity. However, the roles of the other members of the RhopH complex in PSAC/NPP establishment are unknown and it is unclear whether any of the RhopH proteins play a role in invasion. Here we demonstrate that RhopH3, the smallest component of the complex, is essential for parasite survival. Conditional truncation of RhopH3 substantially reduces invasive capacity. Those mutant parasites that do invade are defective in nutrient import and die. Our results identify a dual role for RhopH3 that links erythrocyte invasion to formation of the PSAC/NPP essential for parasite survival within host erythrocytes.

Lack of Association of CD55 Receptor Genetic Variants and Severe Malaria in Ghanaian Children.

In a recent report, the cellular receptor CD55 was identified as a molecule essential for the invasion of human erythrocytes by Plasmodium falciparum, the causal agent of the most severe form of malaria. As this invasion process represents a critical step during infection with the parasite, it was hypothesized that genetic variants in the gene could affect severe malaria (SM) susceptibility. We performed high-resolution variant discovery of rare and common genetic variants in the human CD55 gene. Association testing of these variants in over 1700 SM cases and unaffected control individuals from the malaria-endemic Ashanti Region in Ghana, West Africa, were performed on the basis of single variants, combined rare variant analyses, and reconstructed haplotypes. A total of 26 genetic variants were detected in coding and regulatory regions of CD55. Five variants were previously unknown. None of the single variants, rare variants, or haplotypes showed evidence for association with SM or P. falciparum density. Here, we present the first comprehensive analysis of variation in the CD55 gene in the context of SM and show that genetic variants present in a Ghanaian study group appear not to influence susceptibility to the disease.

An essential dual-function complex mediates erythrocyte invasion and channel-mediated nutrient uptake in malaria parasites.

Malaria parasites evade immune detection by growth and replication within erythrocytes. After erythrocyte invasion, the intracellular pathogen must increase host cell uptake of nutrients from plasma. Here, we report that the parasite-encoded RhopH complex contributes to both invasion and channel-mediated nutrient uptake. As rhoph2 and rhoph3 gene knockouts were not viable in the human P. falciparum pathogen, we used conditional knockdowns to determine that the encoded proteins are essential and to identify their stage-specific functions. We exclude presumed roles for RhopH2 and CLAG3 in erythrocyte invasion but implicate a RhopH3 contribution either through ligand-receptor interactions or subsequent parasite internalization. These proteins then traffic via an export translocon to the host membrane, where they form a nutrient channel. Knockdown of either RhopH2 or RhopH3 disrupts the entire complex, interfering with organellar targeting and subsequent trafficking. Therapies targeting this complex should attack the pathogen at two critical points in its cycle.

Structure of the malaria vaccine candidate antigen CyRPA and its complex with a parasite invasion inhibitory antibody.

Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here, we present the crystal structures of PfCyRPA and its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. PfCyRPA adopts a 6-bladed β-propeller structure with similarity to the classic sialidase fold, but it has no sialidase activity and fulfills a purely non-enzymatic function. Characterization of the epitope recognized by protective antibodies may facilitate design of peptidomimetics to focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in vaccines.

Structural basis for inhibition of erythrocyte invasion by antibodies to Plasmodium falciparum protein CyRPA.

Plasmodium falciparum causes malaria in humans with over 450,000 deaths annually. The asexual blood stage involves invasion of erythrocytes by merozoites, in which they grow and divide to release daughter merozoites, which in turn invade new erythrocytes perpetuating the cycle responsible for malaria. A key step in merozoite invasion is the essential binding of PfRh5/CyRPA/PfRipr complex to basigin, a step linked to the formation of a pore between merozoites and erythrocytes. We show CyRPA interacts directly with PfRh5. An invasion inhibitory monoclonal antibody to CyRPA blocks binding of CyRPA to PfRh5 and complex formation thus illuminating the molecular mechanism for inhibition of parasite growth. We determined the crystal structures of CyRPA alone and in complex with an antibody Fab fragment. CyRPA has a six-bladed β-propeller fold, and we identify the region that interacts with PfRh5. This functionally conserved epitope is a potential target for vaccines against P. falciparum.

P113 is a merozoite surface protein that binds the N terminus of Plasmodium falciparum RH5

Invasion of erythrocytes by Plasmodium falciparum merozoites is necessary for malaria pathogenesis and is therefore a primary target for vaccine development. RH5 is a leading subunit vaccine candidate because anti-RH5 antibodies inhibit parasite growth and the interaction with its erythrocyte receptor basigin is essential for invasion. RH5 is secreted, complexes with other parasite proteins including CyRPA and RIPR, and contains a conserved N-terminal region (RH5Nt) of unknown function that is cleaved from the native protein. Here, we identify P113 as a merozoite surface protein that directly interacts with RH5Nt. Using recombinant proteins and a sensitive protein interaction assay, we establish the binding interdependencies of all the other known RH5 complex components and conclude that the RH5Nt-P113 interaction provides a releasable mechanism for anchoring RH5 to the merozoite surface. We exploit these findings to design a chemically synthesized peptide corresponding to RH5Nt, which could contribute to a cost-effective malaria vaccine.

Dynamics and Topology of Heparin Interaction with Malaria Parasite-Infected Erythrocytes Revealed by Live Cell Time Lapse Microscopy

Heparin strongly inhibits malaria parasite invasion of human erythrocytes, but the mechanism is poorly defined. Using live cell microscopy we show that egress of Plasmodium falciparum from erythrocytes is also inhibited by heparin in a dose-dependent manner. Heparin blocks host erythrocyte membrane rupture (but not parasite vacuolar membrane rupture) and merozoite dispersion. Fluorescent live-cell microscopy of infected erythrocytes releasing mature merozoites showed that heparin binds different targets independently and sequentially, consistent with the inhibition of both parasite egress and invasion. Heparin enters infected erythrocytes through pores, which we previously showed to form in the erythrocyte membrane right before egress; it binds two targets, the inner surface of the erythrocyte membrane and the surface of mature merozoites. The transient nature of heparin presence on the merozoite surface prompted us to explore the hypothesis that heparin binds the major surface antigen, MSP1 that shed by PfSub2 protease upon merozoite natural invasion of new erythrocytes. Indeed, by blocking protease activity we prevented shedding of heparin complex from merozoites. Binding of heparin with MSP1 can explain also defective merozoite dispersion prior to egress, when merozoites topologically are close and can be glued by heparin. A third heparin target was observed shortly after merozoite egress. It was co-localized with the proteins expelled from the merozoite apical prominence, likely rhoptry material needed for merozoite invasion. Thus, heparin attacks the most vulnerable stage of parasite asexual cycle, i.e. merozoite transition from cell to cell, mimicking the effect by which human antibodies neutralize malaria parasites.

One-step design of a stable variant of the malaria invasion protein RH5 for use as a vaccine immunogen

Many promising vaccine candidates from pathogenic viruses, bacteria, and parasites are unstable and cannot be produced cheaply for clinical use. For instance, Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is essential for erythrocyte invasion, is highly conserved among field isolates, and elicits antibodies that neutralize in vitro and protect in an animal model, making it a leading malaria vaccine candidate. However, functional RH5 is only expressible in eukaryotic systems and exhibits moderate temperature tolerance, limiting its usefulness in hot and low-income countries where malaria prevails. Current approaches to immunogen stabilization involve iterative application of rational or semirational design, random mutagenesis, and biochemical characterization. Typically, each round of optimization yields minor improvement in stability, and multiple rounds are required. In contrast, we developed a one-step design strategy using phylogenetic analysis and Rosetta atomistic calculations to design PfRH5 variants with improved packing and surface polarity. To demonstrate the robustness of this approach, we tested three PfRH5 designs, all of which showed improved stability relative to wild type. The best, bearing 18 mutations relative to PfRH5, expressed in a folded form in bacteria at >1 mg of protein per L of culture, and had 10-15 °C higher thermal tolerance than wild type, while also retaining ligand binding and immunogenic properties indistinguishable from wild type, proving its value as an immunogen for a future generation of vaccines against the malaria blood stage. We envision that this efficient computational stability design methodology will also be used to enhance the biophysical properties of other recalcitrant vaccine candidates from emerging pathogens.

Antibody profiles to wheat germ cell-free system synthesized Plasmodium falciparum proteins correlate with protection from symptomatic malaria in Uganda

The key targets of protective antibodies against Plasmodium falciparum remain largely unknown. In this study, we determined immunoreactivity to 1827 recombinant proteins derived from 1565 genes representing ∼30% of the entire P. falciparum genome, for identification of novel malaria vaccine candidates. The recombinant proteins were expressed by wheat germ cell-free system, a platform that can synthesize quality plasmodial proteins that elicit biologically active antibodies in animals. Sera were obtained from indigenous residents of a malaria endemic region in Northern Uganda who were enrolled at the start of a rainy season and prospectively monitored for symptomatic malaria episodes for a year. Immunoreactivity to sera was determined by AlphaScreen; a homogeneous high-throughput system that detects protein interactions. Our analysis revealed antibody responses to 128 proteins that significantly associated with protection from symptomatic malaria. From 128 proteins, 53 were down-selected as the most plausible targets of host protective immune response by virtue of having a predicted signal peptide and/or transmembrane domain(s), or confirmed localization on the parasite surface. The 53 proteins comprised of not only previously characterized vaccine candidates but also uncharacterized proteins. Proteins involved in erythrocyte invasion; RON4, RON2 and CLAG3.1 and pre-erythrocytic proteins; SIAP-2, TRAP and CelTOS, were recommended for prioritization for further evaluation as vaccine candidates. The findings clearly demonstrate that generation of the protein library using the wheat germ cell-free system coupled with high throughput immunoscreening with AlphaScreen offers new options for rational discovery and selection of potential malaria vaccine candidates.

Disrupting the Allosteric Interaction between the Plasmodium falciparum cAMP-dependent Kinase and Its Regulatory Subunit

The ubiquitous second messenger cAMP mediates signal transduction processes in the malarial parasite that regulate host erythrocyte invasion and the proliferation of merozoites. In Plasmodium falciparum, the central receptor for cAMP is the single regulatory subunit (R) of protein kinase A (PKA). To aid the development of compounds that can selectively dysregulate parasite PKA signaling, we solved the structure of the PKA regulatory subunit in complex with cAMP and a related analogue that displays antimalarial activity, (Sp)-2-Cl-cAMPS. Prior to signaling, PKA-R holds the kinase's catalytic subunit (C) in an inactive state by exerting an allosteric inhibitory effect. When two cAMP molecules bind to PKA-R, they stabilize a structural conformation that facilitates its dissociation, freeing PKA-C to phosphorylate downstream substrates such as apical membrane antigen 1. Although PKA activity was known to be necessary for erythrocytic proliferation, we show that uncontrolled induction of PKA activity using membrane-permeable agonists is equally disruptive to growth.

Progress in imaging methods: insights gained into Plasmodium biology.

Over the past decade, major advances in imaging techniques have enhanced our understanding of Plasmodium spp. parasites and their interplay with mammalian hosts and mosquito vectors. Cryoelectron tomography, cryo-X-ray tomography and super-resolution microscopy have shifted paradigms of sporozoite and gametocyte structure, the process of erythrocyte invasion by merozoites, and the architecture of Maurer's clefts. Intravital time-lapse imaging has been revolutionary for our understanding of pre-erythrocytic stages of rodent Plasmodium parasites. Furthermore, high-speed imaging has revealed the link between sporozoite structure and motility, and improvements in time-lapse microscopy have enabled imaging of the entire Plasmodium falciparum erythrocytic cycle and the complete Plasmodium berghei pre-erythrocytic stages for the first time. In this Review, we discuss the contribution of key imaging tools to these and other discoveries in the malaria field over the past 10 years.

A recombinant multi-antigen vaccine formulation containing Babesia bovis merozoite surface antigens MSA-2a1, MSA-2b and MSA-2c elicits invasion-inhibitory antibodies and IFN-γ producing cells.

Background Babesia bovis is a tick-transmitted protozoan hemoparasite and the causative agent of bovine babesiosis, a potential risk to more than 500 million cattle worldwide. The vaccines currently available are based on attenuated parasites, which are difficult to produce, and are only recommended for use in bovines under one year of age. When used in older animals, these vaccines may cause life-threatening clinical symptoms and eventually death. The development of a multi-subunit recombinant vaccine against B. bovis would be attractive from an economic standpoint and, most importantly, could be recommended for animals of any age. In the present study, recombinant ectodomains of MSA-2a1, MSA-2b and MSA-2c antigens were expressed in Pichia pastoris yeast as secreted soluble peptides.ResultsThe antigens were purified to homogeneity, and biochemically and immunologically characterized. A vaccine formulation was obtained by emulsifying a mixture of the three peptides with the adjuvant Montanide ISA 720, which elicited high IgG antibody titers against each of the above antigens. IgG antibodies generated against each MSA-antigen recognized merozoites and significantly inhibited the invasion of bovine erythrocytes. Cellular immune responses were also detected, which were characterized by splenic and lymph node CD4+ T cells producing IFN-γ and TNF-α upon stimulation with the antigens MSA-2a1 or MSA-2c.ConclusionsThese data strongly suggest the high protective potential of the presented formulation, and we propose that it could be tested in vaccination trials of bovines challenged with B. bovis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1862-1) contains supplementary material, which is available to authorized users.

Plasmodium Merozoite TRAP Family Protein Is Essential for Vacuole Membrane Disruption and Gamete Egress from Erythrocytes.

SummarySurface-associated TRAP (thrombospondin-related anonymous protein) family proteins are conserved across the phylum of apicomplexan parasites. TRAP proteins are thought to play an integral role in parasite motility and cell invasion by linking the extracellular environment with the parasite submembrane actomyosin motor. Blood stage forms of the malaria parasite Plasmodium express a TRAP family protein called merozoite-TRAP (MTRAP) that has been implicated in erythrocyte invasion. Using MTRAP-deficient mutants of the rodent-infecting P. berghei and human-infecting P. falciparum parasites, we show that MTRAP is dispensable for erythrocyte invasion. Instead, MTRAP is essential for gamete egress from erythrocytes, where it is necessary for the disruption of the gamete-containing parasitophorous vacuole membrane, and thus for parasite transmission to mosquitoes. This indicates that motor-binding TRAP family members function not just in parasite motility and cell invasion but also in membrane disruption and cell egress.

Plasmodium vivax GPI-anchored micronemal antigen (PvGAMA) binds human erythrocytes independent of Duffy antigen status.

Plasmodium vivax, a major agent of malaria in both temperate and tropical climates, has been thought to be unable to infect humans lacking the Duffy (Fy) blood group antigen because this receptor is critical for erythrocyte invasion. Recent surveys in various endemic regions, however, have reported P. vivax infections in Duffy-negative individuals, suggesting that the parasite may utilize alternative receptor-ligand pairs to complete the erythrocyte invasion. Here, we identified and characterized a novel parasite ligand, Plasmodium vivax GPI-anchored micronemal antigen (PvGAMA), that bound human erythrocytes regardless of Duffy antigen status. PvGAMA was localized at the microneme in the mature schizont-stage parasites. The antibodies against PvGAMA fragments inhibited PvGAMA binding to erythrocytes in a dose-dependent manner. The erythrocyte-specific binding activities of PvGAMA were significantly reduced by chymotrypsin treatment. Thus, PvGAMA may be an adhesion molecule for the invasion of Duffy-positive and -negative human erythrocytes.

Size polymorphism and low sequence diversity in the locus encoding the Plasmodium vivaxrhoptry neck protein 4 (PvRON4) in Colombian isolates.

BackgroundDesigning a vaccine against Plasmodium vivax has focused on selecting antigens involved in invasion mechanisms that must have domains with low polymorphism for avoiding allele-specific immune responses. The rhoptry neck protein 4 (RON4) forms part of the tight junction, which is essential in the invasion of hepatocytes and/or erythrocytes; however, little is known about this locus’ genetic diversity.MethodsDNA sequences from 73 Colombian clinical isolates from pvron4 gene were analysed for characterizing their genetic diversity; pvron4 haplotype number and distribution, as well as the evolutionary forces determining diversity pattern, were assessed by population genetics and molecular evolutionary approaches.Results ron4 has low genetic diversity in P. vivax at sequence level; however, a variable amount of tandem repeats at the N-terminal region leads to extensive size polymorphism. This region seems to be exposed to the immune system. The central region has a putative esterase/lipase domain which, like the protein’s C-terminal fragment, is highly conserved at intra- and inter-species level. Both regions are under purifying selection.Conclusions pvron4 is the locus having the lowest genetic diversity described to date for P. vivax. The repeat regions in the N-terminal region could be associated with immune evasion mechanisms while the central region and the C-terminal region seem to be under functional or structural constraint. Bearing such results in mind, the PvRON4 central and/or C-terminal portions represent promising candidates when designing a subunit-based vaccine as they are aimed at avoiding an allele-specific immune response, which might limit vaccine efficacy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1563-4) contains supplementary material, which is available to authorized users.