Parasite Surface Proteins Research Articles

Exploring malaria parasite surface proteins to devise highly immunogenic multi-epitope subunit vaccine for Plasmodium falciparum

BackgroundMalaria has remained a major health concern for decades among people living in tropical and sub-tropical countries. Plasmodium falciparum is one of the critical species that cause severe malaria and is responsible for major mortality. Moreover, the parasite has generated resistance against all WHO recommended drugs and therapies. Therefore, there is an urgent need for preventive measures in the form of reliable vaccines to achieve the target of a malaria-free world. Surface proteins are the preferable choice for subunit vaccine development because they are rapidly detected and engaged by host immune cells and vaccination-induced antibodies. Additionally, abundant surface or membrane proteins may contribute to the opsonization of pathogens by vaccine-induced antibodies. ResultsIn our study, we have listed all those surface proteins from the literature that could be functionally important and essential for infection and immune evasion of the malaria parasite. Eight Plasmodium surface and membrane proteins from the pre-erythrocyte and erythrocyte stages were shortlisted. Thirty-seven epitopes (B-cell, CTL, and HTL epitopes) from these proteins were predicted using immune-informatic tools and joined with suitable peptide linkers to design a vaccine construct. A TLR-4 agonist peptide adjuvant was added at the N-terminus of the multi-epitope series, followed by the PADRE sequence and EAAAK linker. The TLR-4 receptor was docked with the construct’s anticipated model structure. The complex of vaccine and TLR-4, with the lowest energy −1514, was found to be stable under simulated physiological settings. ConclusionThis study has provided a novel multi-epitope construct that may be exploited further for the development of an efficient vaccine for malaria.

Trypanosoma brucei Invariant Surface Glycoprotein 75 Is an Immunoglobulin Fc Receptor Inhibiting Complement Activation and Antibody-Mediated Cellular Phagocytosis.

Various subspecies of the unicellular parasite Trypanosoma brucei cause sleeping sickness, a neglected tropical disease affecting millions of individuals and domestic animals. Immune evasion mechanisms play a pivotal role in parasite survival within the host and enable the parasite to establish a chronic infection. In particular, the rapid switching of variant surface glycoproteins covering a large proportion of the parasite's surface enables the parasite to avoid clearance by the adaptive immune system of the host. In this article, we present the crystal structure and discover an immune-evasive function of the extracellular region of the T. brucei invariant surface gp75 (ISG75). Structural analysis determined that the ISG75 ectodomain is organized as a globular head domain and a long slender coiled-coil domain. Subsequent ligand screening and binding analysis determined that the head domain of ISG75 confers interaction with the Fc region of all subclasses of human IgG. Importantly, the ISG75-IgG interaction strongly inhibits both activation of the classical complement pathway and Ab-dependent cellular phagocytosis by competing with C1q and host cell FcγR CD32. Our data reveal a novel immune evasion mechanism of T. brucei, with ISG75 able to inactivate the activities of Abs recognizing the parasite surface proteins.

Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes.

Basigin is an essential host receptor for invasion of Plasmodium falciparum into human erythrocytes, interacting with parasite surface protein PfRH5. PfRH5 is a leading blood-stage malaria vaccine candidate and a target of growth-inhibitory antibodies. Here, we show that erythrocyte basigin is exclusively found in one of two macromolecular complexes, bound either to plasma membrane Ca2+-ATPase 1/4 (PMCA1/4) or to monocarboxylate transporter 1 (MCT1). PfRH5 binds to each of these complexes with a higher affinity than to isolated basigin ectodomain, making it likely that these are the physiological targets of PfRH5. PMCA-mediated Ca2+ export is not affected by PfRH5, making it unlikely that this is the mechanism underlying changes in calcium flux at the interface between an erythrocyte and the invading parasite. However, our studies rationalise the function of the most effective growth-inhibitory antibodies targeting PfRH5. While these antibodies do not reduce the binding of PfRH5 to monomeric basigin, they do reduce its binding to basigin-PMCA and basigin-MCT complexes. This indicates that the most effective PfRH5-targeting antibodies inhibit growth by sterically blocking the essential interaction of PfRH5 with basigin in its physiological context.

What Do We Know about Surface Proteins of Chicken Parasites Eimeria?

Poultry is the first source of animal protein for human consumption. In a changing world, this sector is facing new challenges, such as a projected increase in demand, higher standards of food quality and safety, and reduction of environmental impact. Chicken coccidiosis is a highly widespread enteric disease caused by Eimeria spp. which causes significant economic losses to the poultry industry worldwide; however, the impact on family poultry holders or backyard production-which plays a key role in food security in small communities and involves mainly rural women-has been little explored. Coccidiosis disease is controlled by good husbandry measures, chemoprophylaxis, and/or live vaccination. The first live vaccines against chicken coccidiosis were developed in the 1950s; however, after more than seven decades, none has reached the market. Current limitations on their use have led to research in next-generation vaccines based on recombinant or live-vectored vaccines. Next-generation vaccines are required to control this complex parasitic disease, and for this purpose, protective antigens need to be identified. In this review, we have scrutinised surface proteins identified so far in Eimeria spp. affecting chickens. Most of these surface proteins are anchored to the parasite membrane by a glycosylphosphatidylinositol (GPI) molecule. The biosynthesis of GPIs, as well as the role of currently identified surface proteins and interest as vaccine candidates has been summarised. The potential role of surface proteins in drug resistance and immune escape and how these could limit the efficacy of control strategies was also discussed.

Highly potent, naturally acquired human monoclonal antibodies against Pfs48/45 block Plasmodium falciparum transmission to mosquitoes

Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that interrupt malaria parasite development in the mosquito, thereby blocking onward transmission, and provide a much-needed tool for malaria control and elimination. The parasite surface protein Pfs48/45 is a leading TBV candidate. Here, we isolated and characterized a panel of 81 human Pfs48/45-specific monoclonal antibodies (mAbs) from donors naturally exposed to Plasmodium parasites. Genetically diverse mAbs against each of the three domains (D1-D3) of Pfs48/45 were identified. The most potent mAbs targeted D1 and D3 and achieved >80% transmission-reducing activity in standard membrane-feeding assays, at 10 and 2μg/mL, respectively. Co-crystal structures of D3 in complex with four different mAbs delineated two conserved protective epitopes. Altogether, these Pfs48/45-specific human mAbs provide important insight into protective and non-protective epitopes that can further our understanding of transmission and inform the design of refined malaria transmission-blocking vaccine candidates.

Anti-TRAP/SSP2 monoclonal antibodies can inhibit sporozoite infection and may enhance protection of anti-CSP monoclonal antibodies

Vaccine-induced sterilizing protection from infection by Plasmodium parasites, the pathogens that cause malaria, will be essential in the fight against malaria as it would prevent both malaria-related disease and transmission. Stopping the relatively small number of parasites injected by the mosquito before they can migrate from the skin to the liver is an attractive means to this goal. Antibody-eliciting vaccines have been used to pursue this objective by targeting the major parasite surface protein present during this stage, the circumsporozoite protein (CSP). While CSP-based vaccines have recently had encouraging success in disease reduction, this was only achieved with extremely high antibody titers and appeared less effective for a complete block of infection (i.e., sterile protection). While such disease reduction is important, these and other results indicate that strategies focusing on CSP alone may not achieve the high levels of sterile protection needed for malaria eradication. Here, we show that monoclonal antibodies (mAbs) recognizing another sporozoite protein, TRAP/SSP2, exhibit a range of inhibitory activity and that these mAbs may augment CSP-based protection despite conferring no sterile protection on their own. Therefore, pursuing a multivalent subunit vaccine immunization is a promising strategy for improving infection-blocking malaria vaccines.

The malaria parasite sheddase SUB2 governs host red blood cell membrane sealing at invasion.

Red blood cell (RBC) invasion by malaria merozoites involves formation of a parasitophorous vacuole into which the parasite moves. The vacuole membrane seals and pinches off behind the parasite through an unknown mechanism, enclosing the parasite within the RBC. During invasion, several parasite surface proteins are shed by a membrane-bound protease called SUB2. Here we show that genetic depletion of SUB2 abolishes shedding of a range of parasite proteins, identifying previously unrecognized SUB2 substrates. Interaction of SUB2-null merozoites with RBCs leads to either abortive invasion with rapid RBC lysis, or successful entry but developmental arrest. Selective failure to shed the most abundant SUB2 substrate, MSP1, reduces intracellular replication, whilst conditional ablation of the substrate AMA1 produces host RBC lysis. We conclude that SUB2 activity is critical for host RBC membrane sealing following parasite internalisation and for correct functioning of merozoite surface proteins.

Gene expression network analyses during infection with virulent and avirulent Trypanosoma cruzistrains unveil a role for fibroblasts in neutrophil recruitment and activation.

Chagas disease is caused by Trypanosoma cruzi, a protozoan parasite that has a heterogeneous population composed of a pool of strains with distinct characteristics, including variable levels of virulence. In previous work, transcriptome analyses of parasite genes after infection of human foreskin fibroblasts (HFF) with virulent (CL Brener) and non-virulent (CL-14) clones derived from the CL strain, revealed a reduced expression of genes encoding parasite surface proteins in CL-14 compared to CL Brener during the final steps of the intracellular differentiation from amastigotes to trypomastigotes. Here we analyzed changes in the expression of host genes during in vitro infection of HFF cells with the CL Brener and CL-14 strains by analyzing total RNA extracted from cells at 60 and 96 hours post-infection (hpi) with each strain, as well as from uninfected cells. Similar transcriptome profiles were observed at 60 hpi with both strains compared to uninfected samples. However, at 96 hpi, significant differences in the number and expression levels of several genes, particularly those involved with immune response and cytoskeleton organization, were observed. Further analyses confirmed the difference in the chemokine/cytokine signaling involved with the recruitment and activation of immune cells such as neutrophils upon T. cruzi infection. These findings suggest that infection with the virulent CL Brener strain induces a more robust inflammatory response when compared with the non-virulent CL-14 strain. Importantly, the RNA-Seq data also exposed an unexplored role of fibroblasts as sentinel cells that may act by recruiting neutrophils to the initial site of infection. This role for fibroblasts in the regulation of the inflammatory response during infection by T. cruzi was corroborated by measurements of levels of different chemokines/cytokines during in vitro infection and in plasma from Chagas disease patients as well as by neutrophil activation and migration assays.

Theileria annulata surface protein (TaSP) is a target of cyclin-dependent kinase 1 phosphorylation in Theileria annulata-infected cells.

Leucoproliferative Theileria parasites possess the unique capability to transform their bovine host cell, resulting in tumour-like characteristics like uncontrolled proliferation. The molecular mechanisms underlying this parasite-dependent process are only poorly understood. In the current study, bioinformatic analysis of the Theileria annulata surface protein (TaSP) from different T.annulata isolates identified a conserved CDK1 phosphorylation motif T131 PTK within the extracellular, polymorphic domain of TaSP. Phosphorylation assays with radioactively labelled ATP as well as ELISA-based experiments using a phospho-threonine-proline (pThr-Pro) antibody revealed, that CDK1-cyclin B specifically phosphorylates T131 , identifying TaSP as a substrate in vitro. Confocal microscopy and proximity ligation assays suggest an interaction between CDK1 and TaSP in T.annulata-infected cells. Further studies demonstrated a nearly complete co-localization of the pThr-Pro signal and TaSP only in cells in interphase, pointing towards a cell cycle-dependent event. Immunostainings of isolated, non-permeabilized schizonts confirmed the presence of the pThr-Pro epitope on the schizont's surface. Lambda phosphatase treatment abolished the pThr-Pro signal of the schizont, which was reconstituted by the addition of CDK1-cyclin B. Treatment of T.annulata-infected cells with the CDK1 inhibitor purvalanol A resulted in morphological changes characterized by tubulin-rich cell protrusions and an extension of the schizont, and a dose-dependent reduction of BrdU incorporation and Ki67 staining of T.annulata-infected cells, demonstrating a clear impact on the Theileria-dependent proliferation of the bovine host cell. Our data reveal the parasite surface protein TaSP as a target for the host cell kinase CDK1, a major player during cell division. Targeting the uncontrolled proliferation of Theileria-infected cells is a novel and reasonable approach to limit parasite load in order to facilitate a successful cellular immune response against the parasite.

The Plasmodium yoelii microgamete surface antigen (PyMiGS) induces anti-malarial transmission blocking immunity that reduces microgamete motility/release from activated male gametocytes

Malaria transmission-blocking vaccines aim to inhibit the development of malaria parasites in mosquitoes by inducing antibodies targeting surface proteins of sexual stage parasites. We have recently identified PyMiGS, a protein specifically expressed in the osmiophilic body of male gametocytes of Plasmodium yoelii (Py). PyMiGS is translocated to the surface of microgametes, and potent transmission-blocking activity was observed in mosquitoes fed on mice passively immunized with antibodies against PyMiGS. Here we demonstrate using a direct feeding assay that recombinant PyMiGS successfully induces anti-PyMiGS antibodies in mice and that the antibodies block parasite development in mosquitoes. We also show using the membrane-feeding assay that rabbit anti-PyMiGS antibody inhibits parasite development in mosquitoes in a dose-dependent manner without complement involvement. To investigate the mode of action of anti-PyMiGS antibodies against parasite development, we observed exflagellation after mixing Py gametocytes with activation medium containing anti-PyMiGS or anti-GST control antibodies. Whereas most microgametes were released from activated male gametocytes in the control group, a significantly reduced number of microgametes were released in the anti-PyMiGS group, with most of the microgametes left attached to the activated male gametocytes. Moreover, anti-PyMiGS antibodies shortened the duration of the active movement of microgametes after the onset of exflagellation. Taken together, these findings suggest that anti-PyMiGS antibodies bind to the microgamete surface immediately after exflagellation, thereby reducing microgamete motility and inhibiting microgamete release from the activated male gametocytes. These results strongly suggest that PyMiGS orthologues in Plasmodium falciparum and Plasmodium vivax can be promising TBV candidates.

Development of cross-protective Eimeria-vectored vaccines based on apical membrane antigens

Recently, the availability of protocols supporting genetic complementation of Eimeria has raised the prospect of generating transgenic parasite lines which can function as vaccine vectors, expressing and delivering heterologous proteins. Complementation with sequences encoding immunoprotective antigens from other Eimeria spp. offers an opportunity to reduce the complexity of species/strains in anticoccidial vaccines. Herein, we characterise and evaluate EtAMA1 and EtAMA2, two members of the apical membrane antigen (AMA) family of parasite surface proteins from Eimeria tenella. Both proteins are stage-regulated, and the sporozoite-specific EtAMA1 is effective at inducing partial protection against homologous challenge with E. tenella when used as a recombinant protein vaccine, whereas the merozoite-specific EtAMA2 is not. In order to test the ability of transgenic parasites to confer heterologous protection, E. tenella parasites were complemented with EmAMA1, the sporozoite-specific orthologue of EtAMA1 from E. maxima, coupled with different delivery signals to modify its trafficking and improve antigen exposure to the host immune system. Vaccination of chickens using these transgenic parasites conferred partial protection against E. maxima challenge, with levels of efficacy comparable to those obtained using recombinant protein or DNA vaccines. In the present work we provide evidence for the first known time of the ability of transgenic Eimeria to induce cross protection against different Eimeria spp. Genetically complemented Eimeria provide a powerful tool to streamline the complex multi-valent anticoccidial vaccine formulations that are currently available in the market by generating parasite lines expressing vaccine targets from multiple eimerian species.

Towards an anti-disease malaria vaccine.

Human infective parasites, such as those that cause malaria, are highly adapted to evade clearance by the immune system. In situations where they must maintain prolonged interactions with molecules of their host, they often use parasite surface protein families. These families are highly diverse to prevent immune recognition, and yet, to promote parasite survival, their members must retain the ability to interact with specific human receptors. One of the best understood of the parasite surface protein families is the PfEMP1 proteins of Plasmodium falciparum. These molecules cause infected erythrocytes to adhere to human receptors found on blood vessel and tissue surfaces. This protects the parasite within from clearance by the spleen and also causes symptoms of severe malaria. The PfEMP1 are exposed to the immune system during infection and are therefore excellent vaccine candidates for use in an approach to prevent severe disease. A key question, however, is whether their extensive diversity precludes them from forming components of the malaria vaccines of the future?

Malaria parasite LIMP protein regulates sporozoite gliding motility and infectivity in mosquito and mammalian hosts.

Gliding motility allows malaria parasites to migrate and invade tissues and cells in different hosts. It requires parasite surface proteins to provide attachment to host cells and extracellular matrices. Here, we identify the Plasmodium protein LIMP (the name refers to a gliding phenotype in the sporozoite arising from epitope tagging of the endogenous protein) as a key regulator for adhesion during gliding motility in the rodent malaria model P. berghei. Transcribed in gametocytes, LIMP is translated in the ookinete from maternal mRNA, and later in the sporozoite. The absence of LIMP reduces initial mosquito infection by 50%, impedes salivary gland invasion 10-fold, and causes a complete absence of liver invasion as mutants fail to attach to host cells. GFP tagging of LIMP caused a limping defect during movement with reduced speed and transient curvature changes of the parasite. LIMP is an essential motility and invasion factor necessary for malaria transmission.

Resistance to malaria through structural variation of red blood cell invasion receptors.

The malaria parasite Plasmodium falciparum invades human red blood cells by a series of interactions between host and parasite surface proteins. By analyzing genome sequence data from human populations, including 1269 individuals from sub-Saharan Africa, we identify a diverse array of large copy-number variants affecting the host invasion receptor genes GYPA and GYPB We find that a nearby association with severe malaria is explained by a complex structural rearrangement involving the loss of GYPB and gain of two GYPB-A hybrid genes, which encode a serologically distinct blood group antigen known as Dantu. This variant reduces the risk of severe malaria by 40% and has recently increased in frequency in parts of Kenya, yet it appears to be absent from west Africa. These findings link structural variation of red blood cell invasion receptors with natural resistance to severe malaria.

How Antiparasitic Drugs Work-And Sometimes Stop Working!

How Antiparasitic Drugs Work-And Sometimes Stop Working!

Allelic Diversity and Geographical Distribution of the Gene Encoding Plasmodium falciparum Merozoite Surface Protein-3 in Thailand.

Merozoite surface proteins (MSPs) of malaria parasites play critical roles during the erythrocyte invasion and so are potential candidates for malaria vaccine development. However, because MSPs are often under strong immune selection, they can exhibit extensive genetic diversity. The gene encoding the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum displays 2 allelic types, K1 and 3D7. In Thailand, the allelic frequency of the P. falciparum msp-3 gene was evaluated in a single P. falciparum population in Tak at the Thailand and Myanmar border. However, no study has yet looked at the extent of genetic diversity of the msp-3 gene in P. falciparum populations in other localities. Here, we genotyped the msp-3 alleles of 63 P. falciparum samples collected from 5 geographical populations along the borders of Thailand with 3 neighboring countries (Myanmar, Laos, and Cambodia). Our study indicated that the K1 and 3D7 alleles coexisted, but at different proportions in different Thai P. falciparum populations. K1 was more prevalent in populations at the Thailand-Myanmar and Thailand-Cambodia borders, whilst 3D7 was more prevalent at the Thailand-Laos border. Global analysis of the msp-3 allele frequencies revealed that proportions of K1 and 3D7 alleles of msp-3 also varied in different continents, suggesting the divergence of malaria parasite populations. In conclusion, the variation in the msp-3 allelic patterns of P. falciparum in Thailand provides fundamental knowledge for inferring the P. falciparum population structure and for the best design of msp-3 based malaria vaccines.

Shear Forces Enhance Toxoplasma gondii Tachyzoite Motility on Vascular Endothelium

ABSTRACTToxoplasma gondii is a highly successful parasite that infects approximately one-third of the human population and can cause fatal disease in immunocompromised individuals. Systemic parasite dissemination to organs such as the brain and eye is critical to pathogenesis. T. gondii can disseminate via the circulation, and both intracellular and extracellular modes of transport have been proposed. However, the processes by which extracellular tachyzoites adhere to and migrate across vascular endothelium under the conditions of rapidly flowing blood remain unknown. We used microfluidics and time-lapse fluorescence microscopy to examine the interactions between extracellular T. gondii and primary human endothelial cells under conditions of physiologic shear stress. Remarkably, tachyzoites adhered to and glided on human vascular endothelium under shear stress conditions. Compared to static conditions, shear stress enhanced T. gondii helical gliding, resulting in a significantly greater displacement, and increased the percentage of tachyzoites that invaded or migrated across the endothelium. The intensity of the shear forces (from 0.5 to 10 dynes/cm2) influenced both initial and sustained adhesion to endothelium. By examining tachyzoites deficient in the T. gondii adhesion protein MIC2, we found that MIC2 contributed to initial adhesion but was not required for adhesion strengthening. These data suggest that under fluidic conditions, T. gondii adhesion to endothelium may be mediated by a multistep cascade of interactions that is governed by unique combinations of adhesion molecules. This work provides novel information about tachyzoite interactions with vascular endothelium and contributes to our understanding of T. gondii dissemination in the infected host.

Plasmodium falciparum Erythrocyte Invasion: Combining Function with Immune Evasion

All the symptoms and pathology of malaria are caused by the intraerythrocytic stages of the Plasmodium parasite life cycle. Because Plasmodium parasites cannot replicate outside a host cell, their ability to recognize and invade erythrocytes is an essential step for both parasite survival and malaria pathogenesis. This makes invasion a conceptually attractive vaccine target, especially because it is one of the few stages when the parasite is directly exposed to the host humoral immune system. This apparent vulnerability, however, has been countered by the parasite, which has evolved sophisticated molecular mechanisms to evade the host immune response so that parasites asymptomatically replicate within immune individuals. These mechanisms include the expansion of parasite invasion ligands, resulting in multiple and apparently redundant invasion "pathways", highly polymorphic parasite surface proteins that are immunologically distinct, and parasite proteins which are poorly immunogenic. These formidable defences have so far thwarted attempts to develop an effective blood-stage vaccine, leading many to question whether there really is an exploitable chink in the parasite's immune evasion defences. Here, we review recent advances in the molecular understanding of the P. falciparum erythrocyte invasion field, discuss some of the challenges that have so far prevented the development of blood-stage vaccines, and conclude that the parasite invasion ligand RH5 represents an essential pinch point that might be vulnerable to vaccination.

Sequence variation and structural conservation allows development of novel function and immune evasion in parasite surface protein families

Trypanosoma and Plasmodium species are unicellular, eukaryotic pathogens that have evolved the capacity to survive and proliferate within a human host, causing sleeping sickness and malaria, respectively. They have very different survival strategies. African trypanosomes divide in blood and extracellular spaces, whereas Plasmodium species invade and proliferate within host cells. Interaction with host macromolecules is central to establishment and maintenance of an infection by both parasites. Proteins that mediate these interactions are under selection pressure to bind host ligands without compromising immune avoidance strategies. In both parasites, the expansion of genes encoding a small number of protein folds has established large protein families. This has permitted both diversification to form novel ligand binding sites and variation in sequence that contributes to avoidance of immune recognition. In this review we consider two such parasite surface protein families, one from each species. In each case, known structures demonstrate how extensive sequence variation around a conserved molecular architecture provides an adaptable protein scaffold that the parasites can mobilise to mediate interactions with their hosts.

The role played by alternative splicing in antigenic variability in human endo-parasites

Endo-parasites that affect humans include Plasmodium, the causative agent of malaria, which remains one of the leading causes of death in human beings. Despite decades of research, vaccines to this and other endo-parasites remain elusive. This is in part due to the hyper-variability of the parasites surface proteins. Generally these surface proteins are encoded by a large family of genes, with only one being dominantly expressed at certain life stages. Another layer of complexity can be introduced through the alternative splicing of these surface proteins. The resulting isoforms may differ from each other with regard to cell localisation, substrate affinities and functions. They may even differ in structure to the extent that they are no longer recognised by the host’s immune system. In many cases this leads to changes in the N terminus of these proteins. The geographical localisation of endo-parasitic infections around the tropics and the highest incidences of HIV-1 infection in the same areas, adds a further layer of complexity as parasitic infections affect the host immune system resulting in higher HIV infection rates, faster disease progression, and an increase in the severity of infections and complications in HIV diagnosis. This review discusses some examples of parasite surface proteins that are alternatively spliced in trypanosomes, Plasmodium and the parasitic worm Schistosoma as well as what role alternate splicing may play in the interaction between HIV and these endo-parasites.