Genome-wide association study indicates novel associations of annexin A13 to secretory and GAS2L2 with mucous otitis media.

To evaluate the genetics of chronic nonsuppurative otitis media (OM). We performed a genome-wide association study of 429,599 individuals included in the FinnGen study using three different case definitions: combined chronic nonsuppurative OM (7034 cases) (included serous and mucous chronic OM), mucous chronic OM (5953 cases), and secretory chronic OM (1689 cases). Individuals without otitis media were used as controls (417,745 controls). We used immunohistochemistry (IHC) of the murine middle ear to evaluate the expression of annexin A13. Four loci were significantly associated (p < 1.7 × 10-8) with nonsuppurative OM. Three out of the four association signals included missense variants in genes that may play a role in otitis media pathobiology. According to our subtype-specific analyses, one novel locus, located near ANXA13, was associated with secretory OM. Three loci (near TNFRSF13B, GAS2L2, and TBX1) were associated with mucous OM. Immunohistochemistry of murine middle ear samples revealed annexin A13 expression at the apical pole of the Eustachian tube epithelium as well as variable intensity of the secretory cells of the glandular structure in proximity to the Eustachian tube. We demonstrated that secretory and mucous OM have distinct and shared genetic associations. The association of GAS2L2 with ciliary epithelium function and the pathogenesis of dysfunctional mucosa in mucous OM is suggested. The abundant expression of annexin A13 in the Eustachian tube epithelium, along with its role in apical transport for the binding and transfer of phospholipids, indicates the role of annexin A13 and phospholipids in Eustachian tube dysfunction.

Talin2 binds to non-muscle myosin IIa and regulates cell attachment and fibronectin secretion.

Talin2 is localized to large focal adhesions and is indispensable for traction force generation, invadopodium formation, cell invasion as well as metastasis. Talin2 has a higher affinity toward ß-integrin tails than talin1. Moreover, disruption of the talin2-ß-integrin interaction inhibits traction force generation, invadopodium formation and cell invasion, indicating that a strong talin2-ß-integrin interaction is required for talin2 to fulfill these functions. Nevertheless, the role of talin2 in mediation of these processes remains unknown. Here we show that talin2 binds to the N-terminus of non-muscle myosin IIA (NMIIA) through its F3 subdomain. Moreover, talin2 co-localizes with NMIIA at cell edges as well as at some cytoplasmic spots. Talin2 also co-localizes with cortactin, an invadopodium marker. Furthermore, overexpression of NMIIA promoted the talin2 head binding to the ß1-integrin tail, whereas knockdown of NMIIA reduced fibronectin and matrix metalloproteinase secretion as well as inhibited cell attachment on fibronectin-coated substrates. These results suggest that talin2 binds to NMIIA to control the secretion of extracellular matrix proteins and this interaction modulates cell adhesion.

Talin-1 variants associated with spontaneous coronary artery dissection (SCAD) highlight how even subtle changes in multi-functional scaffold proteins can manifest in disease.

Variants of talin-1 (TLN1) have recently been linked with spontaneous coronary artery dissection (SCAD) a condition where a tear can form in the wall of a heart artery necessitating immediate medical care. One talin-1 variant, A2013T, has an extensive familial pedigree of SCAD, which led to the screening for, and identification of, further talin-1 variants in SCAD patients. Here we evaluated these variants with commonly used pathogenicity prediction tools and found it challenging to reliably classify SCAD-associated variants, even A2013T where the evidence of a causal role is strong. Using biochemical and cell biological methods, we show that SCAD-associated variants in talin-1, which would typically be classified as non-pathogenic, still cause a measurable impact on protein structure and cell behaviour, including cell movement and wound healing capacity. Together, this indicates that even subtle variants in central mechanosensitive adapter proteins, can give rise to significant health impacts at the individual level, suggesting the need for a possible re-evaluation of the scoring criteria for pathogenicity prediction for talin variants.

Molecular dynamics simulations reveal how vinculin refolds partially unfolded talin rod helices to stabilize them against mechanical force.

Vinculin binds to specific sites of mechanically unfolded talin rod domains to reinforce the coupling of the cell's exterior to its force generation machinery. Force-dependent vinculin-talin complexation and dissociation was previously observed as contraction or extension of the unfolded talin domains respectively using magnetic tweezers. However, the structural mechanism underlying vinculin recognition of unfolded vinculin binding sites (VBSs) in talin remains unknown. Using molecular dynamics simulations, we demonstrate that a VBS dynamically refolds under force, and that vinculin can recognize and bind to partially unfolded VBS states. Vinculin binding enables refolding of the mechanically strained VBS and stabilizes its folded α-helical conformation, providing resistance against mechanical stress. Together, these results provide an understanding of a recognition mechanism of proteins unfolded by force and insight into the initial moments of how vinculin binds unfolded talin rod domains during the assembly of this mechanosensing meshwork.

Tensin-2 interactomics reveals interaction with GAPDH and a phosphorylation-mediated regulatory role in glycolysis.

Integrin adaptor proteins, like tensin-2, are crucial for cell adhesion and signaling. However, the function of tensin-2 beyond localizing to focal adhesions remain poorly understood. We utilized proximity-dependent biotinylation and Strep-tag affinity proteomics to identify interaction partners of tensin-2 in Flp-In 293 T-REx cells. Interactomics linked tensin-2 to known focal adhesion proteins and the dystrophin glycoprotein complex, while also uncovering novel interaction with the glycolytic enzyme GAPDH. We demonstrated that Y483-phosphorylation of tensin-2 regulates the glycolytic rate in Flp-In 293 T-REx and MEF cells and found that pY483 tensin-2 is enriched in adhesions in MEF cells. Our study unveils novel interaction partners for tensin-2 and further solidifies its speculated role in cell energy metabolism. These findings shed fresh insight on the functions of tensin-2, highlighting its potential as a therapeutic target for diseases associated with impaired cell adhesion and metabolism.

Acetyl-NPKY of integrin-ß1 binds KINDLIN2 to control endothelial cell proliferation and junctional integrity.

Integrin-dependent crosstalk between cell-matrix adhesions and cell-cell junctions is critical for controlling endothelial permeability and proliferation in cancer and inflammatory diseases but remains poorly understood. Here, we investigated how acetylation of the distal NPKY-motif of Integrin-ß1 influences endothelial cell physiology and barrier function. Expression of an acetylation-mimetic ß1-K794Q-GFP mutant led to the accumulation of immature cell-matrix adhesions accompanied by a transcriptomic reprograming of endothelial cells, involving genes associated with cell adhesion, proliferation, polarity, and barrier function. ß1-K794Q-GFP induced constitutive MAPK signaling, junctional impairment, proliferation, and reduced contact inhibition at confluence. Structural analysis of Integrin-ß1 interaction with KINDLIN2, biochemical pulldown assay, and binding energy determination by using molecular dynamics simulation showed that acetylation of K794 and the K794Q-mutant increased KINDLIN2 binding affinity to the Integrin-ß1. Thus, enhanced recruitment of KINDLIN2 to Lysine-acetylated Integrin-ß1 and resulting modulation of barrier function, offers new therapeutic possibilities for controlling vascular permeability and disease conditions.

Production of norovirus-, rotavirus-, and enterovirus-like particles in insect cells is simplified by plasmid-based expression.

Insect cells have long been the main expression host of many virus-like particles (VLP). VLPs resemble the respective viruses but are non-infectious. They are important in vaccine development and serve as safe model systems in virus research. Commonly, baculovirus expression vector system (BEVS) is used for VLP production. Here, we present an alternative, plasmid-based system for VLP expression, which offers distinct advantages: in contrast to BEVS, it avoids contamination by baculoviral particles and proteins, can maintain cell viability over the whole process, production of alphanodaviral particles will not be induced, and optimization of expression vectors and their ratios is simple. We compared the production of noro-, rota- and entero-VLP in the plasmid-based system to the standard process in BEVS. For noro- and entero-VLPs, similar yields could be achieved, whereas production of rota-VLP requires some further optimization. Nevertheless, in all cases, particles were formed, the expression process was simplified compared to BEVS and potential for the plasmid-based system was validated. This study demonstrates that plasmid-based transfection offers a viable option for production of noro-, rota- and entero-VLPs in insect cells.

Visible Light-Induced Specific Protein Reaction Delineates Early Stages of Cell Adhesion.

Light is well-established for control of bond breakage but not for control of specific bond formation in complex environments. We previously engineered the diffusion-limited reactivity of the SpyTag003 peptide with its protein partner SpyCatcher003 through spontaneous isopeptide bond formation. This system enables precise and irreversible assembly of biological building blocks with applications from biomaterials to vaccines. Here we establish a system for the rapid control of this amide bond formation with visible light. We have generated a caged SpyCatcher003, which allows light triggering of covalent bond formation to SpyTag003 in mammalian cells. Photocaging is achieved through site-specific incorporation of an unnatural coumarin-lysine at the reactive site of SpyCatcher003. We showed a uniform specific reaction in cell lysate upon light activation. We then used the spatiotemporal precision of a 405 nm confocal laser for uncaging in seconds, probing the earliest events in mechanotransduction by talin, the key force sensor between the cytoskeleton and the extracellular matrix. Reconstituting talin induced rapid biphasic extension of lamellipodia, revealing the kinetics of talin-regulated cell spreading and polarization. Thereafter we determined the hierarchy of the recruitment of key components for cell adhesion. Precise control over site-specific protein reaction with visible light creates diverse opportunities for cell biology and nanoassembly.

Thrombus Aspirates From Patients With Acute Ischemic Stroke Are Infiltrated by Viridans Streptococci.

BACKGROUND: Acute ischemic stroke may be due to embolism from ruptured atherosclerotic carotid arteries. DNA of oral bacteria, mainly the viridans streptococci group, has been detected in thrombus aspirates of patients with ischemic stroke as well as in carotid endarterectomy samples. Because viridans streptococci are known to possess thrombogenic properties, we studied whether their presence in thrombus aspirates and in carotid artery specimens can be confirmed using bacterial immunohistochemistry. METHODS AND RESULTS: Thrombus aspirates from 61 patients with ischemic stroke (70.5% men; mean age, 66.8 years) treated with mechanical thrombectomy, as well as carotid endarterectomy samples from 20 symptomatic patients (65.0% men; mean age, 66.2 years) and 48 carotid artery samples from nonstroke autopsy cases (62.5% men; mean age, 66.4 years), were immunostained with an antibody cocktail against 3 species (Streptococcus sanguinis, Streptococcus mitis, and Streptococcus gordonii) of viridans streptococci. Of the thrombus aspirates, 84.8% were immunopositive for viridans streptococci group bacteria, as were 80.0% of the carotid endarterectomy samples, whereas immunopositivity was observed in 31.3% of the carotid artery samples from nonstroke autopsies. Most streptococci were detected inside neutrophil granulocytes, but there were also remnants of bacterial biofilm as well as free bacterial infiltrates in some samples. CONCLUSIONS: Oral streptococci were found in aspirated thrombi of patients with acute ischemic stroke as well as in carotid artery samples. Our results suggest that viridans streptococci group bacteria may play a role in the pathophysiology of ischemic stroke.

DNA-origami-directed virus capsid polymorphism.

Viral capsids can adopt various geometries, most iconically characterized by icosahedral or helical symmetries. Importantly, precise control over the size and shape of virus capsids would have advantages in the development of new vaccines and delivery systems. However, current tools to direct the assembly process in a programmable manner are exceedingly elusive. Here we introduce a modular approach by demonstrating DNA-origami-directed polymorphism of single-protein subunit capsids. We achieve control over the capsid shape, size and topology by employing user-defined DNA origami nanostructures as binding and assembly platforms, which are efficiently encapsulated within the capsid. Furthermore, the obtained viral capsid coatings can shield the encapsulated DNA origami from degradation. Our approach is, moreover, not limited to a single type of capsomers and can also be applied to RNA-DNA origami structures to pave way for next-generation cargo protection and targeting strategies.

SpyTag/SpyCatcher display of influenza M2e peptide on norovirus-like particle provides stronger immunization than direct genetic fusion.

Introduction: Virus-like particles (VLPs) are similar in size and shape to their respective viruses, but free of viral genetic material. This makes VLP-based vaccines incapable of causing infection, but still effective in mounting immune responses. Noro-VLPs consist of 180 copies of the VP1 capsid protein. The particle tolerates C-terminal fusion partners, and VP1 fused with a C-terminal SpyTag self-assembles into a VLP with SpyTag protruding from its surface, enabling conjugation of antigens via SpyCatcher. Methods: To compare SpyCatcher-mediated coupling and direct peptide fusion in experimental vaccination, we genetically fused the ectodomain of influenza matrix-2 protein (M2e) directly on the C-terminus of norovirus VP1 capsid protein. VLPs decorated with SpyCatcher-M2e and VLPs with direct M2 efusion were used to immunize mice. Results and discussion: We found that direct genetic fusion of M2e on noro-VLP raised few M2e antibodies in the mouse model, presumably because the short linker positions the peptide between the protruding domains of noro-VLP, limiting its accessibility. On the other hand, adding aluminum hydroxide adjuvant to the previously described SpyCatcher-M2e-decorated noro-VLP vaccine gave a strong response against M2e. Surprisingly, simple SpyCatcher-fused M2e without VLP display also functioned as a potent immunogen, which suggests that the commonly used protein linker SpyCatcher-SpyTag may serve a second role as an activator of the immune system in vaccine preparations. Based on the measured anti-M2e antibodies and cellular responses, both SpyCatcher-M2e as well as M2e presented on the noro-VLP via SpyTag/Catcher show potential for the development of universal influenza vaccines.

Visible light-induced specific protein reaction delineates early stages of cell adhesion.

Light is well established for control of bond breakage, but not for control of specific bond formation in complex environments. We previously engineered diffusion-limited reactivity of SpyTag003 peptide with its protein partner SpyCatcher003 through spontaneous transamidation. This system enables precise and irreversible assembly of biological building blocks, with applications from biomaterials to vaccines. Here, we establish a system for rapid control of this amide bond formation with visible light. We have generated a caged SpyCatcher003, which allows light triggering of covalent bond formation to SpyTag003 in mammalian cells. Photocaging is achieved through site-specific incorporation of an unnatural coumarin-lysine at the reactive site of SpyCatcher003. We showed uniform specific reaction in cell lysate upon light activation. We then used the spatiotemporal precision of a 405 nm confocal laser for uncaging in seconds, probing the earliest events in mechanotransduction by talin, the key force sensor between the cytoskeleton and extracellular matrix. Reconstituting talin induced rapid biphasic extension of lamellipodia, revealing the kinetics of talin-regulated cell spreading and polarization. Thereafter we determined the hierarchy of recruitment of key components for cell adhesion. Precise control over site-specific protein reaction with visible light creates diverse opportunities for cell biology and nanoassembly.

ConFERMing the role of talin in integrin activation and mechanosignaling.

Talin (herein referring to the talin-1 form), is a cytoskeletal adapter protein that binds integrin receptors and F-actin, and is a key factor in the formation and regulation of integrin-dependent cell-matrix adhesions. Talin forms the mechanical link between the cytoplasmic domain of integrins and the actin cytoskeleton. Through this linkage, talin is at the origin of mechanosignaling occurring at the plasma membrane-cytoskeleton interface. Despite its central position, talin is not able to fulfill its tasks alone, but requires help from kindlin and paxillin to detect and transform the mechanical tension along the integrin-talin-F-actin axis into intracellular signaling. The talin head forms a classical FERM domain, which is required to bind and regulate the conformation of the integrin receptor, as well as to induce intracellular force sensing. The FERM domain allows the strategic positioning of protein-protein and protein-lipid interfaces, including the membrane-binding and integrin affinity-regulating F1 loop, as well as the interaction with lipid-anchored Rap1 (Rap1a and Rap1b in mammals) GTPase. Here, we summarize the structural and regulatory features of talin and explain how it regulates cell adhesion and force transmission, as well as intracellular signaling at integrin-containing cell-matrix attachment sites.

Structural mechanism for inhibition of PP2A-B56α and oncogenicity by CIP2A.

The protein phosphatase 2A (PP2A) heterotrimer PP2A-B56α is a human tumour suppressor. However, the molecular mechanisms inhibiting PP2A-B56α in cancer are poorly understood. Here, we report molecular level details and structural mechanisms of PP2A-B56α inhibition by an oncoprotein CIP2A. Upon direct binding to PP2A-B56α trimer, CIP2A displaces the PP2A-A subunit and thereby hijacks both the B56α, and the catalytic PP2Ac subunit to form a CIP2A-B56α-PP2Ac pseudotrimer. Further, CIP2A competes with B56α substrate binding by blocking the LxxIxE-motif substrate binding pocket on B56α. Relevant to oncogenic activity of CIP2A across human cancers, the N-terminal head domain-mediated interaction with B56α stabilizes CIP2A protein. Functionally, CRISPR/Cas9-mediated single amino acid mutagenesis of the head domain blunted MYC expression and MEK phosphorylation, and abrogated triple-negative breast cancer in vivo tumour growth. Collectively, we discover a unique multi-step hijack and mute protein complex regulation mechanism resulting in tumour suppressor PP2A-B56α inhibition. Further, the results unfold a structural determinant for the oncogenic activity of CIP2A, potentially facilitating therapeutic modulation of CIP2A in cancer and other diseases.

Microfluidics-Based Force Spectroscopy Enables High-Throughput Force Experiments with Sub-Nanometer Resolution and Sub-Piconewton Sensitivity.

Several techniques have been established to quantify the mechanicals of single molecules. However, most of them show only limited capabilities of parallelizing the measurement by performing many individual measurements simultaneously. Herein, a microfluidics-based single-molecule force spectroscopy method, which achieves sub-nanometer spatial resolution and sub-piconewton sensitivity and is capable of simultaneously quantifying hundreds of single-molecule targets in parallel, is presented. It relies on a combination of total internal reflection microscopy and microfluidics, in which monodisperse fluorescent beads are immobilized on the bottom of a microfluidic channel by macromolecular linkers. Application of a flow generates a well-defined shear force acting on the beads, whereas the nanomechanical linker response is quantified based on the force-induced displacement of individual beads. To handle the high amount of data generated, a cluster analysis which is capable of a semi-automatic identification of measurement artifacts and molecular populations is implemented. The method is validated by probing the mechanical response polyethylene glycol linkers and binding strength of biotin-NeutrAvidin complexes. Two energy barriers (at 3 and 5.7 Å, respectively) in the biotin-NeutrAvidin interaction are resolved and the unfolding behavior of talin's rod domain R3 in the force range between 1 to ≈10 pN is probed.

The mechanical cell - the role of force dependencies in synchronising protein interaction networks.

The role of mechanical signals in the proper functioning of organisms is increasingly recognised, and every cell senses physical forces and responds to them. These forces are generated both from outside the cell or via the sophisticated force-generation machinery of the cell, the cytoskeleton. All regions of the cell are connected via mechanical linkages, enabling the whole cell to function as a mechanical system. In this Review, we define some of the key concepts of how this machinery functions, highlighting the critical requirement for mechanosensory proteins, and conceptualise the coupling of mechanical linkages to mechanochemical switches that enables forces to be converted into biological signals. These mechanical couplings provide a mechanism for how mechanical crosstalk might coordinate the entire cell, its neighbours, extending into whole collections of cells, in tissues and in organs, and ultimately in the coordination and operation of entire organisms. Consequently, many diseases manifest through defects in this machinery, which we map onto schematics of the mechanical linkages within a cell. This mapping approach paves the way for the identification of additional linkages between mechanosignalling pathways and so might identify treatments for diseases, where mechanical connections are affected by mutations or where individual force-regulated components are defective.

Coxsackievirus B infections are common in Cystic Fibrosis and experimental evidence supports protection by vaccination.

Viral respiratory tract infections exacerbate airway disease and facilitate life-threatening bacterial colonization in cystic fibrosis (CF). Annual influenza vaccination is recommended and vaccines against other common respiratory viruses may further reduce pulmonary morbidity risk. Enteroviruses have been found in nasopharyngeal samples from CF patients experiencing pulmonary exacerbations. Using serology tests, we found that infections by a group of enteroviruses, Coxsackievirus Bs (CVBs), are prevalent in CF. We next showed that a CVB vaccine, currently undergoing clinical development, prevents infection and CVB-instigated lung damage in a murine model of CF. Finally, we demonstrate that individuals with CF have normal vaccine responses to a similar, commonly used enterovirus vaccine (inactivated poliovirus vaccine). Our study demonstrates that CVB infections are common in CF and provides experimental evidence indicating that CVB vaccines could be efficacious in the CF population. The role of CVB infections in contributing to pulmonary exacerbations in CF should be further studied.

Biochemical and Biophysical Characterization of Carbonic Anhydrase VI from Human Milk and Saliva.

Carbonic anhydrases (CA, EC 4.2.1.1) catalyze the hydration of carbon dioxide and take part in many essential physiological processes. In humans, 15 CAs are characterized, including the only secreted isoenzyme CA VI. CA VI has been linked to specific processes in the mouth, namely bitter taste perception, dental caries, and maintenance of enamel pellicle, and implicated in several immunity-related phenomena. However, little is known of the mechanisms of the above. In this study, we characterized human CA VI purified from saliva and milk with biophysical methods and measured their enzyme activities and acetazolamide inhibition. Size-exclusion chromatography showed peaks of salivary and milk CA VI corresponding to hexameric state or larger at pH 7.5. At pH 5.0 the hexamer peaks dominated. SDS- PAGE of milk CA VI protein treated with a bifunctional crosslinker further confirmed that a majority of CA VI is oligomers of similar sizes in solution. Mass spectrometry experiments confirmed that both of the two putative N-glycosylation sites, Asn67 and Asn256, are heterogeneously glycosylated. The attached glycans in milk CA VI were di- and triantennary complex-type glycans, carrying both a core fucose and 1 to 2 additional fucose units, whereas the glycans in salivary CA VI were smaller, seemingly degraded forms of core fucosylated complex- or hybrid-type glycans. Mass spectrometry also verified the predicted signal peptide cleavage site and the terminal residue, Gln 18, being in pyroglutamate form. Thorough characterization of CA VI paves way to better understanding of the biological function of the protein.

Talin variant P229S compromises integrin activation and associates with multifaceted clinical symptoms.

Adhesion of cells to the extracellular matrix (ECM) must be exquisitely coordinated to enable development and tissue homeostasis. Cell-ECM interactions are regulated by multiple signalling pathways that coordinate the activation state of the integrin family of ECM receptors. The protein talin is pivotal in this process, and talin's simultaneous interactions with the cytoplasmic tails of the integrins and the plasma membrane are essential to enable robust, dynamic control of integrin activation and cell-ECM adhesion. Here, we report the identification of a de novo heterozygous c.685C>T (p.Pro229Ser) variant in the TLN1 gene from a patient with a complex phenotype. The mutation is located in the talin head region at the interface between the F2 and F3 domains. The characterization of this novel p.P229S talin variant reveals the disruption of adhesion dynamics that result from disturbance of the F2-F3 domain interface in the talin head. Using biophysical, computational and cell biological techniques, we find that the variant perturbs the synergy between the integrin-binding F3 and the membrane-binding F2 domains, compromising integrin activation, adhesion and cell migration. Whilst this remains a variant of uncertain significance, it is probable that the dysregulation of adhesion dynamics we observe in cells contributes to the multifaceted clinical symptoms of the patient and may provide insight into the multitude of cellular processes dependent on talin-mediated adhesion dynamics.

A reverse vaccinology approach on transmembrane carbonic anhydrases from Plasmodium species as vaccine candidates for malaria prevention.

BACKGROUND: Malaria is a significant parasitic infection, and human infection is mediated by mosquito (Anopheles) biting and subsequent transmission of protozoa (Plasmodium) to the blood. Carbonic anhydrases (CAs) are known to be highly expressed in the midgut and ectoperitrophic space of Anopheles gambiae. Transmembrane CAs (tmCAs) in Plasmodium may be potential vaccine candidates for the control and prevention of malaria. METHODS: In this study, two groups of transmembrane CAs, including α-CAs and one group of η-CAs were analysed by immunoinformatics and computational biology methods, such as predictions on transmembrane localization of CAs from Plasmodium spp., affinity and stability of different HLA classes, antigenicity of tmCA peptides, epitope and proteasomal cleavage of Plasmodium tmCAs, accessibility of Plasmodium tmCAs MHC-ligands, allergenicity of Plasmodium tmCAs, disulfide-bond of Plasmodium tmCAs, B cell epitopes of Plasmodium tmCAs, and Cell type-specific expression of Plasmodium CAs. RESULTS: Two groups of α-CAs and one group of η-CAs in Plasmodium spp. were identified to contain tmCA sequences, having high affinity towards MHCs, high stability, and strong antigenicity. All putative tmCAs were predicted to contain sequences for proteasomal cleavage in antigen presenting cells (APCs). CONCLUSIONS: The predicted results revealed that tmCAs from Plasmodium spp. can be potential targets for vaccination against malaria.