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The monoclonal antibody CIS43 targets the Plasmodium falciparum circumsporozoite protein (PfCSP) and prevents malaria infection in humans for up to 9 mo following a single intravenous administration. To enhance the potency and clinical utility of CIS43, we used iterative site-saturation mutagenesis and DNA shuffling to screen precise gene-variant yeast display libraries for improved PfCSP antigen recognition. We identified several mutations that improved recognition, predominately in framework regions, and combined these to produce a panel of antibody variants. The most improved antibody, CIS43_Var10, had three mutations and showed approximately sixfold enhanced protective potency in vivo compared to CIS43. Co-crystal and cryo-electron microscopy structures of CIS43_Var10 with the peptide epitope or with PfCSP, respectively, revealed functional roles for each of these mutations. The unbiased site-directed mutagenesis and screening pipeline described here represent a powerful approach to enhance protective potency and to enable broader clinical use of antimalarial antibodies.

Female mosquitoes require blood meals for egg development. The saliva of blood feeding arthropods contains biochemically active molecules, whose anti-hemostatic and anti-inflammatory properties facilitate blood feeding on vertebrate hosts. While transcriptomics has presented new opportunities to investigate the diversity of salivary proteins from hematophagous arthropods, many of these proteins remain functionally undescribed. Previous transcriptomic analysis of female salivary glands from Culex quinquefasciatus, an important vector of parasitic and viral infections, uncovered a 12-member family of putatively secreted proteins of unknown function, named the Cysteine and Tryptophan-Rich (CWRC) proteins. Here, we present advances in the characterization of two C. quinquefasciatus CWRC family members, CqDVP-2 and CqDVP-4, including their enrichment in female salivary glands, their specific localization within salivary gland tissues, evidence that these proteins are secreted into the saliva, and their native crystal structures, at 2.3 ​A and 1.87 ​A, respectively. The β-trefoil fold common to CqDVP-2 and CqDVP-4 is similar to carbohydrate-binding proteins, including the B subunit of the AB toxin, ricin, from the castor bean Ricinus communis. Further, we used a glycan array approach, which identifies carbohydrate ligands associated with inflammatory processes and signal transduction. Glycan array 300 testing identified 100 carbohydrate moieties with positive binding to CqDVP-2, and 77 glycans with positive binding to CqDVP-4. The glycan with the highest relative fluorescence intensities, which exhibited binding to both CqDVP-2 and CqDVP-4, was used for molecular docking experiments. We hypothesize that these proteins bind to carbohydrates on the surface of cells important to host immunology. Given that saliva is deposited into the skin during a mosquito bite, and acts as the vehicle for arbovirus inoculation, understanding the role of these proteins in pathogen transmission is of critical importance. This work presents the first solved crystal structures of C. quinquefasciatus salivary proteins with unknown function. These two molecules are the second and third structures reported from salivary proteins from C. quinquefasciatus, an important, yet understudied disease vector.

Aedes aegypti saliva facilitates blood meal acquisition through pharmacologically active compounds that prevent host hemostasis. Among these salivary proteins are the D7s, which are highly abundant and have been shown to act as scavengers of biogenic amines and eicosanoids. In this work, we performed comparative structural modeling, characterized the binding capabilities, and assessed the physiological functions of the Ae. aegypti salivary protein AeD7L2 compared to the well-characterized AeD7L1. AeD7L1 and AeD7L2 show different binding affinities to several biogenic amines and biolipids involved in host hemostasis. Interestingly, AeD7L2 tightly binds U-46619, the stable analog of thromboxane A(2) (K(D) = 69.4 nm), which is an important platelet aggregation mediator, while AeD7L1 shows no binding. We tested the ability of these proteins to interfere with the three branches of hemostasis: vasoconstriction, platelet aggregation, and blood coagulation. Pressure myography experiments showed these two proteins reversed isolated resistance artery vasoconstriction induced by either norepinephrine or U-46619. These proteins also inhibited platelet aggregation induced by low doses of collagen or U-46619. However, D7 long proteins did not affect blood coagulation. The different ligand specificity and affinities of AeD7L1 and AeD7L2 matched our experimental observations from studying their effects on vasoconstriction and platelet aggregation, which confirm their role in preventing host hemostasis. This work highlights the complex yet highly specific biological activities of mosquito salivary proteins and serves as another example of the sophisticated biology underlying arthropod blood feeding.

L9 is a potent human monoclonal antibody (mAb) that preferentially binds two adjacent NVDP minor repeats and cross-reacts with NANP major repeats of thePlasmodium falciparumcircumsporozoite protein (PfCSP) on malaria-infective sporozoites. Understanding this mAb’s ontogeny and mechanisms of binding PfCSP to neutralize sporozoites will facilitate vaccine development. Here, we isolated mAbs clonally related to L9 and showed that this B-cell lineage has baseline NVDP affinity and evolves to acquire NANP reactivity. Pairing the L9 kappa light chain (L9κ) with clonally-related heavy chains resulted in chimeric mAbs that cross-linked two NVDP, cross-reacted with NANP, and more potently neutralized sporozoites compared to their original light chain. Structural analyses revealed that chimeric mAbs bound the minor repeat motif in a type-1 β-turn seen in other repeat-specific antibodies. These data highlight the importance of L9κ in binding NVDP on PfCSP to neutralize SPZ and suggest that PfCSP-based immunogens might be improved by presenting ≥2 NVDP.

L9 is a potent human monoclonal antibody (mAb) that preferentially binds two adjacent NVDP minor repeats and cross-reacts with NANP major repeats of the Plasmodium falciparum circumsporozoite protein (PfCSP) on malaria-infective sporozoites. Understanding this mAb's ontogeny and mechanisms of binding PfCSP will facilitate vaccine development. Here, we isolate mAbs clonally related to L9 and show that this B cell lineage has baseline NVDP affinity and evolves to acquire NANP reactivity. Pairing the L9 kappa light chain (L9κ) with clonally related heavy chains results in chimeric mAbs that cross-link two NVDPs, cross-react with NANP, and more potently neutralize sporozoites in vivo compared with their original light chain. Structural analyses reveal that the chimeric mAbs bound minor repeats in a type-1 β-turn seen in other repeat-specific antibodies. These data highlight the importance of L9κ in binding NVDP on PfCSP to neutralize sporozoites and suggest that PfCSP-based immunogens might be improved by presenting ≥2 NVDPs.

Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.

Female mosquitoes require blood meals for egg development. The saliva of blood feeding arthropods contains biochemically active molecules, whose anti-hemostatic and anti-inflammatory properties facilitate blood feeding on vertebrate hosts. While transcriptomics has presented new opportunities to investigate the diversity of salivary proteins from hematophagous arthropods, many of these proteins remain functionally undescribed. Previous transcriptomic analysis of female salivary glands from Culex quinquefasciatus, an important vector of parasitic and viral infections, uncovered a 12-member family of putatively secreted proteins of unknown function, named the Cysteine and Tryptophan-Rich (CWRC) proteins. Here, we present advances in the characterization of two C. quinquefasciatus CWRC family members, CqDVP-2 and CqDVP-4, including their enrichment in female salivary glands, their specific localization within salivary gland tissues, evidence that these proteins are secreted into the saliva, and their native crystal structures, at 2.3 ​Å and 1.87 ​Å, respectively. The β-trefoil fold common to CqDVP-2 and CqDVP-4 is similar to carbohydrate-binding proteins, including the B subunit of the AB toxin, ricin, from the castor bean Ricinus communis. Further, we used a glycan array approach, which identifies carbohydrate ligands associated with inflammatory processes and signal transduction. Glycan array 300 testing identified 100 carbohydrate moieties with positive binding to CqDVP-2, and 77 glycans with positive binding to CqDVP-4. The glycan with the highest relative fluorescence intensities, which exhibited binding to both CqDVP-2 and CqDVP-4, was used for molecular docking experiments. We hypothesize that these proteins bind to carbohydrates on the surface of cells important to host immunology. Given that saliva is deposited into the skin during a mosquito bite, and acts as the vehicle for arbovirus inoculation, understanding the role of these proteins in pathogen transmission is of critical importance. This work presents the first solved crystal structures of C. quinquefasciatus salivary proteins with unknown function. These two molecules are the second and third structures reported from salivary proteins from C. quinquefasciatus, an important, yet understudied disease vector., Graphical abstract Image 1, Highlights • Saliva of blood feeding mosquitoes contains biochemically active molecules that facilitate blood feeding and pathogen transmission. • CqDVP-2 and CqDVP-4, two major proteins from Culex quinquefasciatus, are specific to adult female salivary glands and are secreted into the saliva. • Sex and tissue specificity of these proteins indicate their possible function(s) in blood feeding and pathogen transmission. • The native crystal structures of CqDVP-2 and CqDVP-4 have a ß-trefoil fold similar to carbohydrate-binding proteins. • CqDVP protein binding to carbohydrates is thought to play a role in inflammation and may be an evolutionary adaptation to blood feeding and help improve mosquito survival.

Adult female mosquitoes require a vertebrate blood meal to develop eggs and continue their life cycle. During blood feeding, mosquito saliva is injected at the bite site to facilitate blood meal acquisition through anti-hemostatic compounds that counteract blood clotting, platelet aggregation, vasoconstriction and host immune responses. D7 proteins are among the most abundant components of the salivary glands of several blood feeding insects. They are members of a family of proteins that have evolved through gene duplication events to encode D7 proteins of several lengths. Here, we examine the ligand binding specificity and physiological relevance of two D7 long proteins, CxD7L1 and CxD7L2, fromCulex quinquefasciatusmosquitoes, the vector of medical and veterinary diseases such as filariasis, avian malaria, and West Nile virus infections. CxD7L1 and CxD7L2 were assayed by microcalorimetry for binding of potential host ligands involved in hemostasis, including bioactive lipids, biogenic amines, and nucleotides/nucleosides. CxD7L2 binds serotonin, histamine, and epinephrine with high affinity as well as the thromboxane A2 analog U-46619 and several cysteinyl leukotrienes, as previously described for other D7 proteins. CxD7L1 does not bind any of the ligands that are bound by CxD7L2. Unexpectedly, CxD7L1 exhibited high affinity for adenine nucleotides and nucleosides, a binding capacity not reported in any D7 family member. We solved the crystal structure of CxD7L1 in complex with bound ADP to 1.97 Å resolution. The binding pocket for ADP is located between the two domains of CxD7L1, whereas all known D7s bind ligands either within the N-terminal or the C-terminal domains. We demonstrated that these two CxD7 long proteins inhibit human platelet aggregation inex vivoexperiments. CxD7L1 and CxD7L2 help blood feeding in mosquitoes by scavenging host molecules that promote vasoconstriction, platelet aggregation, itch, and pain at the bite site. The novel ADP-binding function acquired by CxD7L1 evolved to enhance blood feeding in mammals where ADP plays a key role in platelet aggregation.

During blood-feeding, mosquito saliva is injected into the skin to facilitate blood meal acquisition. D7 proteins are among the most abundant components of the mosquito saliva. Here we report the ligand binding specificity and physiological relevance of two D7 long proteins from Culex quinquefasciatus mosquito, the vector of filaria parasites or West Nile viruses. CxD7L2 binds biogenic amines and eicosanoids. CxD7L1 exhibits high affinity for ADP and ATP, a binding capacity not reported in any D7. We solve the crystal structure of CxD7L1 in complex with ADP to 1.97 Å resolution. The binding pocket lies between the two protein domains, whereas all known D7s bind ligands either within the N- or the C-terminal domains. We demonstrate that these proteins inhibit hemostasis in ex vivo and in vivo experiments. Our results suggest that the ADP-binding function acquired by CxD7L1 evolved to enhance blood-feeding in mammals, where ADP plays a key role in platelet aggregation., D7 proteins are highly abundant in the salivary glands of several blood feeding insects. Here, the authors study the ligand binding specificity and physiological roles of the mosquito D7 proteins CxD7L1 and CxD7L2, showing that CxD7L1 acquired ADP-binding properties to enhance blood feeding in mammals.